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Heterogeneous nuclear ribonucleoprotein C promotes non-small cell lung cancer progression by enhancing XB130 mRNA stability and translation

Cancer Cell International volume 25, Article number: 10 (2025) Cite this article

Abstract

Background

XB130, a classical adaptor protein, exerts a critical role in diverse cellular processes. Aberrant expression of XB130 is closely associated with tumorigenesis and aggressiveness. However, the mechanisms governing its expression regulation remain poorly understood. Heterogeneous nuclear ribonucleoprotein C (hnRNPC), as an RNA-binding protein, is known to modulate multiple aspects of RNA metabolism and has been implicated in the pathogenesis of various cancers. We have previously discovered that hnRNPC is one of the candidate proteins that interact with the 3’ untranslated region (3’UTR) of XB130 in non-small cell lung cancer (NSCLC). Therefore, this study aims to comprehensively elucidate how hnRNPC regulates the expression of XB130 in NSCLC.

Materials and methods

We evaluated the expression of hnRNPC in cancer and assessed the correlation between hnRNPC expression and prognosis in cancer patients using public databases. Subsequently, several stable cell lines were constructed. The proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of these cells were detected through Real-time cellular analysis, adherent colony formation, wound healing assay, invasion assay, and Western blotting. The specific regulatory manner between hnRNPC and XB130 was investigated by Real-time quantitative PCR, Western blotting, RNA pull‑down assay, dual‑luciferase reporter assay, RNA immunoprecipitation, and Co-Immunoprecipitation.

Results

We identified that hnRNPC expression is significantly elevated in NSCLC and correlates with poor prognosis in patients with lung adenocarcinoma. HnRNPC overexpression in NSCLC cells increased the expression of XB130, subsequently activating the PI3K/Akt signaling pathway and ultimately promoting cell proliferation and EMT. Additionally, overexpressing XB130 in hnRNPC-silenced cells partially restored cell proliferation and EMT. Mechanistically, hnRNPC specifically bound to the 3’UTR segments of XB130 mRNA, enhancing mRNA stability by inhibiting the recruitment of nucleases 5’-3’ exoribonuclease 1 (XRN1) and DIS3-like 3’-5’ exoribonuclease 2 (DIS3L2). Furthermore, hnRNPC simultaneously interacted with the eukaryotic initiation factor 4E (eIF4E), a component of the eIF4F complex, facilitating the circularization of XB130 mRNA and thereby increasing its translation efficiency.

Conclusions

HnRNPC overexpression promotes NSCLC progression by enhancing XB130 mRNA stability and translation, suggesting that hnRNPC might be a potential therapeutic and prognostic target for NSCLC.

Background

Lung cancer represents one of the primary causes of cancer-related mortality worldwide [1, 2]. It is classified into two major histological subtypes, among which non-small cell lung cancer (NSCLC) accounts for approximately 85% [3]. In recent years, despite the remarkable advancements in lung cancer treatment modalities such as surgery, chemotherapy, biotherapy, immunotherapy, and targeted therapy, the Overall Survival (OS) rate remains at around 25% [1, 4]. Therefore, to improve the OS rate of NSCLC, it is imperative for us to further explore the molecular mechanisms underlying the progression of NSCLC, which will contribute to identifying novel potential molecular targets for the early diagnosis and treatment of this malignancy.

XB130, a classical adaptor protein also known as actin filament-associated protein 1 like 2, plays a critical role in various cellular processes, including cell polarization, cytoskeletal organization, proliferation, migration, invasion, and differentiation [5, 6]. Research has shown that XB130 mediates interactions between the microfilament and microtubule systems in thyroid cells, which are essential for maintaining cell polarity, apical membrane structure, and function [6, 7]. Additionally, phosphorylated XB130 can bind to proteins containing src-homology 2 or src-homology 3 domains, such as the P85α subunit of PI3K, thereby influencing their activity and downstream signaling pathways (e.g., PI3K/Akt) and ultimately regulating processes such as cell proliferation, migration, invasion, differentiation, inflammation, and innate immune responses [8, 9]. Under normal conditions, XB130 is evenly distributed throughout the cytoplasm. However, when stimulated by factors such as injury, epidermal growth factor, or nicotine-derived nitrosamine ketone, XB130 translocates to the cell periphery, co-localizing with membrane-associated actin to promote the formation of lamellipodia, thus enhancing cell migration and invasion [10,11,12].

Ingenuity Pathway Analysis suggests that XB130 is closely associated with various cancers [13]. Previous studies have confirmed that XB130 is overexpressed in several cancers, including cholangiocarcinoma, liver hepatocellular carcinoma, and esophageal squamous cell carcinoma, where it facilitates cell proliferation, migration, and invasion [14,15,16]. Conversely, XB130 expression is significantly downregulated in basal cell carcinoma compared to normal human skin [17]. Notably, XB130 deficiency has been shown to increase the proliferation of epithelial tumor cells in mice and enhance inflammatory responses, creating a favorable environment for tumorigenesis [18]. This suggests that XB130 may play distinct roles across different types of tumors.

Atsushi Shiozaki et al. have shown that the interference of XB130 suppresses the proliferation of A549 cells [9]. Moreover, our research has indicated that silencing XB130 inhibits the migration, invasion, and epithelial-mesenchymal transition (EMT) of NSCLC cells [19]. These results suggest that targeted modulation of XB130 expression may have the potential to impede the progression of NSCLC. However, the mechanisms regulating XB130 expression remain largely unexplored at present.

Our previous research has demonstrated that the segments 113–230 and 503–660 of XB130 mRNA 3’ untranslated region (3’UTR) (1–1218 bp) play crucial roles in regulating mRNA stability and translational efficiency [20]. Through RNA pull-down assays combined with mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein C (hnRNPC) as a potential binding protein for these regions within XB130 mRNA 3’UTR [20].

HnRNPs are involved in various aspects of mRNA metabolism, including transcription, alternative splicing, nuclear transport, stability, and translation [21]. Aberrant expression of hnRNPs has been associated with numerous malignancies [21, 22]. As a member of the hnRNP family, hnRNPC, functioning as an m6A reader and an RNA-binding protein (RBP), exerts a significant influence on the development and progression of tumors [23,24,25]. For example, by binding to β-catenin mRNA, hnRNPC is capable of enhancing the stability of β-catenin mRNA, resulting in an elevation of β-catenin expression and consequently promoting the metastasis of colorectal cancer [25]. Moreover, hnRNPC promotes the stability of interleukin 1 receptor associated kinase 1 mRNA in an m6A-dependent manner, which subsequently activates the mitogen-activated protein kinase signaling pathway and facilitates the malignant transformation of glioma [24]. In the case of lung adenocarcinoma, hnRNPC interacts with TGF-β-upregulated lncRNA 1 to stabilize growth factor receptor bound protein 2 mRNA in an m6A-dependent way, thereby triggering the EMT [23].

In this study, we identified hnRNPC as a novel regulatory factor for XB130 expression in NSCLC. Our results indicate that hnRNPC directly interacts with the 3’UTR of XB130 mRNA, increasing XB130 mRNA stability by inhibiting the binding of nucleases 5’-3’ exoribonuclease 1 (XRN1) and DIS3 like 3’-5’ exoribonuclease 2 (DIS3L2). Furthermore, hnRNPC enhances the translational efficiency of XB130 mRNA by simultaneously interacting with the eukaryotic initiation factor 4E (eIF4E), which is a critical component of the eIF4F complex. This increase in XB130 expression activates the PI3K/Akt signaling pathway, facilitating the proliferation and EMT of NSCLC cells. In summary, this study demonstrates that hnRNPC enhances XB130 expression at the post-transcriptional level, thereby modulating the biological function of NSCLC cells.

Materials and methods

Gene expression analysis

Differential expression of hnRNPC between tumor and normal tissues was analyzed employing the Gene_DE function within the Cancer Exploration module of the TIMER2.0 database (http://timer.cistrome.org/), an analytical platform that primarily utilizes data from The Cancer Genome Atlas (TCGA). The correlation between hnRNPC expression and OS across various tumor types was assessed using the Survival Analysis functionality available in the GEPIA2 database (http://gepia2.cancer-pku.cn/#survival), which primarily relies on TCGA and Genotype-Tissue Expression (GTEx) data. The median expression value of hnRNPC served as the threshold for classifying samples into high-expression and low-expression groups. Survival curves were generated using the Kaplan-Meier method, and comparisons were performed using the log-rank test.

Cell culture and transfection

Human NSCLC cell lines PC‑9 and NCI-H292, along with the human renal epithelial cell line 293T were used in this study. PC‑9 was purchased from FuHeng Biology Company (Shanghai, China), and NCI-H292 and 293T were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences (Kunming, China). All cell lines were tested to eliminate the possibility of mycoplasma contamination. The passage number of all cell lines was no more than 15. These cells were cultured in RPMI-1640 medium or Dulbecco’s Modified Eagle Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibco). The cultures were maintained in a humidified incubator (Thermo Fisher Scientific) at 37 °C in an atmosphere containing 5% CO2. For DNA and RNA transfections, Entranster™-H4000 and Entranster™-R4000 (Engreen Biosystem, Beijing, China) were utilized, respectively. Co-transfections with DNA and RNA oligos (Table S1) were performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). Transfection procedures were carried out following the manufacturer’s protocol. The transfection efficiency regarding DNA or RNA surpassed 70%. Unless otherwise specified, all cells were collected for subsequent analyses 48 h post-transfection.

Plasmid construction

To silence hnRNPC gene, control shRNA (NC shRNA) and hnRNPC shRNA (hnRNPC shRNA-1 and -2) primers were designed and synthesized (Table S2). All shRNA sequences undergo BLAST analysis to confirm their sequence specificity. Each single-strand shRNA primer was dissolved in Tris-EDTA buffer to a final concentration of 100 µM. The sense and antisense single-strand shRNA primers were mixed in a 1:1 ratio and subjected to a PCR program (95 °C for 2 min, 72 °C for 2 min, 37 °C for 2 min, and 25 °C for 2 min) to generate double-stranded shRNA. This product was then cloned into the psi-LVRU6GP vector (GeneCopoeia, Germantown, MD, USA) to create NC or hnRNPC silencing recombinant vectors.

For the overexpression of hnRNPC or XB130, specific primers for amplifying the open reading frame of hnRNPC or XB130 were designed and synthesized (Table S2). Using cDNA derived from PC-9 cells as a template, hnRNPC and XB130 open reading frames were amplified according to the following PCR protocol: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 2 min. The amplified PCR products were subsequently inserted into the EX-NEG-Lv201 and EX-NEG-Lv151 vectors (GeneCopoeia), respectively. The hnRNPC overexpression recombinant vector was designed to express hnRNPC protein with a His tag at the C-terminus. The DIS3L2 overexpression recombinant vector was obtained from Hunan Keai Medical Equipment Co., Ltd. (Hunan, China) and encodes the DIS3L2 protein with a Flag tag at its C-terminus.

Dual-luciferase reporter recombinant vectors containing mRNA 3’UTR sequences were constructed as previously described [20]. Additionally, a 3290 bp fragment located upstream of the XB130 transcription start site was amplified and cloned into the dual-luciferase reporter vector PEZX-FR01 (GeneCopoeia) to assess XB130 promoter activity. All recombinant vectors were verified by DNA sequencing.

Establishment of stable cell lines

293T cells were seeded in 6-well plates and incubated until they reached 80% confluence after 24 h. Gene silencing or overexpression recombinants were mixed with lentivirus packaging plasmids (psPASX2 and PMD2G) at a ratio of 4:3:1, and this mixture was co-transfected into the 293T cells. After 48 h, the cell supernatant was collected as viral solution. NSCLC cells were then plated in 6-well plates and allowed to achieve 40% ~ 60% confluence within 24 h. The collected viral solution was mixed with an appropriate volume of lentivirus infection reagent (Engreen Biosystem) and then added to the NSCLC cells. Following incubation for 8 h, the viral solution was replaced with fresh complete culture medium. After 72 h of viral infection, puromycin was added to select for cells containing the psi-LVRU6GP or EX-NEG-Lv201 recombinant vector.

Real-time quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The isolated RNA was reverse transcribed into cDNA using the HyperScript III 1st Strand cDNA Synthesis Kit with gDNA Remover (NovaBio, Shanghai, China). Quantitative PCR was conducted with the 2× SYBR Green qPCR MasterMix (Bimake, Shanghai, China) on a Bio-Rad CFX Connect™ Real-Time System (Hercules, CA, USA). Relative gene expression levels were calculated using the 2‑ΔΔCq method. To quantify exogenous luciferase mRNA, the level of Renilla luciferase (hRluc) mRNA in each sample was normalized to the corresponding amount of Firefly luciferase (hluc). For assessing endogenous mRNA expression, GAPDH served as the internal reference. The RT-qPCR primers are listed in Table S2.

Western blotting

Cell pellets were resuspended in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride and incubated on ice for 30 min. After centrifugation at 14,000 × g for 10 min at 4 °C, the supernatant was collected. An appropriate volume of 5× SDS-PAGE Loading Buffer was added to the supernatant, which was then vortexed and heated at 100 °C for 10 min. The protein samples were separated by electrophoresis on 12% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Merck, Billerica, MA, USA). After blocking with 5% BSA for 1 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-hnRNPC (1:2000; ProteinTech, Wuhan, China), anti-XB130 (1:2000; ProteinTech), anti-AKT (1:8000; ProteinTech), anti-p-AKT (Ser473) (1:5000; ProteinTech), anti-Hsp90 (1:5000; ProteinTech), anti-Flag (1:1000; ProteinTech), anti-N-cadherin (1:5000; ProteinTech), anti-β-catenin (1:5000; ProteinTech), anti-SNAI1 (1:2000; ProteinTech), anti-eIF4E (1:2000; ABclonal, Wuhan, China), anti-hnRNPR (1:2000; ABclonal), and anti-GAPDH (1:5000; ProteinTech). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000; ProteinTech). Target bands were visualized using ECL chemiluminescence detection (Millipore) on a GeneGnome XRQ Chemiluminescence Imaging System (Syngene, Cambridge, England).

Real-time cellular analysis (RTCA)

A volume of 50 µL of complete culture medium was added to each well of the E-Plate, and the background value was measured after incubation for 10 min. Cells were digested with trypsin and counted. A total of 120 µL of cell suspension containing 1.5 × 104 cells was seeded into each well. The E-Plate was then placed on the xCELLigence RTCA instrument (Agilent, Santa Clara, CA, USA), and the program was initiated to monitor cell growth dynamics in real-time.

Adherent colony formation

Cells were seeded at a density of 1000 cells per well in a 6-well plate and cultured in complete medium for 2 weeks. After incubation, cells were washed twice with 1× PBS and fixed with 4% paraformaldehyde for 20 min. Subsequently, cells were stained with 0.01% crystal violet for 30 min. Following rinsing with running water to remove excess dye, the number of colonies was counted.

Wound healing assay

Cells were seeded into 6-well plates and allowed to reach confluence after 24 h. A 200 µL pipette tip was used to create a scratch in the monolayer. Cells were then washed three times with 1× PBS, and a medium containing 4% fetal bovine serum was added for continued culture. Photographs were taken immediately after scratching (0 h) and at 24 h post-scratch for the same fields. The area of scratches is calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA). The scratch healing rate was calculated using the formula: [(scratch area at 0 h - scratch area at 24 h)/scratch area at 0 h] × 100%. Four fields within each well were selected for imaging, and the average scratch healing rate across these four regions was utilized as a replicate measure for statistical analysis.

Invasion assay

A total of 100 µL of 10% Matrigel matrix (BD Biosciences, San Jose, CA, USA) was evenly added to the upper chamber of the Transwell apparatus (Corning Costar, Tewksbury, MA, USA) and incubated at 37 °C for 2 h. Cells were digested with trypsin and resuspended in a serum-free medium. 100 µL of the suspension containing 2 × 104 cells was then seeded into the upper chamber. Subsequently, 700 µL of complete medium was added to the lower chamber. After incubation for 48 h, the remaining cells in the upper chamber were removed. The cells that adhered to the lower surface of the chamber were fixed with 4% paraformaldehyde for 20 min, stained with 0.01% crystal violet for 30 min, and rinsed with running water to remove excess dye. Photographs were taken, and the number of cells that migrated through the upper chamber was quantified using ImageJ. For each chamber, three randomly selected regions were imaged, and the average cell count from these regions was used as a measure of the overall invasion level of the cells within that chamber.

Dual‑luciferase reporter assay

Cells were seeded in a 48-well plate and transfected with a dual-luciferase reporter vector. After 48 h, dual-luciferase activity was measured using the Luc‑Pair™ Duo‑Luciferase HS Assay Kit (GeneCopoeia) according to the manufacturer’s instructions. Briefly, the cells were washed twice with 1× PBS, and then 65 µL of 1× Luc-Lysis II Buffer was added to each well for 10 min of lysis at room temperature. Subsequently, 20 µL of the lysate from each well was transferred to a new black 96-well plate, and 100 µL of hluc and hRluc detection reagents were added in succession. Light output was measured immediately following each reagent addition using a BioTek Synergy2 Multimode Microplate Reader (Biotek Winooski, Vermont, USA). hRluc and hluc served as internal controls for the PEZX-FR01 and psiCHECK-2 (Promega, Madison, WI, USA) constructs, respectively.

mRNA decay assay

Cells were treated with actinomycin D (10 µg/mL) to inhibit mRNA synthesis, and samples were collected at 0, 2, 4, 8, and 10 h post-treatment. Total RNA was extracted from each sample, and RT-qPCR was performed to assess the mRNA abundance of the target gene and internal control. The relative abundance of the target gene mRNA at 0 h was set to 1, and fold changes in relative abundance at subsequent time points were calculated accordingly. A graph was generated to illustrate changes in target gene mRNA abundance over time, and the half-life of the target gene mRNA was determined.

RNA immunoprecipitation (RIP) and Co-Immunoprecipitation (Co-IP)

A total of 2 × 106 cells were suspended in 200 µL of Pierce™ IP Buffer (Thermo Scientific, Thermo Fisher Scientific) supplemented with a 1× proteinase inhibitor cocktail (ProteinTech) and lysed on ice for 15 min. The resulting cell lysate was centrifuged at 14,000 × g for 10 min at 4 °C to separate the supernatant, which was then incubated overnight at 4 °C with 4 µg of primary antibody. For bead preparation, 20 µL of protein G Dynabeads (NEB, Ipswich, MA, USA) were incubated with 5% BSA at 4 °C for 30 min. The beads were collected and added to the supernatant-antibody mixture, followed by an additional incubation at 4 °C for 2 h. After incubation, the beads were washed and collected. For RIP, RNA was extracted from the immunoprecipitated complexes for quantitative analysis via RT-qPCR. For Co-IP, the protein within the bead-precipitated complexes was identified through Western blotting.

RNA pull‑down assay

A DNA fragment containing a T7 promoter was generated through PCR amplification and subsequently transcribed into RNA in vitro using a T7 RNA polymerase kit (Roche, Indianapolis, IN, USA). The RNA products were labeled using a Pierce RNA 3’ End Desthiobiotinylation Kit (Invitrogen) according to the manufacturer’s instructions. The biotinylated RNA was then incubated with streptavidin-coated magnetic beads at room temperature for 15 ~ 30 min. Following this, the beads were collected and incubated with cell lysate at 4 °C for 30 ~ 60 min. The target protein present in the bead-precipitated complexes was subsequently detected by Western blotting.

Statistical analysis

Statistical analyses were performed using SPSS 26.0 software (IBM, Armonk, NY, USA). Unless otherwise specified, the differences between two groups were assessed using Student’s t-test, while comparisons among more than two groups were conducted using analysis of variance with the Tukey test. All experiments were carried out in triplicate. Data are presented as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant.

Results

HnRNPC expression is significantly elevated in NSCLC and correlates with poor prognosis in patients with lung adenocarcinoma

To investigate the potential role of hnRNPC in cancer, we analyzed its expression across 33 different human malignancies. Our findings revealed a substantial increase in hnRNPC expression levels in 14 cancer types compared to normal tissue, including ‌bladder urothelial carcinoma, breast invasive carcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, stomach adenocarcinoma, and thyroid carcinoma (Fig. 1A). Subsequently, we assessed the correlation between hnRNPC expression and prognosis in patients with these cancer types. The analysis revealed a correlation between elevated hnRNPC expression and poor prognosis in patients with adrenocortical carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney renal papillary cell carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, and sarcoma. Conversely, high hnNRPC expression levels were significantly associated with improved prognosis in patients with kidney renal clear cell carcinoma and thymoma (Fig. 1B). Collectively, these findings suggest that the elevated hnRNPC expression may contribute to the progression of various cancers, including NSCLC. Moreover, hnNRPC may serve as a potential prognostic marker for several cancers, such as lung adenocarcinoma.

Fig. 1
figure 1

HnRNPC expression is elevated in NSCLC and correlates with poor prognosis in lung adenocarcinoma patients. A: The expression levels of hnRNPC in 33 types of cancer tissues were analyzed using the TIMER2.0 database. The statistical significance was determined using the Wilcoxon test. B: The correlation between hnRNPC expression and OS in patients across different cancer types was analyzed with the GEPIA2 database. Comparisons were performed using the log-rank test. ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: esophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukemia; LGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; UCS: uterine carcinosarcoma; UVM: uveal melanoma; TPM: transcripts per million. * p < 0.05, ** p < 0.01, *** p < 0.001

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HnRNPC overexpression significantly enhances cell proliferation and EMT in NSCLC

To investigate the role of hnRNPC dysregulation in the progression of NSCLC, we established stable NSCLC cell lines with either silenced or overexpressed hnRNPC (Figs. 2A and 3A). We subsequently assessed the effects of hnRNPC dysregulation on NSCLC cell proliferation, migration, invasion, and EMT. As shown in Fig. 2, silencing hnRNPC significantly inhibited the proliferation (the colony number of NCI-H292: pshRNA−1 = 0.000006, pshRNA−2 = 0.000007; PC-9: pshRNA−1 = 0.000004, pshRNA−2 = 0.000005), migration (NCI-H292: pshRNA−1 = 0.001823, pshRNA−2 = 0.012732; PC-9: pshRNA−1 = 0.000647, pshRNA−2 = 0.000163), and invasion (NCI-H292: pshRNA−1 = 7.5087 × 10− 7, pshRNA−2 = 9.605 × 10− 7; PC-9: pshRNA−1 = 0.000256, pshRNA−2 = 0.001886) of NSCLC cells compared to the control group (Fig. 2B-E). Meanwhile, the expression levels of EMT-related markers, including N-cadherin, β-catenin, and SNAI1, were significantly decreased in the hnRNPC-silenced group (Fig. 2F). Additionally, although silencing hnRNPC did not affect the total protein levels of Akt in NSCLC cells, it led to a significant reduction of phosphorylated Akt at Ser473 (p-Akt (Ser473)) (Fig. 2G).

Fig. 2
figure 2

Silencing hnRNPC inhibits cell proliferation and EMT of NSCLC by suppressing the Akt signaling pathway. Control (NC shRNA) and hnRNPC-silenced (hnRNPC shRNA-1 and -2) NCI-H292 and PC-9 stable cell lines were constructed. A: HnRNPC expression levels in cells were assessed using RT-qPCR and Western blotting. B: RTCA assay was utilized to monitor cell growth. Cell index is a dimensionless parameter calculated from the impedance values measured by the RTCA system using standard formulas. It indicates cell viability within the detection well. C: The colony-forming ability of cells was examined through adherent colony formation assays. D: Wound healing assays investigated cell migration. Scale bars represent 100 μm. E: Invasion assays assessed the invasive capability of cells. Scale bars represent 100 μm. F-G: Western blotting analyzed expression levels of EMT-related markers (F), total Akt, and p-Akt (Ser473) (G) in the cells. GAPDH was used as an internal control for both RT-qPCR and Western blotting. Multiple membranes from the same sample were exposed simultaneously during Western blotting to detect different proteins, resulting in the presence of more than one GAPDH loading control. * p < 0.05, ** p < 0.01, *** p < 0.001

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Fig. 3
figure 3

HnRNPC overexpression promotes NSCLC cell proliferation and EMT by activating the Akt signaling pathway. Control (OV-NC) and hnRNPC-overexpressing (OV-hnRNPC) NCI-H292 and PC-9 stable cell lines were constructed. A: Western blotting was performed to assess the expression of hnRNPC in the cells. B: Cell growth was assessed by RTCA analyses. C: Adherent colony formation assays analyzed the colony-forming ability of the cells. D: Wound healing assays were performed to assess cell migration. Scale bars represent 100 μm. E: Invasion assays measured the invasive capability of the cells. Scale bars represent 100 μm. F-G: Western blotting was conducted to detect the expression levels of EMT-related markers (F), as well as total Akt and p-Akt (Ser473) (G) in the cells. GAPDH served as the loading control. * p < 0.05, ** p < 0.01, *** p < 0.001

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Conversely, overexpression of hnRNPC dramatically enhanced the proliferation (the colony number of NCI-H292: p = 0.001828; PC-9: p = 0.000252), migration (NCI-H292: p = 0.00011; PC-9: p = 0.012811), and invasion (NCI-H292: p = 0.000358; PC-9: p = 0.00645) of NSCLC cells (Fig. 3B-E) and increased the expression levels of EMT-related markers such as N-cadherin, β-catenin, and SNAI1 (Fig. 3F). Furthermore, p-Akt (Ser473) protein levels were significantly elevated in response to hnRNPC overexpression (Fig. 3G). Collectively, these findings indicate that hnRNPC promotes NSCLC cell proliferation and EMT through the activation of the Akt signaling pathway.

HnRNPC upregulates XB130 expression by binding to the 3’UTR of XB130 mRNA

To explore the relationship between hnRNPC and XB130, we analyzed the expression levels of XB130 mRNA and protein in NSCLC cells with either silenced or overexpressed hnRNPC. The results demonstrated that silencing hnRNPC resulted in a significant decrease in both XB130 mRNA (NCI-H292: pshRNA−1 = 0.005908, pshRNA−2 = 0.027352; PC-9: pshRNA−1 = 0.006882, pshRNA−2 = 0.005533) and protein levels compared to the control group (Fig. 4A). Conversely, overexpression of hnRNPC led to an upregulation of XB130 expression (XB130 mRNA level of NCI-H292: p = 0.000426; PC-9: p = 0.000132) (Fig. 4B). To determine whether hnRNPC affects XB130 expression at the transcriptional level, we constructed a dual-luciferase reporter recombinant vector containing XB130 promoter and transfected it into control and hnRNPC-silenced cells. Data analysis revealed no significant differences in luciferase activity between the control and hnRNPC-silenced groups, indicating that hnRNPC does not impact the transcription of XB130 (Fig. 4C). Therefore, we further investigated how hnRNPC regulates XB130 expression at the post-transcriptional level.

Fig. 4
figure 4

HnRNPC promotes XB130 expression post-transcriptionally by binding to XB130 mRNA 3’UTR. A-B: RT-qPCR and Western blotting were conducted to assess the expression of XB130 in control and hnRNPC-silenced (A) or -overexpressing (B) cells. GAPDH was used as an internal control. C: A dual-luciferase reporter recombinant vector (PEZX-XB130 promoter) containing the XB130 promoter was constructed and transfected into control and hnRNPC-silenced cells. Dual-luciferase activity was measured 48 h post-transfection. hRluc was used as an internal control. D: The wild-type full-length XB130 3’UTR (3’UTR), along with the 3’UTR segments 113–230 and 503–660, and a mutated XB130 3’UTR lacking the segments 113–230 and 503–660 (MU-3’UTR), were generated through in vitro transcription. RNA pull-down assays were performed to investigate the interactions of these RNA fragments with hnRNPC and Hsp90 in NCI-H292 cells. E: RIP assays verified the interactions of hnRNPC protein with XB130 and Hsp90 mRNA in hnRNPC-overexpressing cells. IgG-bound RNA served as a negative control. F: A series of dual-luciferase reporter recombinant vectors containing different segments of XB130 mRNA 3’UTR were constructed and transfected into control and hnRNPC-silenced cells. Dual-luciferase activity was assessed 48 h after transfection. hluc served as an internal control. * p < 0.05, ** p < 0.01, *** p < 0.001

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RNA pull-down assays demonstrated that hnRNPC specifically bound to the 3’UTR of XB130 mRNA, particularly to the 3’UTR segments 113–230 and 503–660. Notably, deletion of these segments completely abolished the interaction between hnRNPC and XB130 mRNA 3’UTR (Fig. 4D). Furthermore, RIP assays confirmed that hnRNPC could bind to XB130 mRNA (NCI-H292: p = 0.003798; PC-9: p = 0.000827) (Fig. 4E). To determine whether hnRNPC regulates XB130 expression through its interaction with the 3’UTR segments 113–230 and 503–660 of XB130 mRNA, we generated a series of dual-luciferase reporter recombinant vectors containing distinct regions of XB130 mRNA 3’UTR, which were then transfected into control and hnRNPC-silenced cells. Our findings revealed that silencing hnRNPC significantly decreased luciferase expression in the recombinant vectors containing the 113–230 (NCI-H292: pshRNA−1 = 0.010408, pshRNA−2 = 0.01006; PC-9: pshRNA−1 = 0.005875, pshRNA−2 = 0.0099) or 503–660 (NCI-H292: pshRNA−1 = 0.000749, pshRNA−2 = 0.000793; PC-9: pshRNA−1 = 0.035416, pshRNA−2 = 0.000994) fragment compared to the control group. In contrast, no significant effect was observed on luciferase expression in recombinant vectors containing other regions of XB130 mRNA 3’UTR following hnRNPC silencing (Fig. 4F). These results collectively indicate that hnRNPC increases XB130 expression by binding to the 3’UTR segments 113–230 and 503–660 of XB130 mRNA.

HnRNPC increases XB130 mRNA stability and translation efficiency by binding to XB130 mRNA 3’UTR

The 3’UTR segments 113–230 and 503–660 of XB130 mRNA play critical roles in regulating mRNA stability and translation [20]. Therefore, we subsequently investigated the impact of hnRNPC binding to these specific regions on the stability and translation of XB130 mRNA. As illustrated in Fig. 5A, silencing hnRNPC resulted in a significant decrease in the stability of XB130 mRNA in NSCLC cells compared to the control group (the t1/2 of XB130 mRNA in NCI-H292: 14.05 h vs. 7.60 h; PC-9: 12.15 h vs. 6.76 h). Furthermore, we transfected dual-luciferase reporter recombinant vectors containing either the 3’UTR segment 113–230 or 503–660 of XB130 mRNA into control and hnRNPC-silenced cells. The mRNA decay assays demonstrated that silencing hnRNPC significantly downregulated the stability of luciferase mRNA compared to the control group (Fig. 5B, C), suggesting that hnRNPC enhances XB130 mRNA stability by binding to the 3’UTR segments 113–230 and 503–660.

Fig. 5
figure 5

HnRNPC enhances XB130 mRNA stability and translation efficiency by binding to the 3’UTR of XB130 mRNA. A: mRNA decay assays analyzed the stability of XB130 mRNA in control and hnRNPC-silenced cells. B-C: Dual-luciferase reporter recombinant vectors containing the 3’UTR segment 113–230 (B) or 503–660 (C) of XB130 mRNA were transfected into control and hnRNPC-silenced cells, followed by mRNA decay assays to assess the stability of the reporter gene hRluc mRNA. D: RIP assays were performed to examine interactions between eIF4E protein and XB130 and Hsp90 mRNA in control and hnRNPC-silenced cells. IgG-bound RNA was used as a negative control. E: Co-IP experiments were conducted to investigate interactions of hnRNPC with eIF4E and hnRNPR in hnRNPC-overexpressing cells, under RNA-present and RNA-absent conditions. IgG-bound protein was used as a negative control. t1/2: half‑lives of mRNA; ActD: actinomycin D. * p < 0.05, ** p < 0.01, *** p < 0.001

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Additionally, we calculated the ratio of luciferase activity to its mRNA content in cells transfected with the dual-luciferase reporter recombinant vectors containing the 3’UTR segment 113–230 or 503–660 of XB130 mRNA. This allowed us to preliminarily assess whether silencing hnRNPC affected the translation of luciferase mRNA through these specific fragments. Our findings demonstrated a significant reduction in this ratio in the hnRNPC-silenced group compared to the control group, indicating that hnRNPC might promote XB130 mRNA translation by binding to specific regions within XB130 mRNA 3’UTR (Table 1).

Table 1 Comparison of luciferase activity to mRNA content in NSCLC cells
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In eukaryotic cells, the recognition and binding of ribosomal subunits to the 5’ cap structure of mRNA rely on the eIF4F complex [26]. To investigate whether hnRNPC influences XB130 translation by modulating ribosomal binding, we performed RIP using eIF4E, a critical component of the eIF4F complex. Due to the downregulation of XB130 mRNA levels in hnRNPC-silenced cells, we included excess cell lysate in the RIP assays to mitigate potential biases stemming from initial differences in mRNA abundance across the groups. The results revealed a significant reduction in the amount of XB130 mRNA associated with eIF4E antibody in the hnRNPC-silenced group compared to the control group (NCI-H292: p = 0.00075; PC-9: p = 0.00067). Furthermore, hnRNPC protein was pulled down by the eIF4E antibody, suggesting that hnRNPC promoted the translation initiation of XB130 mRNA, potentially through its interactions with the eIF4F complex (Fig. 5D). Co-IP assays further confirmed the interaction between hnRNPC and eIF4E, which is independent of RNA (Fig. 5E). Moreover, our other research indicated that hnRNPR also enhanced the stability of XB130 mRNA by binding to the 3’UTR segments 113–230 and 503–660. Consequently, we investigated whether hnRNPC forms a complex with hnRNPR to contribute to XB130 expression. The results indicated that hnRNPR was absent in the proteins pulled down by His antibody, regardless of RNA presence (Fig. 5E), suggesting that hnRNPC and hnRNPR may competitively bind to the 3’UTR segments 113–230 and 503–660 of XB130 mRNA. Collectively, these results indicate that hnRNPC not only enhances the stability of XB130 mRNA through its binding to the 3’UTR segments but also facilitates the translation of XB130 mRNA by concurrently interacting with the eIF4F complex.

The binding of hnRNPC to XB130 mRNA 3’UTR obstructs the recruitment of nucleases XRN1 and DIS3L2

To elucidate how hnRNPC affects the stability of XB130 mRNA, we silenced eight common nucleases in hnRNPC-silenced cells, including XRN1 (p = 0.000786), decapping mRNA 2 (p = 0.000592), DIS3 like exosome 3’-5’ exoribonuclease (p = 0.000059), exosome component 4 (p = 0.000007), DIS3L2 (p = 0.000423), CCR4-NOT transcription complex subunit 1 (p = 0.001014), poly A-specific ribonuclease (p = 0.000506), and poly A specific ribonuclease subunit PAN3 (p = 0.000371) [27, 28] (Fig. 6A). Following this, we assessed the expression levels of XB130 mRNA and protein in these cells. The results indicated that only the silencing of XRN1 and DIS3L2 could upregulate the expression of both XB130 mRNA (NCI-H292: pXRN1 = 0.000005, pDIS3L2 = 00.000035; PC-9: pXRN1 = 0.000015, pDIS3L2 = 0.000388) and protein in two NSCLC cell lines (Fig. 6B, C). Subsequently, we co-transfected control and hnRNPC-silenced cells with dual-luciferase reporter recombinant vectors containing XB130 mRNA 3’UTR fragments and either control siRNA (NC siRNA) or specific siRNA targeting XRN1 or DIS3L2. The dual‑luciferase reporter assays demonstrated a significant decrease in luciferase activity in hnRNPC-silenced cells compared to the control group. Conversely, luciferase activity was markedly increased in cells where either XRN1 or DIS3L2 was silenced. Notably, the luciferase activity in groups double-silenced for hnRNPC and either XRN1 or DIS3L2 was significantly higher than that in the hnRNPC-silenced group but lower than that in the XRN1 or DIS3L2-silenced group, respectively. In contrast, cells transfected with an empty dual-luciferase reporter vector showed no significant differences in luciferase activity across the experimental groups (Fig. 6D). This observation suggests that nuclease XRN1 and DIS3L2 may be involved in regulating XB130 expression by hnRNPC.

Fig. 6
figure 6

HnRNPC binding to XB130 mRNA impedes recruitment of nucleases XRN1 and DIS3L2. A-C: Either control siRNA (NC siRNA) or gene-specific siRNA was transfected into hnRNPC-silenced cells. RT-qPCR and Western blotting assessed the silencing levels of each nuclease (A) and expression levels of XB130 mRNA (B) and protein (C) in the cells. GAPDH was used as an internal control. D: Control (psiCHECK-2) or dual-luciferase reporter recombinant vectors containing the 3’UTR segment 113–230 or 503–660 of XB130 mRNA, along with either NC siRNA or specific siRNA targeting XRN1 or DIS3L2, were co-transfected into control and hnRNPC-silenced cells. Dual-luciferase activity was measured 48 h post-transfection. hluc served as an internal control. E: A recombinant vector overexpressing Flag-tagged DIS3L2 was transfected into control or hnRNPC-silenced cells. RIP assays were then performed to evaluate interactions of DIS3L2 protein with XB130 and Hsp90 mRNA. IgG-bound RNA was used as a negative control. DCP2: decapping mRNA 2; DIS3L: DIS3 like exosome 3’-5’ exoribonuclease; EXOSC4: exosome component 4; CNOT1: CCR4-NOT transcription complex subunit 1; PARN: poly A-specific ribonuclease; PAN3: poly A specific ribonuclease subunit PAN3. * p < 0.05, ** p < 0.01, *** p < 0.001

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Based on these findings, we hypothesized that hnRNPC binds to the 3’UTR segments 113–230 and 503–660 of XB130 mRNA, thereby inhibiting the recruitment of XRN1 and DIS3L2 to XB130 mRNA. To validate this hypothesis, we selected DIS3L2 for further analysis. Flag-tagged DIS3L2 protein was overexpressed in both control and hnRNPC-silenced cells, and RIP assays using Flag antibody were performed. The results revealed a significant increase in the amount of XB130 mRNA enriched by the Flag antibody in hnRNPC-silenced cells compared to the control group (NCI-H292: p = 0.000002; PC-9: p = 0.000002), suggesting that hnRNPC silencing enhanced the binding of DIS3L2 to XB130 mRNA (Fig. 6E). In conclusion, our findings provide evidence that the binding of hnRNPC to the 3’UTR segments 113–230 and 503–660 of XB130 mRNA impeded the recruitment of nucleases XRN1 and DIS3L2, thereby protecting XB130 mRNA from degradation.

The silencing of nuclease XRN1 or DIS3L2 reverses the effect of hnRNPC silencing on XB130 mRNA stability

To verify the effect of XRN1 and DIS3L2 on the stability of XB130 mRNA, we transfected control or hnRNPC-silenced cells with either NC siRNA or specific siRNA targeting XRN1 or DIS3L2, followed by mRNA decay assays to assess XB130 mRNA stability. The results indicated that silencing hnRNPC significantly decreased the stability of XB130 mRNA compared to the control group. Notably, in the context of hnRNPC silencing, further silencing of XRN1 or DIS3L2 led to an increase in XB130 mRNA stability (Fig. 7A, B). Additionally, we co-transfected control or hnRNPC-silenced cells with either NC siRNA or specific siRNA targeting XRN1 or DIS3L2, along with a dual-luciferase reporter construct containing XB130 mRNA 3’UTR fragment. The mRNA decay assays demonstrated that, compared to the control group, hnRNPC silencing significantly reduced luciferase mRNA stability. However, under conditions of hnRNPC silencing, additional silencing of XRN1 or DIS3L2 resulted in increased luciferase mRNA stability (Fig. 7C-F). These findings suggest that hnRNPC protects XB130 mRNA from degradation by the nucleases XRN1 and DIS3L2, thereby enhancing the stability of XB130 mRNA.

Fig. 7
figure 7

Silencing XRN1 or DIS3L2 reverses the effects of hnRNPC silencing on XB130 mRNA stability. A-B: NC siRNA, XRN1 siRNA (A), or DIS3L2 siRNA (B) were transfected into control and hnRNPC-silenced cells. mRNA decay assays analyzed the stability of XB130 mRNA in these groups. C-D: Dual-luciferase reporter recombinant vectors containing the 3’UTR segment 113–230 of XB130 mRNA were co-transfected with NC siRNA, XRN1 siRNA (C), or DIS3L2 siRNA (D) into control and hnRNPC-silenced cells, followed by mRNA decay assays to assess the stability of the reporter gene hRluc mRNA in cells. E-F: Dual-luciferase reporter recombinant vectors containing the 3’UTR segment 503–660 of XB130 mRNA were co-transfected with NC siRNA, XRN1 siRNA (E), or DIS3L2 siRNA (F) into control and hnRNPC-silenced cells. mRNA decay assays were conducted to evaluate the stability of the reporter gene hRluc mRNA in cells. * means significantly different from the control group (NC siRNA + NC shRNA); # means significantly different from the hnRNPC-silenced group (NC siRNA + hnRNPC shRNA-2). t1/2: half‑lives of mRNA; ActD: actinomycin D. * p < 0.05, ** p < 0.01, *** p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001

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XB130 overexpression reverses the effects of hnRNPC silencing on NSCLC cell proliferation and EMT

To investigate the role of XB130 in NSCLC cell proliferation and EMT, we transfected control and hnRNPC-silenced cells with either control vector (Lv151) or XB130 overexpression recombinant (Lv151-XB130) (Fig. 8A). Various cellular assays demonstrated that, compared to the control group, silencing hnRNPC significantly reduced cell proliferation, migration, invasion, and the expression of EMT-related markers (Fig. 8B-F). Additionally, hnRNPC silencing led to decreased levels of p-Akt (Ser473) without affecting total Akt protein expression (Fig. 8G). However, in the context of hnRNPC silencing, overexpression of XB130 partially restored cell proliferation, migration, invasion, and the expression levels of EMT-related markers and p-Akt (Ser473) (Fig. 8B-G). These findings indicate that hnRNPC promotes the proliferation and EMT of NSCLC cells by regulating XB130 expression.

Fig. 8
figure 8

Overexpression of XB130 reverses effects of hnRNPC silencing on cell proliferation and EMT. Control (Lv151) or XB130 overexpression recombinant vectors (Lv151-XB130) were transfected into control or hnRNPC-silenced cells. A: Western blotting assessed protein expression levels of XB130 and hnRNPC. B: RTCA assays monitored cell growth. C: Adherent colony formation assays evaluated the cloning ability of cells. D: Wound healing assays investigated cell migration. E: Invasion assays assessed the invasive capability of the cells. F-G: Western blotting analyzed the expression of EMT-related markers (F), as well as total Akt and p-Akt (Ser473) (G) in cells. GAPDH was used as a loading control. * p < 0.05, ** p < 0.01, *** p < 0.001

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Discussion

XB130 is a multifunctional adaptor protein that plays a crucial role in influencing cellular structure by interacting with the cytoskeleton and regulating signaling pathways through its associations with downstream proteins [5, 9, 12]. Dysregulation of XB130 expression has been implicated in tumorigenesis and progression of various cancers [13, 14, 16,17,18]. However, the regulatory mechanisms governing XB130 expression remain poorly understood. Our previous studies revealed that specific regions in the 3’UTR of XB130 mRNA, particularly segments 113–230 and 503–660, significantly impact mRNA stability and translation [20]. Moreover, we identified hnRNPC as a potential binding protein for these regions [20]. In the present study, we demonstrated that hnRNPC is overexpressed in NSCLC, and its high expression is closely related to the poor prognosis of lung adenocarcinoma patients. In NSCLC cells, hnRNPC directly interacts with the 3’UTR of XB130 mRNA. By impeding the recruitment of the nucleases XRN1 and DIS3L2, hnRNPC increases the stability of XB130 mRNA. Furthermore, hnRNPC stimulates the translation of XB130 mRNA by simultaneously interacting with the eIF4F complex. This consequent upregulation of XB130 expression activates the PI3K/Akt signaling pathway, thereby facilitating the proliferation and EMT of NSCLC cells.

The 3’UTR of mRNA contains various elements participating in the regulation of eukaryotic gene expression [29]. Among these elements, GU/AU/CU/U-rich elements (G/A/C/UREs) are essential components involved in multiple processes, including mRNA processing, splicing, polyadenylation, nuclear transport, stability, and translation [30,31,32]. Typically, G/A/C/UREs exert their regulatory effects through interactions with specific RBPs [33]. In tumor cells, the expression or localization of these RBPs is often abnormal, closely associated with several aspects of cancer, including tumor progression, metabolic dysregulation, drug resistance, stem cell self-renewal, and immune evasion [34]. In our analysis, we identified three typical AREs (AUUUA sequences) along with multiple potential G/A/C/UREs within the 3’UTR of XB130 mRNA [35]. Notably, the absence of each ARE did not significantly impact gene expression. Conversely, segments 113–230 and 503–660 in the 3’UTR exhibited significant effects on mRNA stability and translation. Through systematic screening, we identified hnRNPC as a candidate binding protein for these specific segments [20]. Previous research has demonstrated that hnRNPC expression is aberrantly regulated in diverse cancer tissues and exhibits a close correlation with tumorigenic progression [36,37,38]. In this study, the analysis of TCGA data has established that hnRNPC exhibited overexpression in 14 cancer types. Moreover, a significant correlation was observed between the aberrant expression of hnRNPC and the prognosis of patients with 8 distinct cancers. Notably, the results regarding the expression of hnRNPC were derived from bulk data analysis, which might potentially present certain biases, such as the presence of cell-specific expression patterns [39]. The upregulation of hnRNPC in NSCLC cells promotes the proliferation and EMT by enhancing XB130 expression, which will need to be validated in vivo in future studies. Regarding the molecular mechanisms underlying hnRNPC-mediated regulation of XB130 expression, we demonstrated that hnRNPC directly binds to the 3’UTR segments 113–230 and 503–660 of XB130 mRNA, thereby increasing both the stability and translation efficiency of XB130 mRNA. While hnRNPC is known to interact with UREs in RNA [40, 41], we did not identify any potential binding sites for hnRNPC in the 3’UTR segments 113–230 and 503–660 of XB130 mRNA. Consequently, we hypothesize that these segments may comprise non-conserved sequences or form specific secondary structures that facilitate hnRNPC binding.

To date, the manners by which hnRNPC regulates mRNA stability remain incompletely understood. Previous studies have shown that RBPs interact with mRNA in ways that either potentially stabilize it by protecting it from nuclease-mediated degradation or facilitate mRNA decay by recruiting nucleases [27, 42, 43]. Therefore, we conducted a screening of nucleases involved in the regulation of XB130 expression. Our results indicated that silencing either XRN1 or DIS3L2 resulted in a significant upregulation of both XB130 mRNA and protein levels. Furthermore, the regulation of XB130 expression by XRN1 and DIS3L2 is mediated through the 3’UTR segments 113–230 and 503–660. RIP assays validated that hnRNPC silencing enhances the binding of DIS3L2 to XB130 mRNA. Additionally, silencing XRN1 or DIS3L2 effectively reversed the effects of hnRNPC silencing on XB130 mRNA stability. It should be emphasized that in our study, only one siRNA was employed to silence the relevant nuclease. This approach potentially gives rise to off-target effects [44]. Consequently, it is advisable to conduct further validation by synthesizing two or more siRNA sequences to enhance the reliability and specificity of the observed results. DIS3L2 is known to specifically recognize uridylated RNA and initiate its degradation in the 3’ to 5’ direction, suggesting that the 3’ UTR segments 113–230 and 503–660 of XB130 mRNA may contain sequences that mediate mRNA uridylation [45,46,47]. The binding of hnRNPC to these 3’ UTR segments may suppress mRNA uridylation, thereby enhancing the stability of XB130 mRNA. XRN1, functioning as a 5’ to 3’ exoribonuclease, primarily associates with the 5’ end of RNA, implying that its interaction with RNA 3’UTR may be indirect [48, 49]. Thus, we propose that the 3’UTR segments 113–230 and 502–660 may mediate the specificity of XB130 mRNA degradation by XRN1, while hnRNPC binding may impede XRN1 recruitment to the 5’ end of XB130 mRNA, ultimately enhancing its stability.

HnRNPC regulates mRNA translation through multiple mechanisms [50,51,52]. For example, hnRNPC can modulate translation by influencing the length of the 3’UTR of the associated mRNA [41, 51]. Additionally, it interacts with the complex of Internal Ribosome Entry Site trans-acting factors, which recruits the ribosomal subunit to facilitate the translation of mRNAs [50]. In this study, we observed that hnRNPC interacts with the 3’UTR segments 113–230 and 503–660 of XB130 mRNA, as well as with the translation initiation factor eIF4E, thereby promoting proximity between the two ends of XB130 mRNA. This specific structural arrangement improves ribosome recycling and consequently increases the efficiency of translation initiation [53, 54]. Our findings reveal a novel mechanism by which hnRNPC regulates mRNA translation.

HnRNPC serves as an m6A reader or an RBP, playing a pro-oncogenic role in various tumors by regulating the activity of multiple signaling pathways [23,24,25]. The Akt signaling pathway, a downstream pathway regulated by hnRNPC, contributes significantly to the proliferation and metastasis of cancer cells [55]. Currently, Akt inhibitors have been utilized as anti-tumor agents in the research and treatment of various cancers. However, due to the complexity of tumorigenesis and progression, targeted therapies focusing on a single signaling protein often show limited efficacy [55]. Consequently, the development of hnRNPC inhibitors in combination with Akt inhibitors may overcome the limitations associated with targeting a single pathway, representing a promising therapeutic strategy for various tumors, including NSCLC. At present, data from public databases are predominantly derived from large-scale tumor tissue samples, which may obscure tumor heterogeneity and impede the clinical application of new therapeutic targets [39]. However, emerging technologies such as single-cell analysis may hold promise for providing novel insights into the treatment and prognosis of NSCLC [39, 56].

Conclusions

Our findings demonstrate that XB130 expression is regulated post-transcriptionally by hnRNPC. The overexpression of hnRNPC in NSCLC promotes cell proliferation and EMT. Mechanistically, hnRNPC binds to the 3’UTR of XB130 mRNA, stabilizing the mRNA by inhibiting the recruitment of the nucleases XRN1 and DIS3L2. Moreover, hnRNPC simultaneously interacts with eIF4E, a component of the eIF4F complex, enhancing the translation efficiency of XB130 mRNA by promoting its cyclization. The resulting increase in XB130 expression activates the PI3K/Akt signaling pathway, ultimately facilitating NSCLC cell proliferation and EMT (Fig. 9). Overall, this study suggests that hnRNPC may represent a potential therapeutic and prognostic target for NSCLC.

Fig. 9
figure 9

Mechanisms underlying hnRNPC/XB130-promoted NSCLC progression. In NSCLC cells, elevated levels of hnRNPC bind to XB130 mRNA 3’UTR. This interaction performs a dual function: first, hnRNPC enhances XB130 mRNA stability by inhibiting the recruitment of nucleases XRN1 and DIS3L2. Second, hnRNPC simultaneously interacts with eIF4E, a component of the eIF4F complex, thereby facilitating the proximity between the 5’ and 3’ ends of XB130 mRNA. This spatial arrangement supports the efficient recycling of ribosomes, thereby increasing the translation efficiency of XB130 mRNA. Together, these mechanisms result in the upregulation of XB130 expression, which subsequently activates the PI3K/Akt signaling pathway, thereby promoting cell proliferation and EMT in NSCLC

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Data availability

No datasets were generated or analysed during the current study.

Abbreviations

hnRNPC:

heterogeneous nuclear ribonucleoprotein C

NSCLC:

non-small cell lung cancer

EMT:

epithelial-mesenchymal transition

XRN1:

5’-3’ exoribonuclease 1

DIS3L2:

DIS3-like 3’-5’ exoribonuclease 2

RBP:

RNA-binding protein

TCGA:

The Cancer Genome Atlas

GTEx:

Genotype-Tissue Expression

OS:

Overall Survival

RT-qPCR:

Real-time quantitative PCR

hRluc:

Renilla luciferase

hluc:

Firefly luciferase

RTCA:

Real-time cellular analysis

RIP:

RNA immunoprecipitation

Co-IP:

Co-Immunoprecipitation

p-Akt (Ser473):

phosphorylated Akt at Ser473

eIF4E:

eukaryotic initiation factor 4E

G/A/C/URE:

GU/AU/CU/U-rich element

SD:

standard deviation

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (grant number 82360466), the Guizhou Provincial Basic Research Program (Natural Science) (grant number ZK[2022]041, ZK[2022]372), the Academic Seedling Project of Guizhou Medical University (grant number 21NSFCP04), the Excellent Young Talents Plan of Guizhou Medical University (grant number [2023]101), and 2024 College Student Innovation and Entrepreneurship Training Program of Guizhou Medical University.

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Author notes

  1. Qinrong Wang, Xuanjing Gou, Lingling Liu, and Daolan Deng contributed equally to this work and share the first authorship.

Authors and Affiliations

  1. Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education & Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, 9 Beijing Road, Guiyang, Guizhou, 550004, P. R. China

    Qinrong Wang, Xuanjing Gou, Lingling Liu, Daolan Deng, Yan Zhao, Jianjiang Zhou, Yuan Xie, Yinhui Jiang & Ying Liu

  2. Department of Thoracic Surgery, The Affiliated Hospital of Guizhou Medical University, 28 Beijing Road, Guiyang, Guizhou, 550004, P. R. China

    Jianglun Li & Jian Zhang

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  1. Qinrong Wang
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Contributions

QR W: Writing-original draft, Methodology, Data curation, Conceptualization, Funding acquisition. XJ G, LL L, and DL D: Methodology, Investigation. Y Z and JJ Z: Formal analysis. Y X: Methodology. YH J: Supervision, Funding acquisition. JL L and J Z: Project administration, Conceptualization. Y L: Writing-review & editing, Supervision, Conceptualization, Funding acquisition.

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Correspondence to Jianglun Li, Jian Zhang or Ying Liu.

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Wang, Q., Gou, X., Liu, L. et al. Heterogeneous nuclear ribonucleoprotein C promotes non-small cell lung cancer progression by enhancing XB130 mRNA stability and translation. Cancer Cell Int 25, 10 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03638-9

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Cancer Cell International

ISSN: 1475-2867

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