Non-coding RNAs, a double-edged sword in breast cancer prognosis

Abstract

Cancer is a rising issue worldwide, and numerous studies have focused on understanding the underlying reasons for its occurrence and finding proper ways to defeat it. By applying technological advances, researchers are continuously uncovering and updating treatments in cancer therapy. Their vast functions in the regulation of cell growth and proliferation and their significant role in the progression of diseases, including cancer. This review provides a comprehensive analysis of ncRNAs in breast cancer, focusing on long non-coding RNAs such as HOTAIR, MALAT1, and NEAT1, as well as microRNAs such as miR-21, miR-221/222, and miR-155. These ncRNAs are pivotal in regulating cell proliferation, metastasis, drug resistance, and apoptosis. Additionally, we discuss experimental approaches that are useful for studying them and highlight the advantages and challenges of each method. We then explain the results of these clinical trials and offer insights for future studies by discussing major existing gaps. On the basis of an extensive number of studies, this review provides valuable insights into the potential of ncRNAs in cancer therapy. Key findings show that even though the functions of ncRNAs are vast and undeniable in cancer, there are still complications associated with their therapeutic use. Moreover, there is an absence of sufficient experiments regarding their application in mouse models, which is an area to work on. By emphasizing the crucial role of ncRNAs, this review underscores the need for innovative approaches and further studies to explore their potential in cancer therapy.

Graphical Abstract

Introduction

Breast cancer is a global issue that affects women around the world. In 2020, the World Health Organization reported that 2.3 million women were diagnosed with it and that 685,000 of those women lost their lives to it [1]. Moreover, it is forecasted that by 2040, these rates will increase to 3 million new cases and 1 million deaths [2]. The statistics can be observed in Fig. 1. These rates highlight the undeniable importance of research into the underlying mechanisms and precursors contributing to the rise of breast cancer and the need to develop effective strategies to overcome it.

Fig. 1

Breast cancer statistics

Breast cancer is classified into four main subtypes: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-positive, and triple-negative breast cancer (TNBC). These subtypes differ in their molecular profiles, prognoses, and treatment strategies (Fig. 2). Luminal subtypes, which are characterized by hormone receptor positivity, are often treated with hormonal therapies, whereas the HER2-positive and TNBC subtypes are more aggressive and resistant to standard treatments [3,4,5,6,7,8,9,10,11,12]. Understanding the molecular characteristics of these subtypes is essential for identifying therapeutic targets that could improve outcomes.

Fig. 2

Classification of breast cancer and their characteristics

Current breast cancer treatments, including surgery, radiation, chemotherapy, and targeted therapies, face significant challenges, such as therapy resistance and adverse side effects [13, 14]. For example, resistance to conventional therapies often arises from changes in key regulatory pathways, such as phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR), which lead to drug resistance [15]. Moreover, side effects such as heart disease, bone issues, and ovarian failure following treatment highlight the limitations of existing approaches [16, 17]. Therefore, there is a need to identify new and effective therapeutic targets for treating different stages of breast cancer. Understanding and investigating the factors involved in its formation and progression is crucial for designing more practical and targeted treatments.

There are different types of RNAs in cells, and they can be coding or non-coding. In higher organisms, less than 3% of the genes that are transcribed are encoded to proteins, and projects such as the Encyclopedia of DNA Elements (ENCODE) suggest that 80% of the genome is transcribed to non-coding RNA (ncRNA). Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are two types of well-known ncRNAs [18]. These RNAs can play a significant role in breast cancer progression and drug resistance [19] and therefore have emerged as promising candidates for breast cancer treatment. Non-coding RNAs are instrumental in various cancer pathways [20] because of their wide range of interactions with materials such as DNA, mRNA proteins, and other non-coding RNAs [21,22,23]. Dysregulated miRNAs can play a role in maintaining proliferative signals, evading growth suppressors, resisting cell death, activating invasion and metastasis, and promoting the formation of new blood vessels (angiogenesis) [24,25,26,27,28]. Recent research has shown that secreted miRNAs can act as ligands to activate premetastatic inflammatory responses inside the tumor microenvironment [29]. MicroRNA-21’s (miR-21) dysregulation in various organs, such as the breast and brain, is linked to apoptosis (the process of programmed cell death), metastasis, and cell growth and differentiation pathways [30,31,32].

Another type of non-coding RNA known as PIWI-interacting RNAs (piRNAs) regulate cancer cell metastasis, tumor cell proliferation, apoptosis, and invasion [33, 34]. In addition, the levels of piR-4987 in breast cancer are positively associated with lymph node metastases [35]. piR-823 affects transcriptional activity, apoptosis, DNA methylation, metastasis, and cell proliferation [36,37,38].

Several lncRNAs have been studied and linked with cancer through their roles in angiogenesis, posttranscriptional gene regulation, metastasis, cell proliferation, and treatment response [26, 39]. For example, HOX transcript antisense intergenic RNA’s (HOTAIR) dysregulation in the thyroid, prostate, ovarian, endometrial, and lung can lead to methylation and subsequent silencing of tumor suppressor genes [30, 40, 41]. In response to stress, H19 induces survival pathways and epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions, and this non-coding RNA is dysregulated in the bladder, ovarian, endometrial, breast, prostate, and colorectal regions [42]. The long non-coding RNA X-inactivation-specific transcript (XIST) is an important factor in the development of breast cancer [43]. Moreover, some studies have indicated that the overexpression of XIST can repress cancer cell growth and invasion [44, 45].

As highlighted above, the role of ncRNAs in cancer is vast and complex and is constantly being updated and identified. Given the importance of their function in various serious diseases, our aim in this review is to provide a general classification of non-coding RNAs, highlight the timeline of their discovery, and explain their role in breast cancer. Later, explore the advantages of targeting non-coding RNAs, discuss experimental approaches, and finally, review clinical trials involving non-coding RNAs were reviewed to present prospects and challenges.

Brief history of non-coding RNAs

The discovery of ribosomal RNA (rRNA) was first documented by Georges Palade in 1955 via micrographs obtained from electron microscopy [46]. Two years later, Francis Crick proposed a fundamental framework, the Central Dogma, which explains how genetic information flows from DNA to RNA to protein during gene expression [47, 48]. Later, in 1958, Paul Zamecnik and colleagues led groundbreaking research on transfer RNA (tRNA), a crucial element in the translation process [49]. In 1968, small nuclear RNAs (snRNAs) were identified in the laboratories of Harris Busch and Sheldon Penman [50, 51], increasing the complexity of our understanding of RNA regulatory activities. Sanger et al. reported the existence of circular RNA (circRNA) in 1976 [52], and the first lncRNA, H19, was discovered in 1990 by Brannan et al. [53], which was a great milestone in the investigation of ncRNA. In 1993, another revolutionary ncRNA, microRNA (miRNA), was discovered in the roundworm Caenorhabditis elegans by Lee and colleagues [54,55,56]. In 1998, Andrew Fire and Craig Mello described RNA interference (RNAi), which inactivates genes by corrupting the corresponding mRNAs in the nematode Caenorhabditis elegans [57]. The journey of ncRNAs continues, and important discoveries were made in the late 1990s and early 2000s. David Baulcombe's group published a paper in 1999 suggesting the existence of small interfering RNA (siRNA) in plants [58]. Three years later, Calin and Croce reported a connection between dysregulated miR-16–1 and miR-15a and chronic lymphocytic leukemia (CLL) [59], providing insights into the complex interactions of ncRNAs and cancer. In 2003, Lee et al. discovered that human Drosha is the core nuclease responsible for initiating miRNA processing in the nucleus [60], providing more knowledge on the molecular mechanisms involved. Two years later, a technical breakthrough occurred when researchers presented an integrated system that could efficiently handle large sequences, leading to advances in whole-genome sequencing [61]. In the coming years, scientists have performed different studies with different RNA-seq methodologies. 454 Sequencing was employed for deep sequencing, making it easier to identify uncommon and rare transcripts despite a limited sequence portion [62]. Building on these innovations, in 2006, the first comprehensive DNA methylation map of an entire genome was reported for Arabidopsis thaliana with 35 base pair resolutions [63]. In 2012, the National Human Genome Institute (NHGI) and the ENCODE project announced a significant discovery: three-quarters of the human genome can be transcribed [64], revealing a new landscape in the genome world. Our understanding became more complicated than ever, as we discovered the range, expression levels, localization, and processing fates of both known and unknown RNAs [65]. Following academic exploration, ncRNAs entered medicinal approaches as miRNA therapeutics in Austin, Texas, which launched phase 1 studies for liposomal miR-34a mimic (MRX34), the first microRNA mimic, in April 2013 [66]. The non-coding RNA timeline is shown in Table 1.

Table 1 Non-coding RNA timeline

Classification of non-coding RNAs

Non-coding RNAs can be divided into two groups: housekeeping and regulatory [67]. Housekeeping ncRNAs, such as rRNA and tRNA, are involved in messenger RNA (mRNA) translation, whereas regulatory ncRNAs are created in response to an external trigger [68]. In terms of transcript size, ncRNAs can be divided into two domains: small ncRNAs (200 nucleotides) and long ncRNAs (> 200 nucleotides) [69]. RNA maturation, RNA processing, signaling, gene expression, and protein synthesis are important areas in which ncRNAs play a role [70,71,72].

lncRNAs are important non-coding RNAs that contain at least 20,000 distinct genes [73]. Some lncRNAs specifically interact with RNA, DNA, and proteins in a sequence-dependent manner (e.g., non-coding RNA activated by DNA damage (NORAD), nuclear enriched abundant transcript 1 (NEAT1), and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)) [74]. LncRNAs can overlap with or cis-regulate DNA regulatory elements such as enhancers to harness PCGs [75, 76]. In general, HOX transcript antisense RNA (HOTAIR), nuclear enriched abundant transcript 1 (NEAT1), and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) are among the most researched long non-coding RNAs. HOTAIR is a transcript of the homeobox C (HOXC) gene and has multiple functions in cancer and metastasis [77]. NEAT plays a role as a tumor oncogene in several cancers [78,79,80]. MALAT1 has been found to increase proliferation and hinder cell death and apoptosis [81].

Among small non-coding RNAs, microRNAs (miRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) are the most studied. miRNAs can activate gene expression [82]. Recent studies suggest that miRNAs shuttle between subcellular compartments to regulate translation and transcription [83]. Abnormal expression of microRNAs is linked with numerous human diseases [84, 85]. In addition, extracellular miRNAs, which serve as signaling molecules, have been widely reported as potential biomarkers for a variety of diseases [86,87,88].

A biomarker, or biological marker, is an objective indicator that reflects the biological activity of a cell or organism at a specific time. Biomarkers improve our understanding of the connections between environmental chemicals and human diseases and improve our ability to diagnose, monitor, and predict disease risk, including cancers [89]. Therefore, ncRNAs such as miRNAs can be considered reliable indicators for various pathological conditions.

Eukaryotic cells contain small, highly abundant, nucleus-localized non-coding RNAs (snRNAs), which play important roles in the splicing of introns from primary genomic transcripts [90]. snRNAs are ribonucleoprotein particles (snRNPs) along with additional proteins that create a large particulate complex (spliceosome) bound to unsliced primary RNA transcripts to facilitate the process [91]. In addition to splicing, snRNPs also function in the nuclear maturation of primary transcripts in mRNAs, the splicing of donors in noncanonical systems, the regulation of gene expression, and 3′-end processing of replication-dependent histone mRNAs [92]. Small nucleolar RNAs (snoRNAs) are a type of non-coding RNA that are ubiquitous in the nucleoli of eukaryotic cells. They play an essential role in modifying ribosomal RNA (rRNA), which is critical for the proper functioning of the ribosome and protein synthesis. Studies have shown that snoRNAs, both those that promote and suppress tumors, not only affect tumors but also have a significant effect on the prognosis of patients [93]. Piwi-interacting RNAs (piRNAs) are short non-coding RNAs that play important roles in genome integrity by regulating transposons [94]. They are also linked to major cancer hallmarks, including proliferation, invasion, and chemoresistance, and have the potential to be used as biomarkers for cancer diagnosis and prognosis [95]. Small interfering RNAs (siRNAs) are double-stranded RNA molecules that play important roles in eukaryotic gene regulation after transcription [96]. Recently, they have been known as a naturally occurring gene-silencing mechanism [97] Table 2.

Table 2 Classification of non-coding RNAs

Role of non-coding RNAs in breast cancer

Our review focused on the role of non-coding RNAs in breast cancer. The two most investigated ncRNAs in breast cancer are miRNAs and long non-coding RNAs (lncRNAs), which we discuss below.

Long non-coding RNAs

Data obtained from The Cancer Genome Atlas (TCGA) revealed approximately 1059 dysregulated lncRNAs in breast cancer tissues [98]. Studies over the last few decades have shown a link between patient survival rates and the recurrence of breast cancer and the lncRNAs involved in the disease [99, 100], highlighting its crucial impact and the need for further research. Below, we briefly discuss several lncRNAs and their molecular pathways in breast cancer.

HOTAIR

HOX transcript antisense intergenic RNA (HOTAIR) is involved in various signaling pathways in breast cancer. The expression of HOTAIR is significantly greater in breast cancer tissues than in normal tissues, and through its inhibition, the migration and proliferation of cancer cells can be reduced [101]. HOTAIR impacts breast cancer progression by restricting miRNAs, which can act as tumor suppressors and activate pro-oncogenic miRNAs, leading to the expression of epithelial-to-mesenchymal transition (EMT)-related proteins that are important in metastasis. EMT is a complex biological process that results in epithelial cells transitioning to mesenchymal cells; it is linked with cancer because it provides motility for cancer cells and aids in their spread [102, 103]. Wenxing He et al. reported that HOTAIR reduces the level of miR-130a-3p, leading to the regulation of the suppressor of variegation 3–9 homolog 1 (Suv39H1)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in BC cells, which promotes the growth and metastasis of BC cells [104]. HOTAIR promotes EMT by regulating the miR-129-5p/Frizzled-7 (FZD7) axis [105] and E-cadherin, N-cadherin and vimentin [106]. It also activates AKT, which promotes metastasis by activating the miR-601/zinc finger E-box-binding homeobox 1 (ZEB1) axis [107]. Furthermore, HOTAIR can activate angiogenesis via activation of the glucose-regulated protein 78 (GRP78)/angiopoietin-2 (Ang2) axis [105]. Additionally, it can be correlated with autophagy, a cellular process that degrades and recycles damaged components to maintain homeostasis under stress, which is essential for cancer cell viability [108]. HOTAIR is detectable in circulating exosomes and has been linked to erythroblastic oncogene B2 (ErbB2)/HER2-positive breast cancer, making it a potential biomarker for liquid biopsy, a minimally invasive method to detect tumor-related materials in blood [109]. A study in 2022 revealed that HOTAIR increases resistance to breast cancer radiation by increasing the expression of heat shock protein family A (Hsp70) member 1A (HSPA1A). MiR-449b-5p inhibits HSPA1A expression by targeting its mRNA, but HOTAIR interferes with this process by sponging miR-449b-5p, leading to the recovery of HSPA1A expression during radiation [110]. Finally, HOTAIR has been identified as a potential target for medication [111], and its expression is related to drug resistance [19]. For example, Tianwen Chen et al. highlighted the upregulation of HOTAIR with resistance against trastuzumab drug in SK-BR-3-TR cell lines and suggested that blocking its expression can restore sensitivity [112]. HOTAIR is known as a promising specific biomarker for breast cancer, and its overexpression has been documented in both primary and metastatic cases. Studies suggest that detecting elevated HOTAIR levels in blood or tissue samples could serve as an indicator of the presence of breast cancer and may offer insights into its aggressiveness and metastatic potential. These findings highlight the potential of HOTAIR as a critical diagnostic and prognostic tool in breast cancer management [105, 113, 114].

MALAT1

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is another long non-coding RNA that plays an instrumental role in cancer progression. MALAT1 is often linked to poor outcomes in many types of cancer, including breast cancer, making it a potential prognostic marker. Studies have shown that MALAT1 is most highly expressed in the later stages of breast cancer (stages III and IV), where it is mostly associated with increased metastasis and worse outcomes. These findings indicate that its levels tend to increase as breast cancer becomes more advanced [115, 116]. A study in 2015 demonstrated that MALAT 1 is downregulated in breast cancer cells and tissue and that its knockdown can increase EMT through the phosphatidylinositide-3 kinase (PI3K)-AKT pathway [117]. Research in 2021 revealed that the expression of MALAT1 is increased in breast cancer cells and that MALAT1 can target miR-570–3p, which is important in the development of resistance in cancer cells to doxorubicin [118]. The results of another study correlate with previous research and indicate that when MALAT1 is knocked down, proliferation is reduced, suggesting that MALAT1 trans-targets the Eukaryotic Translation Elongation Factor 1 Alpha 1 (EEF1A1) promoter, which leads to increased tumor progression in breast cancer cells [119]. MALAT1 can regulate breast cancer progression by downregulating miR-101-3p and stimulating the expression of the mTOR/pyruvate kinase M2 (PKM2) pathway, which is negatively associated with the level of miR-101-3p, suggesting that MALAT1 might control breast cancer progression via upregulation of the mTOR/PKM2 pathway through the sponging of miR-101-3p. This process leads to the proliferation, migration and invasion of breast cancer cells [120]. MALAT1 can act as a competing endogenous RNA (ceRNA) for miR-26a/26b in breast cancer cells and weaken its repressive impact on ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 (ST8SIA4) gene expression. The MALAT1/miR-26a/26b/ST8SIA4 axis significantly influences the proliferation, invasion, and migration of breast cancer cells [121]. Furthermore, it has been discovered that MALAT1 activates angiogenesis in breast cancer, possibly as a result of its interaction with miR-145 [122]. Additionally, genetic variations in MALAT1 have been related to an increased risk of breast cancer, whereas other variants have been linked to a reduced risk of the disease [123]. MALAT1 has been shown to play a role in the growth and metastasis of triple-negative and Her-2-positive breast tumors, suggesting that it might be a useful prognostic indicator and therapeutic target [124].

NEAT1

Various studies have highlighted the impact of nuclear paraspeckle assembly transcript 1 (NEAT1) in breast cancer [125,126,127]. NEAT1 targets miR-146b-5p, which stimulates EMT, proliferation and invasion, suggesting that, by silencing the expression of NEAT1, the invasion of cancer cells can be stopped [128]. NEAT1 can also participate in the regulation of the miR-410-3p/cyclin D1 (CCND1) axis and increase breast cancer progression [129]. It plays a role in the inhibition of miR-448 and the increase in ZEB1, leading to breast cancer development [130]. The overexpression of NEAT1 impacts the Wingless-related integration site (Wnt)/beta-catenin (β-catenin) regulatory network by sponging microRNA-148a-3p, resulting in tumor progression in breast cancer [131]. NEAT1 sponges miR-218-5p and stops its repressive function toward TPD52; in other words, NEAT1 upregulates tumor protein D52 (TPD52). This can result in increased migration and proliferation of breast cancer cells [132]. The upregulation of Krüppel-like transcription factor 12 (KLF12), a transcriptional regulator, through the connection of NEAT1 to miR-141-3p is another pathway that promotes metastasis in breast cancer cells and can lead to resistance to chemotherapy [133]. C-X-C motif chemokine ligand 12 (CXCL12) is another factor important for cell growth and metastasis and is activated by NEAT1; it also connects to miR-133b and stimulates drug resistance in cancer cells [134]. NEAT1 is linked to Taxol resistance in breast cancer through NEAT1-mediated miRNA-23a-3p downregulation, and NEAT1 targets miR-23a-3p, which affects Forkhead box A1 (FOXA1), which is a participant in tumor growth. Interestingly, restoring FOXA1 reversed miR-23a-3p-induced Taxol sensitization [126]. Moreover, reducing the expression of NEAT1 increases the susceptibility of cells to chemotherapy, which demonstrates its role in chemoresistance [135]. In conclusion, NEAT1 is a promising biomarker for breast cancer due to its noteworthy overexpression in most stages of breast cancer in breast cancer cells compared with normal tissue. Its elevated expression is closely associated with tumor progression, invasion, metastasis, and poor outcomes, which makes it a valuable indicator for monitoring breast cancer. Additionally, its role as a "microRNA sponge," regulating various genes involved in cancer cell behavior, further shows its potential as a biomarker [136,137,138]. LncRNAs and their interactions are shown in Fig. 3.

Fig. 3

LncRNAs and their interactions with various microRNAs resulting in different pathways including EMT, tumor expansion, proliferation, and growth

More information about lncRNAs and their associations with cancer can be found in Table 3.

Table 3 LncRNAs associated with cancer

MiRNAs

MicroRNAs are a class of non-coding RNAs that are significantly dysregulated in cancer. They are typically 22 nucleotides long and transcribed from DNA into primary miRNAs (pri-miRNAs), which are then transformed into precursor miRNAs (pre-miRNAs) and mature miRNAs [139]. They can regulate gene expression via a posttranscriptional mechanism [140, 141] and play a role in DNA methylation via argonaute proteins [142]. Additionally, they are instrumental in processes such as proliferation, metabolism and apoptosis [143] (Fig. 4). Furthermore, miRNAs can act as tumor suppressors (tumor suppressor miRNAs), but they also have the potential to act as oncogenes [144]. Several miRNA subtypes and their pathways are discussed below.

Fig. 4

microRNAs biogenesis and their roles in cancer

Tumor suppressors

MiR-34a

MicroRNA-34a (miR-34a) is a tumor suppressor that has recently gained much popularity [145, 146]. Research by Shaban et al. revealed that the expression levels of miR-34a, BRCA1, BRCA2, and p53 were significantly lower in stage III patients than in stage I and II patients both before and after chemoradiotherapy [147]. The ability of miR-34a to distinguish between early and advanced stages of cancer shows its potential as a biomarker for disease progression. MiR-34a has also been linked to the regulation of cancer stem cell functions across different cancer types, such as pancreatic cancer, prostate cancer, glioblastoma, and medulloblastoma [148]. In breast cancer stem cells, miR-34a can reduce the regulation of the neurogenic locus Notch homolog protein 1 pathway (NOTCH) pathway, which is essential for cell maintenance, inhibits proliferation, and can augment sensitivity to paclitaxel (PTX) [149]. Rui Han et al. reported that the overexpression of miR-34a leads to a decrease in 3D spheroid cell mass in both the T-47D and MDA-MB-231 cell lines, demonstrating its potential to diminish spheroid formation by inhibiting the expression of the breast cancer stem cell (BCSC)-associated transcription factors E2F1 and E2F3 and that E2F1 and E2F3 regulate CASP3 and control apoptosis [150]. Proteins of the miR-34 family have various functions, such as inhibiting cell migration and proliferation and regulating the p53 pathway. p53 is a transcription factor important in DNA damage responses and oncogene activation [151, 152]. One of the important functions of miR-34 is the regulation of EMT, which occurs via the targeting of key genes and signaling pathways involved in EMT control [153]. Research has shown that miR-34a affects CD4 + T-cell infiltration indirectly by changing variables such as the tumor microenvironment, immunological checkpoint molecules, and macrophage polarization [154, 155]. By synthesizing poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying miR-34a, scientists were able to effectively suppress triple-negative breast cancer (TNBC) cell proliferation and induce cell cycle arrest [156]. Ectopic expression of miR-34a can downregulate survivin, an inhibitor of apoptosis [157], in TNBC cells, stimulate caspase-3 activation and reduce cell growth and migration, and in the MDA-MB-231 TNBC model, it improves the anti-proliferative function of selinexor. These findings suggest that miR-34a can positively modulate the function of anticancer medications [158]. This highlights its potential in further therapeutic studies and drug delivery systems.

MiR-200

miR-200 is another microRNA that has been investigated in many types of cancer, including breast cancer, and its role as a tumor suppressor has been widely researched [159,160,161,162]. A study by Feng et al. suggested that miR-200 interacts with a specific cell communication system known as the Notch signaling pathway, creating a regulatory loop. The balance of this interaction may play a crucial role in determining tumor stage [163].

MiR-200 targets transcription factors such as ZEB1 and ZEB2, which are involved in cell invasiveness and metastasis and subsequently limit EMT [164, 165]. Additionally, it can reduce the population of cancer stem cells by affecting genes involved in stemness [166, 167]. MiR-200 is a crucial factor in the inhibition of genes vital in cell migration and invasion [168, 169]. Furthermore, it can eliminate cells at risk of becoming malignant or with undesirable alternations by causing them to undergo programmed cell death (apoptosis) [170, 171]. One study showed that interleukin-8 (IL-8) promotes EMT in breast cancer cells and increases migration by downregulating the miR-200 family; controlling this pathway can be an effective way to hinder invasion and metastasis [172]. In ER-positive breast cancer cells, Cellular Myeloblastosis Oncogene (c-MYB), which is a transcription factor, is downregulated by miR-200 (miR-200b and miR-200c), and c-MYB is stabilized by EMT-inducing Transforming Growth Factor-β (TGF-β) [173]. This study revealed that IL-8 promotes EMT in breast cancer cells and enhances cell migration and invasion by downregulating the expression of the miR-200 family. This pathway plays a significant role in regulating EMT in breast cancer cells, and modulating it could be a potential method to inhibit invasion and metastasis. By decreasing B-cell leukemia/lymphoma 2 (Bcl-2) expression, miR-200 inhibits cell proliferation, and through reducing the regulation of the multidrug resistance 1 (MDR1) gene, it can increase the effect of doxorubicin on breast cancer cells [174]. Another study suggested that by targeting fibronectin 1 (FN1), which is an important factor in migration, miR-200 controls EMT and increases vulnerability to doxorubicin in cancer cells [175]. On the basis of these findings, miR-200 shows great potential as a biomarker in breast cancer because of its role in regulating EMT, metastasis, and drug sensitivity. Its involvement in key pathways shows its diagnostic and therapeutic value.

MiR-15a/16-1

The miR-15/16 cluster encodes microRNAs (miRNAs), which are known as tumor suppressors. The expression of these miRNAs can hinder cell growth and induce cancer cell apoptosis [176]. MiR-15a and miR-16-1 are generally downregulated in advanced stages of cancer [176], making them valuable elements for studying and investigating cancer progression. One of the main mechanisms by which miR-15a/16-1 functions in tumor suppression involves targeting the antiapoptotic gene BCL2. BCL2 is downregulated by miR-15a and miR-16-1, resulting in an increase in apoptosis and limiting excessive cell proliferation [177]. MiR-15/16 reduces cell proliferation, augments cancer cell death, and decreases tumorigenicity [178]. It can regulate cell proliferation and hinder EMT in breast cancer cells by influencing the Enhancer of Zeste Homolog 2 (EZH2) and Twist1. By forced expression of miR-15/16, the expression of mTOR and ribosomal protein S6 kinase beta-1 (RPS6KB1) can be reduced, and its overexpression can lead to G1-cell cycle arrest [178]. MiR-15a/16-1 has been shown to target genes involved in angiogenesis, preventing the creation of new blood vessels and reducing the tumor’s nutritional supply [179, 180]. The miR-15 family can increase radiosensitivity by causing unrepaired DNA damage, disrupting radiation-induced G2 arrest, and reducing proliferation. This process involves checkpoint kinase 1 (chk1) and Wee1 [181]. The miR-15a/miR-16 cluster can reduce the expression of the oncogene B-lymphoma Moloney Murine Leukemia Virus Insertion Region 1 (BMI1), impair the repair of damaged DNA through apoptosis and homologous recombination, and increase sensitivity to doxorubicin [182]. A study showed that a decreased level of miR-16 expression in the MCF-7/A breast cancer cell line can lead to adriamycin resistance. Additionally, the chemosensitivity of these cells was increased when miR-16 was overexpressed making Wild-type p53-induced phosphatase 1 (Wip1 or PPM1D) and Bcl-2 expression reduced and the cell death spurred by Adriamycin, augmented. Furthermore, luciferase activity was decreased by the overexpression of miR-16 in cells transfected with BCL-2 3′UTRs and PPM1D [183]. Therefore, miR-15/16 has potential to be a new cancer biomarker and a promising candidate for cancer treatment. Tumor suppressor miRNAs and their interactions are shown in Fig. 5.

Fig. 5

Tumor suppressor miRNAs can aid in hindering migration, proliferation, and angiogenesis, and increase apoptosis through interactions with different molecules

Oncogenes

Genetic mutations in DNA can change gene structure and expression, which drives cancer development and tumor progression. While traditionally linked to mutations in coding regions of oncogenes and tumor-suppressor genes, recent research highlights the role of non-coding RNAs in cancer. Notably, microRNAs regulate key processes such as the cell cycle and apoptosis and are often dysregulated in tumors [184]. While many microRNAs have the ability to control and suppress cancer, many of them are considered oncogenes [185,186,187]. Meaning, that they can drive cancer progression by suppressing tumor suppressor genes or those involved in cell differentiation and apoptosis [188]. We conducted our review on a selection of more researched ones, listed below.

MiR-21

One of the most significant miRNAs in breast cancer, which is linked to increased cell invasion, proliferation, and resistance to apoptosis, is miR-21 [189,190,191,192]. MiR-21 is located on chromosome 21 and in a region that overlaps the gene for the human papillomavirus (HPV16) on chromosome 17 in the tenth intron of the coding gene transmembrane protein 49 [193]. Research has revealed a significant link between high levels of miR-21 expression and advanced breast cancer stages [194], which can potentially serve as a biomarker to differentiate advanced stages from early stages of the disease. Additionally, the overexpression of miR-21 has often been observed in various cancers, such as lung, breast, stomach, prostate, colon, and pancreatic cancers [195]. A study showed that removing miR-21 from MDA-MB-231 breast cancer cells results in the downregulation of mesenchymal markers and a decrease in epithelial‒mesenchymal transition. The findings of this study identified Wnt-11 as an important target of miR-12. Furthermore, the number of extracellular vesicles was reduced in cells with deleted miR-12 [196]. MiR-21 can increase metastasis and proliferation in breast cancer by suppressing leucine zipper transcription factor-like 1 (LZTFL1), which is a vital gene in cancer progression, by removing LZTFL1, these effects can be reversed. Disruption of LZTFL1 can neutralize the effect of miR-21 on the expression of EMT markers through the regulation of β-catenin, snail, and slug by miR-21/LZTFL1 [196]. In triple-negative breast cancer, Nestin (NES) is a protein associated with poor prognosis [197], the thyroid hormone receptor interactor 13 (TRIP13) protein is related to DNA repair [198], and reticulum membrane protein complex subunit 1 (EMC1) is another protein that controls the actin cytoskeleton and enhances angiogenesis [199]. The findings of a previous study indicated that miR-21 upregulates these three proteins and promotes metastasis via an immune cell-independent approach [191]. MiR-21 contributes to breast cancer by downregulating the expression of phosphatase and tensin homolog (PTEN), a tumor suppressor that stimulates TBNC cells to proliferate and invade [200]. These findings suggest that miR-21 may prevent the death of breast cancer cells by encouraging their division and survival. Moreover, miR-21 has been linked to poorer prognosis, lymph node involvement, and larger tumors in individuals with breast cancer [201]. Another study indicated that a high level of miR-21 can lead to a reduction in the expression of PTEN (a tumor suppressor) and trastuzumab resistance in HER2-expressing breast cancer cells. This study further showed that by employing miR-21 antisense oligonucleotides (ASOs) or a PTEN rescue vector via the removal of the miR-21 targeting sequence, it is possible to reverse the resistance created against trastuzumab drugs and reverse PTEN expression [202], demonstrating the therapeutic use of miR-21 in breast cancer treatment.

MiR-221/222

The miR-221/222 cluster is known to act as an oncogene in breast cancer, driving tumor development, inhibiting apoptosis, and fostering medication resistance [203,204,205]. It can be involved in epithelial-to-mesenchymal transition in breast cancer through the negative regulation of Notch3, which is an important factor in limiting EMT [206]. Previous studies reported increased expression levels of miR-221 and miR-222 in breast cancer tissues compared with normal breast tissues, and higher levels of these microRNAs were associated with more advanced clinical stages of the tumor [207]. MiR-221 and miR-222 are highly expressed in various tumors, such as those in the colon, pancreas, and stomach [208]. MiR-221/222 play a role in luminal-like breast cancer by regulating invasion, proliferation, and tumor growth by targeting signal transducer and activator of transcription signal transducer and activator of transcription 5A (STAT5A), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and β4-integrin [209]. These microRNAs are linked to S-phase entry, cellular migration, and the G1/S transition of the cell cycle, and they are overexpressed in highly invasive basal-like breast cancer cells [205]. Additionally, they target the growth arrest-specific 5 (GAS5) tumor suppressor gene, which promotes the growth of tumors [204]. Researchers have shown that, through the inhibition of miR-221/222, sensitivity to tamoxifen in tamoxifen-resistant breast cancer cell lines can be restored. It was confirmed that miR-221/222 inhibitors derepress estrogen receptor alpha (Erα) and PTEN expression, which can result in a decrease in cell growth and cause cell cycle arrest [210]. Another study on resistance to tamoxifen suggested that the repression of miRNA-221/222 enhances the sensitivity of estrogen receptor (ER)-positive MCF-7 breast cancer cells to tamoxifen by increasing the expression of tissue inhibitor of metalloproteinases 3 (TIMP3), which is involved in limiting migration and tumor growth [211]. Another study highlighted the importance of miR-221/222 in breast cancer through the activation of the Akt/nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2) pathway through the downregulation of PTEN [212]. The effect of miR-221/222 on cisplatin drug sensitivity in TNBC was investigated, and it was found that the removal of miR-221/222 can lead to the upregulation of suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibitor 1B (p27), which in turn inhibits signal transducer and activator of transcription 3 (STAT3) expression. This cascade can reduce the expression of c-Myc and Bcl2 and upregulate p53 and Pten, which participate in increased cell apoptosis in response to cisplatin [213]. Given the role of miR-221 and miR-222 in regulating tumor progression, invasion, proliferation, and drug resistance in breast cancer, these non-coding RNAs have potential as biomarkers for this disease. Many studies have highlighted their diagnostic and therapeutic relevance, supporting their use as indicators for breast cancer prognosis and treatment response [214,215,216].

MiR-155

MiR-155 is found inside the B-cell integration cluster (BIC) on chromosome 21 [217]. miR-155 has been linked to the development of several tumors, their growth, and resistance to treatment [218,219,220]. It was previously shown that increased miR-155 expression in breast cancer is linked to increased tumor grade and advanced disease stage. Additionally, higher miR-155 levels are associated with poorer overall survival, indicating its potential as a clinically significant miRNA [221, 222]. MicroRNA-155 is elevated across various cancer types, including breast, prostate, liver, lung, and pancreatic [223]. In breast cancer patients, the level of circulating miR-155 (in the blood) is highly increased, whereas after treatment, this level is decreased [224]. These findings highlight it as an important biomarker. miR-155 can upregulate MMP16, a cytokine that regulates tumor migration and invasion; additionally, miR155 enhances proliferation through negatively regulating SOCS1 (tumor suppressor gene) and turning on the critical inflammatory Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway [225]. The nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome regulates the immune system; once activated, it triggers the release of proinflammatory cytokines [226]. Research has shown that disruption of miRNA-155 can suppress the NLRP3 pathway in MDA-MB-231 cells, increase apoptosis and prevent the proliferation, migration and secretion of inflammatory factors [227]. It was discovered that miR-155 enhances the development of stem cell growth by targeting tetraspanin 5 (TSPAN5), which is important for signaling and cell differentiation, affecting resistance to decitabine drugs in TNBC cells [228]. Another study demonstrated that by inhibiting the expression of miR-155 in ATP-binding cassette subfamily G member 2 (ABCG2), the expression of CD44 and CD90 decreases while that of CD24 increases, and the inhibition of miR-155 enhances the sensitivity of MDA-MB-231 cells to the drug doxorubicin [229]. According to one study, Schizandrin A (SchA) can reduce the migration and proliferation of breast cancer cells by decreasing PI3K/AKT and Wnt/β-catenin through the negative regulation of miR-155 expression [230]. MiR-155 plays an important role in breast cancer progression, regulating processes such as tumor migration, invasion, proliferation, and treatment resistance. Its increased expression in tumor tissues and the circulation, along with its reduction following treatment, shows its potential as a biomarker. Oncogenic miRNAs and their interactions can be observed in Fig. 6.

Fig. 6

Oncogene miRNAs result in migration, proliferation, resistance to drugs, and invasion by interacting with various molecules

More information on miRNAs and their associations with cancer can be found in Table 4.

Table 4 microRNAs associated with cancer

Experimental approaches

To understand the functions, expression patterns and regulatory mechanisms of non-coding RNAs (ncRNAs) in cancer, a variety of experimental methods and approaches have been employed. In this review, we cover some of the most known of them below.

Microarray analysis

Microarray analysis plays a significant role in understanding the function of non-coding RNAs in breast cancer [231]. It can be used to compare the expression of miRNAs and lncRNAs in breast cancer tissues to that in normal tissues. This approach has been utilized in many investigations [231,232,233]. Microarrays consist of glass or silicon slides with DNA probes arranged in a grid-like pattern [234]. Samples of mature miRNAs (reference and test) should be purified and then converted to cDNA via reverse transcription. The samples are labeled with fluorescent dyes, and fluorescent green and red labels are provided for the reference and test samples, respectively. Next, two samples are mixed together and applied to high-affinity probes on the array. By measuring the amount of red and green fluorescence at each location, the relative number of red- and green-labeled fragments can be determined, which allows the comparison of numbers between reference and test samples [235,236,237]. Figure 7. A study focused on epigenetic and genetic factors related to the expression of miRNAs in two populations of Lebanese women and American women. By employing microarray analysis, they identified miRNAs that regulate tumor-related mRNAs, and their results demonstrated ethnic and age-associated differences in miRNA expression [238]. Researchers have used microarrays and unsupervised cluster analysis to measure the expression of miRNAs in breast cancer cell lines. These findings indicate that specific miRNAs are associated with genetic and epigenetic alterations in various subtypes of breast cancer cell lines, such as Erb-B2 receptor tyrosine kinase 2 (ERBB2) overexpression, E-cadherin mutation, and promoter hypermethylation. LessFewer miRNAs are associated with phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)/PTEN, breast cancer type 1 susceptibility protein (BRCA1), and tumor protein p53 (TP53) mutation status [239].

Fig. 7

Microarray. A RNA is converted to cDNA via reverse transcription, B they are labeled with fluorescent dyes, C samples are applied to high-affinity probes on the array, D they are scanned

Another study focused on non-small cell lung cancer (NSCLC) patients who underwent surgery at a Chinese hospital. Tumors and adjacent tissues were collected, preserved, and analyzed for circular RNA (circRNA) profiles. By employing microarray analysis, total RNA was treated with RNase R to remove linear RNAs and to enrich circRNAs. Next, the circRNAs were labeled and hybridized onto Arraystar Human circRNA Arrays. Scanned images were processed, and circRNA profiles were analyzed via quantile normalization, cluster analysis, fold-change filtering, and student’s t-testing. Two differentially expressed circRNAs (hsa_circ_0014130 and hsa_circ_0016760) were validated via quantitative reverse transcription‒polymerase chain reaction (qRT‒PCR). Network analyses revealed possible interactions between circRNAs, microRNAs, and mRNAs, providing insights into the regulatory mechanisms involved. Functional analyses explored the roles of circRNAs and their associated genes. These findings indicate that hsa_circ_0014130 can play a significant role in the development of non-small cell lung cancer (NSCLC) and can be employed as a marker [240].

Microarray analysis is a promising tool in ncRNA discovery and is praised for its ability to monitor transcript levels fully; however, it has some limitations that are worthy of note. Issues include the high cost of the experiment, dependence on low-specificity probes, and low control of the studied transcript pool since most platforms use manufacturer-designed probes; challenges in accuracy, precision, and specificity; and susceptibility to changes in hybridization settings, genetic material quality, and amplification procedures [241,242,243,244].

Next-generation sequencing (NGS)

Sequencing began a lengthy journey with first-generation sequencing and Sangers' work. Next-generation sequencing refers to second- and third-generation sequencing technologies, which include the Roche 454, Illumina Solexa, ABI-SOLiD, Helicos, and PacBio, and the fourth generation consists of Nanopore sequencers offered by Oxford Nanopore Technologies (ONT) [245]. Transcriptomics or expression profiling is the study of the transcriptome, which is the entire collection of RNA [246]. Next-generation sequencing significantly affects transcriptomics and makes it possible to study the complete collection of RNA molecules, including non-coding RNAs. It can be employed in several approaches, such as sequencing mRNAs, analyzing alternative splicing (a process with transcriptome complexity), studying lncRNAs, and assembling the transcriptome [247]. Next-generation sequencing (NGS) technology is a critical step in identifying ncRNAs and their link to breast cancer [248] and plays a significant role in tumor investigation, enabling breakthroughs in tumor genetic profiling [249]. Any NGS technology results in a large amount of output data. The fundamentals of sequence analysis follow a centralized approach that comprises raw read quality control (QC), preprocessing and mapping, postalignment processing, variant annotation, variant calling, and visualization [250]. For example, one of the well-known NGS approaches is Illumina, and its workflow consists of RNA isolation, preparation of the desired non-coding RNA sequencing library, mapping and identification, functional enrichment analysis, qRT‒PCR and statistical analysis [251]. A study created a complementary DNA (cDNA) library of HCT-8 VCR-resistant colon cancer cells and analyzed it via HiSeq 2500 sequencing and bioinformatics methods to identify differentially expressed lncRNAs in drug-resistant cells and nonresistant cells. The results of this research revealed a greater total transcript number in resistant cells, with the majority being high-quality transcripts, and revealed 121 transcripts whose expression significantly differed between the two cell types [252]. In another study, researchers studied microRNAs in 11 normal samples and 104 cancerous breast tissues via next-generation sequencing (NGS), and the results revealed two novel miRNAs, namely, miR-574-3p and miR-660-5p, related to breast cancer and 10 other miRNAs linked to overall survival (OS) and/or recurrence-free survival (RFS) [253]. Krishnan et al. used the Illumina genome analyzer IIx to study snoRNAs in 104 breast cancer and 11 normal breast tissues. Their findings revealed that 13 snoRNAs are associated with patient outcomes. These results indicate that snoRNAs interact with other RNAs involved in cancer development and can indirectly regulate gene expression associated with tumorigenesis [254].

However, this approach is not without limitations. In technologies such as Roche 454 (GS FLX plus), sequencing the homopolymer regions is difficult [255]. In Illumina, uneven representation of DNA fragments with different guanine‒cytosine (GC) contents in the amplified clusters is the main limitation [256], with a higher error rate in placebo, particularly in terms of insertion and deletion [257]. Despite these challenges, NGS stands as a powerful tool for high-throughput sequencing applications.

Quantitative real-time PCR

Polymerase chain reaction (PCR) is an approach employed to amplify DNA and create many copies from it [258]. It has three main repeated steps consisting of melting through high temperature, annealing to designed primers and replication via enzymes such as Taq DNA polymerase [259]. Real-time PCR or quantitative PCR (qPCR) is a PCR-based technique that detects products in real time and does not require gel electrophoresis for analysis [260, 261]. The fluorescent reporter/dye used in qPCR produces fluorescence related to the amount of amplified DNA product (amplicon). The fluorescent molecules utilized in qPCR can be fluorescent tagged probes such as TaqMan or DNA binding dyes such as SYBR Green. The fluorescence emitted in each qPCR cycle will be detected and quantified [260]. Reverse transcription‒quantitative polymerase chain reaction (RT‒qPCR) is used to determine the amount of RNA in a sample, where RNA is converted to cDNA through reverse transcription and then amplified via qPCR [262]. RT‒qPCR can be a useful method for examining non-coding RNAs (ncRNAs) in the context of breast cancer and has been used in several studies [263,264,265]. Breast cancer tumors and adjacent nontumor tissues from female patients were examined, RNA extraction and cDNA synthesis were performed, and a quantitative real-time PCR technique was used to evaluate differentially expressed genes. The results of this study demonstrated that the lncRNA MCM3AP-AS1 was significantly overexpressed in breast tumor tissues compared with adjacent nontumor tissues. This overexpression is related to estrogen and progesterone receptor expression in tumors [266]. Another study employed a mixture of microarray and PCR to analyze the expression of MALAT1 in 195 cases of benign and malignant thyroid neoplasms; with these two approaches, they discovered a role for MALAT1 in EMT in thyroid tumors and reported that the induction of epithelial-to-mesenchymal transition (EMT) in a papillary thyroid carcinoma (PTC) cell line led to augmented MALAT1 expression [267]. Researchers have used RT‒qPCR to evaluate the expression levels of prostate cancer-associated non-coding RNA 1 (PRNCR1) in breast cancer, and their results demonstrated that this lncRNA is expressed at higher levels in breast cancer tissue than in normal tissue. Furthermore, high expression of PRNCR1 was linked to poor patient prognosis and metastasis, and by reducing PRNCR1 gene expression, the aggressive characteristics of breast cancer cells were decreased [268]. There are several drawbacks to the use of RT‒qPCR in the study of non-coding RNA; for example, miRNA is a short RNA molecule and is difficult to quantify by RT‒qPCR, necessitating the use of special kits for cDNA synthesis. In longer RNA molecules, hairpin loops are a challenge in cDNA synthesis, which demands more temperature to make them available for reverse transcriptase [269, 270]. Moreover, ncRNAs such as lncRNAs are less stable and prone to degradation because of their length [271], which can impact their quantification. Additionally, the localization of lncRNAs in the genome influences their transcript stability [272, 273].

Northern blotting

The northern blot technique is a widely used laboratory method for the analysis of RNA and provides essential information about gene expression. This method involves the separation of purified RNA fragments from a biological sample, such as blood or tissue, by passing them through a sieve-like matrix or gel. The separation process is based on the size of the RNA fragments, with smaller fragments moving more quickly through the matrix or gel than larger fragments. The RNA fragments are transported from the gel or matrix to a solid membrane, which is then exposed to a radioactive, fluorescent, or chemically tagged DNA probe [274]. Figure 8. The tag enables the visualization of any RNA fragments bearing complementary sequences to the DNA probe sequence within the Northern blot. Northern blot analysis can be performed to determine whether a gene is overexpressed or underexpressed in cancer cells compared with normal cells [275]. The size and expression levels of non-coding RNAs in breast cancer tissues have been investigated, and these RNAs have been used in several studies [276,277,278]. One study on the lncRNA DIO3 Opposite Strand Upstream RNA (DIO3OS) revealed that this RNA causes glycolytic-dominant metabolic reprogramming to increase aromatase inhibitor resistance in breast cancer. Northern blotting was used as part of this method in the following way: the RNA was loaded onto a gel, divided by electrophoresis, and transferred to a nylon membrane. The membrane was then treated with buffers and incubated with labeled oligonucleotides overnight, and the resulting hybrids were detected via a luminescent detection kit [267]. Another study employed northern blotting on U2OS osteosarcoma cells. Total RNA was separated and transferred to membranes before UV cross-linking. Digoxin-labeled oligonucleotide probes were made and utilized for hybridization, followed by visualization via an imaging analysis system. The results revealed a main long non-coding RNA (Lnc8) transcript at approximately 1.25 kilobases. This transcript was found to decrease under conditions of glucose deprivation [279]. Researchers have shown the differential expression of the lncRNA LINC00908 in triple-negative breast cancer (TNBC) via northern blot analysis. These findings revealed that the protein ASRPS produced by LINC00908 can reduce the phosphorylation of STAT3 and decrease the level of vascular endothelial growth factor (VEGF), which is involved in the growth of blood vessels. By increasing ASRPS levels in mouse models, angiogenesis was reduced, and the expression of ASRPS was decreased in TNBCs [280].

Fig. 8

Northern Blotting. A The RNA is extracted, B divided by gel electrophoresis, C moved to a nylon membrane, D the membrane is then treated with probes, and E) the resulting hybrids are visualized

It is undeniable that northern blotting is an extensively used method for the analysis of genes at the RNA level and guides the detection of transcript size; however, similar to any other approach, it has limitations, such as the need for high-quality RNA and the lengthy time it requires [281, 282].

Fluorescence in situ hybridization (FISH)

Joseph Gall and Mary Lou Pardue pioneered fluorescence in situ hybridization (FISH) in the 1960s [283], which was followed by John et al. in [284]. This method has various applications, such as utilization in gene mapping, locating DNA sequences and recognition of new oncogenes in cancer [285]. RNA-FISH is a powerful method for evaluating RNA transcription and can provide information about degradation rates [286]. RNA fluorescence in situ hybridization (FISH) can be used to determine the cellular localization of lncRNAs by creating a lncRNA-specific antisense chain sequence and tagging it with fluorescent dyes [287]. Therefore, it has been employed in several studies [288,289,290]. The steps involved designing a probe, fixation, permeabilization, hybridization, posthybridization washing, sequence amplification and detection [291]. A study in 2020 revealed that high expression of the lncRNA NONHSAT028712 (Lnc712) is associated with breast cancer. They used RNA-FISH as a method to localize Lnc712 within the cell, and the results demonstrated its existence in the cytoplasm, which might be due to its role in the regulation of cytosolic processes [292]. Hongnan Jiang et al. suggested a correlation between lncRNAs, small nuclear protein RNA host gene 3 (SNHG3), and breast cancer via microRNA-154-3p; they used probes targeting SNHG3 via FISH to identify the location of a given lncRNA, and the results of their experiment confirmed its presence in the cytoplasm within HCC1937 and MCF-7 cells [293]. In another study on the role of small nucleolar RNA host gene 7 (SNHG7) in liver cancer, RNA FISH was performed on hepatocellular carcinoma (HCC) tissue cells and adjacent normal tissues via a fluorescent in situ hybridization kit. The Cy3-conjugated SNHG7 probe was used for detection, and fluorescence images were acquired via a confocal microscope. These results demonstrated that the expression of SNHG7 was increased in hepatocellular carcinoma tissues compared with adjacent normal tissues [294]. The limitations of FISH methods can be considered as follows: long hybridization times are needed for saturation of the signal, making this method time-consuming and demanding experienced personnel. Another challenge is that few commercially available probes and laboratories produce their own probes, and the process of creating, preparing, and labeling these probes still requires much effort. Additionally, adjusting cutoffs for FISH probes can be a technical challenge in laboratories. The cost of probes, especially at a relatively high scale, is relatively high [295, 296]. Enhancing automation and hindering hybridization time are platforms for improving the efficiency of the FISH method.

CRISPR-Cas9

Initially, found in bacteria, clustered regularly interspaced palindromic repeats (CRISPR) is an effective system in bacteria for defending against phages and plasmid DNAs as part of adaptive immunity [297]. Cas9 is a nuclease enzyme in the type II CRISPR system from S. pyogenes that is guided by single guide RNA (sgRNA) to target the DNA site [298]. The sgRNA is composed of two parts, CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA), and it interacts with the enzyme Cas9 [299]. They work together to find the target DNA sequence, break it, and enable gene editing. CRISPR/Cas gene editing can provide knockdown of certain non-coding RNAs by deleting the transcriptional termination or start site and removing the promoter and exon‒exon junctions [300], making it a valuable approach for harnessing the effects of non-coding RNA in cancer. The CRISPR/Cas9 system is known as a molecular scissor and is widely used in a variety of studies, including cancer research, drug development, mental disease therapy, and plant applications [301]. Increasing evidence suggests that CRISPR/Cas9 can target human ncRNAs as well as the protein-coding genome [302, 303]. This system has been used in a variety of studies [304,305,306]. A study by Hebatalla Said Ali et al. revealed that the levels of the genetic network were reversed when CRISPR Cas9 was used to knock out the lncRNA RP11-156p1.3 in a human liver cancer cell line (HepG2), and the proportions of tumor necrosis factor alpha (TNF-α) and NFκβ were reduced as a result of this experiment, highlighting the use of CRISPR-Cas9 in hepatocellular carcinoma [307]. Another study used CRISPR-Cas9 to knock out mature miR-3662 in TNBC cells, resulting in a reduction in the proliferation and migration of tumors in vivo [308]. Researchers have used the CRISPR/Cas9 system to deactivate maternally expressed gene 3 (MEG3) in gene-expressing breast cancer cell lines. They verified the knockout via PCR and RT‒qPCR. These results showed that deleting MEG3 makes cells resistant to doxorubicin drugs. Additionally, it leads to increased levels of certain proteins, such as N‑cadherin and transforming growth factor β, while reducing the levels of factors such as the collagen type III α1 chain, zinc‑finger E‑box binding homeobox 1, and matrix metallopeptidase 2 [309]. Another in vitro and in vivo study revealed that knocking out miR-23b and miR-27b via CRISPR/Cas9 decreased tumorigenesis [185]. It is undeniable that CRISPR/Cas9 is a revolutionary and valuable tool, but its limitations and challenges should be considered to achieve better results. One notable complexity is the gene locus that must be estimated for sgRNA design and data analysis [310], which can cause off-target results and changes in sequences that are not aimed at [311]. This can be due to the untargeted function of Cas9, which cleaves DNA sites other than the intended regions [312].

CLIP-Seq (crosslinking and immunoprecipitation followed by sequencing)

With cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq), we can determine RNA targets connected to a certain RNA-binding protein with significant precision [313]. The method isolates particular RNA‒protein complexes by crosslinking them with UV light and then purifying the resulting mixture under strict conditions. After the RNA fragments have been extracted, techniques such as RNA linker ligation, RT‒PCR amplification, and sequencing can be used to search for native binding sites of RNA-binding proteins [314]. This approach has been applied across several investigations [315,316,317]. For example, Junhao Li et al. used CLIP-seq to construct miRNA–mRNA and RNA–lncRNA interaction networks and identified approximately 10,000 ceRNA pairs from miRNA target sites. Additionally, miRNA-mediated regulatory networks can be used to predict the roles of miRNAs and other non-coding RNAs [318]. Michael Lidschreiber et al. identified protein systems for the creation of mature eukaryotic protein-coding and non-coding RNAs via CLIP-seq and reported that sync1/APT (a specific protein involved in the regulatory mechanisms of the cell cycle and transcription in yeast) and cleavage and polyadenylation factor (CPF) have different functions, which might overlap. Syc1/APT is more involved in sn/snoRNA construction, whereas CPF is responsible for handing out the 3'-ends of protein-coding pre-mRNAs [319]. The results of another study that employed CLIP-Seq data demonstrated that the RNA-binding protein FUS/TLS interacts with NEAT1 and that, by hindering fused in sarcoma/translocated in liposarcoma (FUS/TLS) expression, cell apoptosis can be triggered. Additionally, they suggested that MiR-548ar-3p interacts with NEAT1 and downregulates it [320]. One of the limitations of CLIP-seq is its ability to measure in vivo protein binding occupancy on transcripts because of the challenges in quantifying binding occupancy, which is important in understanding the relevance of binding sites. For example, the regulatory impact of a protein binding to all 20 copies of one transcript in a cell varies from protein binding to 20 out of 200 copies of another transcript. Future improvements are needed to improve protein‒RNA capture efficiency and increase protein capture strength [321].

Bioinformatics tools

The study of non-coding RNAs (ncRNAs) in breast cancer is a complex and evolving field, with a variety of bioinformatic tools being used to understand their role. MiRBase, miRWalk, LNCipedia, and NONCODE are four popular bioinformatic tools for non-coding RNAs related to breast cancer. miRBase (https://mirbase.org/) [322] is a comprehensive database for microRNA sequences and annotations that provides valuable information on the expression patterns and potential functions of non-coding RNAs in breast cancer. This approach can aid in the discovery of biomarkers and medicinal targets. The database also aids in the prediction of novel microRNAs and their targets in cancer [323]. MiRWalk (http://mirwalk.umm.uni-heidelberg.de/) [324] is another comprehensive online resource that is employed for predicting and validating microRNA (miRNA) binding sites in genes from humans, mice, rats, dogs, and cows [325]. It includes a comparison platform of miRNA-binding sites, genetic networks of miRNA-gene pathways, and analytically validated information [326]. Since the database is constantly updated and openly accessible, it is a great resource for researchers studying miRNA‒target interactions [327]. LNCipedia (https://lncipedia.org/) [328] is an annotated human long non-coding RNA (lncRNA) sequencing database that provides a high-confidence list of lncRNA transcripts with low coding potential [329]. It has been used to create a custom microarray for profiling lncRNA expression [330]. NONCODE (http://www.noncode.org/) [331] is a database of non-coding RNAs (ncRNAs), in addition to transfer RNAs and ribosomal RNAs, that covers a wide range of these molecules. It contains detailed information on the sequencing, functions, and cellular roles of ncRNAs and offers an easy-to-use interface [332].

Non-coding RNAs as biomarkers for diagnosis and prognosis

In recent years, significant advancements have been made in the diagnosis, subtyping, and complex treatment of breast cancer. Traditional methods for diagnosing breast cancer have been supplemented by modern molecular methodologies. NcRNAs, including miRNAs, lncRNAs, and circRNAs, are recognized as pivotal in tumorigenesis and have shown promise in the early detection, diagnosis, prognosis and treatment response prediction of multiple cancers [333, 334].

MiRNAs regulate various biological processes, such as proliferation, cell adhesion, motility, and apoptosis, all of which are fundamental to tumorigenesis. Studies suggest that the abnormal expression of miRNAs might be clinically useful as predictive markers, potential therapeutic targets, and suppressors of resistance to certain anticancer chemotherapies. Tumor-derived miRNAs exist in the circulating nucleic acids of cancer patients, making their detection a favorable potential diagnostic method, as it is noninvasive [335, 336]. More than 3000 miRNAs associated with the occurrence and progression of tumors have been identified [334]. Some of these findings are summarized in Table 5.

Table 5 microRNAs as potential biomarkers

Another group of ncRNAs, lncRNAs, are key regulators of tumor development and progression, and their abnormal expression is closely linked to breast cancer initiation and metastasis [337]. Notable lncRNAs, such as H19, MALAT1, and DANCR, influence cell survival, proliferation, metastasis, and resistance therapy, making them promising biomarkers for breast cancer diagnosis, prognosis, and risk management [338, 339]. For example, MALAT1 overexpression is correlated with poor patient survival, and its expression level can be a potential prognostic factor [340]. Similarly, LINC01787 is upregulated in breast cancer tissues and is associated with advanced stages and poor survival [341].

While ncRNAs show significant potential as biomarkers, their clinical utility faces several challenges. Finding a suitable molecular biomarker remains difficult, as it must be stable, with sufficient specificity, easily detectable, and ideally applicable to all patients. Since the content of ncRNAs in body fluids is not high, they are difficult to detect. Additionally, variability between preclinical models and humans, as well as between individuals, complicates their reliability [333]. Addressing these limitations requires further research to validate the potential of ncRNAs as biomarkers.

Clinical trials

Various endeavors have been made to demonstrate the usage of ncRNAs in clinical settings, including cancer therapy. For example, miRNAs can act as either conventional tumor suppressors or oncogenes and target multiple genes simultaneously; therefore, they are promising cancer therapy agents. Before any testing in humans, preclinical studies are conducted in vitro, in vivo, ex vivo, or in silico to determine a drug, procedure, or treatment’s efficacy and safety. In this section, we outline some of the preclinical studies that have investigated the therapeutic potential of non-coding RNAs in breast cancer, followed by clinical trials that were carried out in humans. One study in 2021 was conducted on multiple human breast cancer cell lines using a miR-100 mimic as well as miR-100 inhibitors. Oligonucleotides were transfected into breast cancer cells. Taken together, the results of this study revealed that, by targeting FOXA1 expression, miR-100 inhibits the proliferation, invasion, and migration of breast cancer cells [342]. SUM159PT (a triple-negative breast cancer cell line) was transfected with plasmid expression vector (pEP)-miR-205-miR205 and pEP-empty vectors, followed by a trypan blue exclusion assay, cell lysis and immunoblotting, quantification of cell migration, determination of invasiveness, and calculation of sphere-forming efficiency. MiR-205 drastically reduced the proliferation, migration and invasion properties of the SUM159 cell line. An analysis of the expression of different markers involved in the epithelial–mesenchymal process and the ability of cells to form mammospheres revealed that miR-205 inhibits cancer stem cell renewal and affects several parameters associated with the initial steps of tumorigenesis, suggesting that miR-205 is a potential therapeutic agent for triple-negative breast cancer. [343]. Kim et al. demonstrated that miR-145 can inhibit the growth and motility of breast cancer cells both in vitro and in vivo [344]. Multiple studies have been conducted using miR-21 antagonists in various cell lines and mouse models via different delivery systems, such as transfection and different types of nanoparticles. The results demonstrated that the combination of these drugs suppressed tumor growth and angiogenesis and reduced cancer cell migration while also improving the efficacy of anticancer drugs [345,346,347,348]. In another study, miR34-a-treated tumors from nude mice were significantly smaller than those from control mice [344, 349]. More studies on ncRNAs as treatment agents as well as human clinical trials will aid in the development of effective ncRNA-based therapeutic agents for treating malignant diseases [350].

MiR-34a is a naturally occurring tumor suppressor that is mutated, inactivated, or expressed at low levels in a wide range of different tumors. In the case of breast cancer, miR-34a is downregulated compared with that in healthy tissue. Studies on the exogenous introduction of miR-34a mimics in vitro have shown decreased cell proliferation, migration, and invasion [351, 352]. When combined with other anticancer therapies, miR-34a mimics have shown synergistic effects. Compared with miRNA-34a or docetaxel alone, codelivery of miRNA-34a with nanocarriers prolonged the blood circulation of docetaxel, improved tumor accumulation, and significantly inhibited tumor growth and metastasis in vivo in mouse models [353].

MiR-34a has been administered via different routes in preclinical animal models, where it inhibits primary tumor growth, blocks metastasis, and improves survival.

MRX34 is a liposomal formulation of miR-34a. A clinical trial was conducted on adult patients with solid tumors refractory to standard treatment in 2017. Among the participants, 3 patients (6 percent) suffered from breast cancer. Patients were given MRX34 intravenously twice weekly for three weeks in 4-week cycles. Dexamethasone premedication was administered to manage infusion-related adverse effects. This treatment showed acceptable safety and antitumor activity in a subset of patients with refractory advanced solid tumors. However, the trial faced significant challenges, including serious immune-mediated adverse events, which led to it ending early after four patients died. The maximal tolerated dose for the twice-weekly schedule was 110 mg/m² for nonhepatocellular carcinoma patients and 93 mg/m² for HCC patients. The final report included pharmacodynamics, determination, and evaluation of the recommended phase 2 dose of a different daily treatment schedule (MRX34 daily for 5 days along with dexamethasone premedication twice daily for seven days in week 1 followed by two weeks of rest in 3-week cycles), in which some antitumor activity was observed.

While the trial showed promise, it also highlighted several limitations and challenges that must be addressed in future studies. The unexpected immune-mediated adverse events showed difficulty in translating findings from preclinical models to humans as these events did not occur in preclinical studies. It is important to anticipate toxic effects that may not be evident in preclinical toxicology studies, as such models often fail to capture complex human immune responses. In addition, while MRX34 treatment with dexamethasone premedication showed manageable toxicity in most patients, its effect did not prevent severe reactions in all patients. The lack of adequate preclinical and animal studies on dosing regimens and toxicity profiles made it more difficult to predict MRX34’s safety profile in a clinical setting. While the trial demonstrated that miRNA-based cancer therapy is potentially useful in terms of clinical activity, such as dose-dependent modulation of miR-34a target genes (in patients’ white blood cells) and miR-34a localization in tumors, effectively delivering RNA constructs and further optimizing delivery systems remain critical areas for improvement so that systemic exposure is reduced and side effects become more manageable. Although adverse immune-mediated effects have occurred, the MRX34 trial was the first to study the effects of the administration of a synthetic miRNA to human patients and therefore provides a direction for the application of MRX34 in cancer therapy. Further studies on alternative dosing schedules, combination therapies, and premedication regimens are needed. Overall, the use of RNA constructs requires further development to prevent immune-related toxicity and be clinically useful in humans [352, 354].

Recent clinical trials have been investigating the role of ncRNAs in the diagnosis, prognosis, and treatment of breast cancer. Although results are pending or have yet to be published, these studies significantly contribute to the understanding of ncRNAs as biomarkers for breast cancer Table 6.

Table 6 Clinical trials on ncRNAs to investigate their role in breast cancer prognosis

Discussion

The evolution of ncRNA research

Research on ncRNAs began a long journey, with the discovery of rRNAs in 1955, which led to their employment in clinical platforms in 2013 [46, 66]. During this process, various endeavors have been made to understand their multiple functions in the body. Projects such as ENCODE have advanced the understanding of their role, shedding light on the regulatory mechanism of ncRNAs [18]. With the increasing number of cancers, efforts to find effective treatments are increasing. Most anticancer treatments eventually lead to some level of resistance among patients and involve serious side effects. Advances in technologies such as microarray analysis, next-generation sequencing (NGS), and real-time quantitative reverse transcription (qPCR) have helped researchers determine the delicate functions of ncRNAs in cancer, and numerous studies have investigated their roles.

NcRNAs: a double-edged sword in breast cancer

MiRNAs and lncRNAs are two of the most studied types of ncRNAs. Famous lncRNAs, including MALAT1, NEAT1 and HOTAIR, have been fully studied. They can act as oncogenes and promote cancer via multiple pathways, including EMT, angiogenesis, autophagy, proliferation and metastasis. Moreover, they play an instrumental role in the development of resistance in cancer cells against radiation and anticancer medications [106, 112, 117, 118, 128, 134]. MicroRNAs are other popular ncRNAs in the literature, and their role has been discussed as both oncogenes and tumor suppressors [144]. For example, miR-21 has been considered an oncogene in various studies [189,190,191,192], whereas ncRNAs such as miR-34a are known as tumor suppressors [145, 146].

The study of ncRNAs is a dynamic field; even among the most well-known types of ncRNAs, notable differences in the results of researchers can be observed. For example, MALAT1 has been identified as an oncogene in various cancers, whereas in a study by Jongchan Kim et al., it was demonstrated to be a tumor suppressor. In this study, MALAT1 was reported to bind to the transcriptional enhanced associate domain (TEAD), which is a prometastatic transcription factor, inactivating it and preventing it from interacting with the target gene promoter and coactivator Yes-Associated Protein (YAP), and a negative correlation between MALAT1 and the spread of breast cancer was shown [355]. The results of a study performed by Zhang Yang et al. were consistent and reported the downregulation of MALAT1 in breast cancer cell lines and tissues. Cell proliferation was augmented when its expression was inhibited. Additionally, they demonstrated that in MALAT-depleted cells, a large increase in cyclin D1 (CCND1) mRNA and protein levels can be observed [356]. In a review paper published by Nadine Smith et al., it was mentioned that NEAT1 can play a role in both parts depending on its specific form. They highlighted the importance of studying different isoforms of NEAT1 with respect to cancer progression and usage in therapy [357]. In Chunfu Zhang et al.’s study, miR-21 is considered to play a dual role, as it can affect both breast cancer genes and tumor suppressor genes. Interestingly, when cancer gene suppression is dominant in tumor cells, it prevents tumor growth, and when suppression of tumor suppressor genes is abundant, these genes act as oncogenes and augment cancer progression [190]. A study by Junfeng Wang focused on the levels of miR-155 in breast cancer and reported a positive relationship between higher levels of miR-155 and the antitumor immune profile. Experiments in mice demonstrated that miR-155 overexpression upregulated CXCL9/10/11 production [358].

Future direction

The inconsistency observed in the literature illustrates the importance of studying ncRNA at a more in-depth level. Notably, despite a vast amount of research examining and validating the use of ncRNAs as potential therapeutic agents in cancer treatment, the results of the most recent clinical trial revealed significant adverse effects, which is a crucial aspect to look at. The large number of studies on cancer cell lines and the small number of studies on mouse models highlight the lack of sufficient information on the side effects of ncRNA drug models in entire body systems.

Conclusion

On the basis of numerous studies, the role of ncRNAs in cancer is undeniable. Their function is similar to that of a double-edged sword since many of them are considered tumor suppressors and many are considered oncogenes. LncRNAs can participate in various pathways leading to cancer promotion, such as angiogenesis, EMT, metastasis and unregulated proliferation, through mediators such as miRNAs, which themselves can act as inducers or suppressors of cancer [104, 359]. With recent advances in technology, studying the role of ncRNAs is more feasible than ever, although challenges remain with each approach, highlighting a place for future research and investigation. Furthermore, bridging the gap between preclinical studies and clinical trials is essential to validate the therapeutic potential of ncRNAs. The development of robust animal models and the execution of large-scale clinical trials will play pivotal roles in translating ncRNA-based therapies into clinical practice. By gaining a deeper understanding of the functions and mechanisms of ncRNAs, we may be able to effectively control cancer progression, reverse adverse outcomes, and design more targeted and effective therapeutic strategies. However, significant gaps and unanswered questions remain, necessitating further comprehensive research to fully unlock their potential.