Abstract
Background/Aim: The epidermal growth factor receptor (EGFR) is a key driver in bladder cancer progression. This study investigated the tumor-suppressive role of miR-124-3p and its regulatory effect on EGFR.
Materials and Methods: TSGH8301 and T24 bladder cancer cells were treated with the EGFR inhibitor erlotinib or transfected with an miR-124-3p mimic. Cell viability, proliferation, migration, and invasion were assessed using MTT, colony formation, and transwell assays. EGFR targeting was confirmed via Western blot, immunofluorescence, and luciferase reporter assays.
Results: Erlotinib and miR-124-3p both reduced cell viability and proliferation. miR-124-3p significantly inhibited EGFR phosphorylation and expression, suppressed migration and invasion, and downregulated the EGFR downstream targets MMP2, MMP9, and VEGF-A. Luciferase assays confirmed the direct binding of miR-124-3p to EGFR 3′UTR.
Conclusion: miR-124-3p suppresses bladder cancer cells progression by directly targeting and inactivating EGFR, thereby impairing cell proliferation, migration, and invasion. These findings highlight miR-124-3p as a potential therapeutic agent in EGFR-driven bladder cancer.
Introduction
Bladder cancer refers to various types of tumors that occur within the bladder. It is the most common type of tumor in the urinary system and ranks as the tenth most common cancer worldwide (1). Its incidence is steadily increasing in industrialized countries (1). Treatment strategies for bladder cancer are determined by the stage of the disease. In cases of non-muscle-invasive bladder cancer (NMIBC), management typically involves transurethral resection of the bladder tumor (TURBT), followed by intravesical therapy such as BCG or mitomycin C. For muscle-invasive bladder cancer (MIBC), standard treatment includes radical cystectomy with pelvic lymph node dissection and cisplatin-based neoadjuvant chemotherapy. In selected patients, bladder-sparing approaches–such as partial cystectomy or combined chemoradiation–may also be considered (2-4).
To improve the prognosis of patients with bladder cancer, novel therapeutic strategies are continuously being developed, including targeted therapies aimed at activated oncogenic pathways and enhanced antitumor immune responses, which have demonstrated promising anticancer activity (5, 6). Elevated levels of epidermal growth factor receptor (EGFR) are associated with poor outcomes in bladder cancer (7). Moreover, the EGFR inhibitor erlotinib has been reported to provide beneficial effects when used as neoadjuvant therapy in patients with invasive bladder cancer, improving surgical pathology and short-term clinical outcomes (8).
It has been shown that microRNAs (miRNAs or miRs), as small non-coding RNA molecules, can inhibit or degrade target protein-coding genes. Their expression is often altered in cancers, including bladder cancer, where they are implicated in diagnosis, prognosis, and treatment. Despite these advances, more research is needed to translate these findings into clinical practice (9-11). Several studies indicate that miR-124-3p has the potential to serve as a therapeutic biomarker for bladder cancer. It suppresses the growth, migration, and invasion of bladder cancer cells by targeting multiple molecules involved in tumor progression (12, 13). In addition, miR-124-3p has been reported to inhibit EGFR signaling in lung cancer cells (14). However, its role in modulating EGFR signaling in bladder cancer cells remains unclear. Therefore, the objective of this study is to elucidate whether miR-124-3p interferes with EGFR-mediated growth and metastatic potential in bladder cancer cells.
Materials and Methods
Cell culture. Human bladder cancer cell lines TSGH8301 and T24 were maintained in RPMI-1640 and McCoy’s 5A media, respectively, each supplemented with 10% fetal bovine serum (FBS), and 1% penicillin. The cells will be cultured in a humidified incubator at 37°C with 5% CO2 (15). All culture related reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
miR-124-3p transfection. The cells were mixed thoroughly with jetPRIME® Transfection Reagent (Polyplus, Illkirch, France) and were then seeded into a 6-well plate. Cells were transfected with the miR-124-3p mimic and the negative control miRNA (mimic CT). They were then harvested 48 h post-transfection (16).
EGFR 3′ UTR luciferase reporter assay. The interaction between miR-124-3p and EGFR, along with their predicted binding sequences, was analyzed using the miRDB database (17). To construct the wild-type EGFR (WT-EGFR) reporter, the 3′ untranslated region (3′ UTR) of EGFR (Transcript ID: NM_005228.5; genomic coordinates: chr7:55,086,724–55,087,432) containing the predicted miR-124-3p binding site was amplified by PCR (insert length: 709 bp) and cloned into the pmirGLO dual-luciferase vector (Promega, Madison, WI, USA) using XhoI and NotI restriction sites. The mutant EGFR (mut-EGFR) reporter was generated by site-directed mutagenesis of the miR-124-3p seed-matching region within the 3′ UTR. Specifically, three nucleotides in the predicted binding site were substituted. The wild-type and mutant sequences were as follows:
EGFR-WT: 5′-TCACTGTCTGACTTTAGTCT-3′
EGFR-Mut: 5′-TGACTGTCGTACTTAAGTCT-3′
The resulting mutant fragment was cloned into the same pmirGLO vector. Both constructs were confirmed and supplied as Custom DNA Dual-Luc Reporter Plasmid (PN: PLA1006D, Product: EGFR-hs-3UTR-MU_124-3p, Lot No. T10840-2). TSGH8301 and T24 bladder cancer cells were seeded into 24-well plates (1×105 cells/well) and co-transfected with either the WT or mutant reporter construct (200 ng/well) together with 50 nM miR-124-3p mimic (or negative control mimic, miR-CT) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 48 h, luciferase activity was measured with the Dual-Luciferase® Reporter Assay System (Promega) (18).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). TSGH8301 and T24 cells were seeded into 96-well plates and incubated until reaching approximately 70% confluence. Cells were then treated with varying concentrations of the EGFR inhibitor erlotinib (Sigma-Aldrich, Burlington, MA, USA). After 48 hours of treatment, the medium was removed and 100 μl of fresh medium containing MTT solution (medium:MTT = 9:1, Sigma-Aldrich) was added to each well. The cells were incubated until purple formazan crystals formed. The medium was then removed, and 100 μl of DMSO was added to each well to dissolve the crystals. Optical density (OD) was measured at 570 nm using an ELISA reader (Thermo Fisher Scientific, Fremont, CA, USA).
Real-time quantitative polymerase chain reaction (qPCR). Total RNA was extracted from TSGH8301 and T24 cells following various treatments using the PureLink™ RNA Mini Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was verified by agarose gel electrophoresis. For miRNA analysis, 10 ng of total RNA was reverse-transcribed into cDNA using the TaqMan® Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using the TaqMan® Advanced miRNA Assay specific for hsa-miR-124-3p (Assay ID: 477879_mir) and U6 small nuclear RNA (Assay ID: 001973) as the endogenous control. For mRNA analysis (e.g., EGFR), reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), followed by qPCR with SYBR Green Master Mix (Applied Biosystems). Primer sequences were as follows:
EGFR forward: 5′-GGTGGCATATTCCTGGATCC-3′;
EGFR reverse: 5′-GGCTCACCCTCCAGAAGTAA-3′;
GAPDH forward: 5′-GAAGGTGAAGGTCGGAGT-3′;
GAPDH reverse: 5′-GAAGATGGTGATGGGATTTC-3′.
qPCR reactions were run on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each reaction was performed in triplicate, and no-template controls were included. Cycle threshold (Ct) values were obtained, and relative expression levels were calculated using the comparative ΔΔCt method, normalizing miRNA expression to U6 and mRNA expression to GAPDH (16, 19).
Colony formation assay. TSGH8301 and T24 cells were seeded into 6-well plates at a density of 5,000 cells per well following transfection with either the mimic CT or miR-124-3p mimic. After two weeks of incubation, the medium was removed, and the cells were fixed with 4% neutral buffered formalin for 30 minutes. Cells were then stained with 0.01% crystal violet and dried in an oven. Images were captured using a UVP imaging system for subsequent analysis (20).
Western blotting. Total cellular proteins were extracted from TSGH8301 and T24 cells transfected with the miR-124-3p mimic using RIPA lysis buffer. The proteins were separated by 10% SDS-PAGE based on molecular weight and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated overnight with primary antibodies, followed by incubation with appropriate secondary antibodies. Protein expression levels of EGFR (#4267, Cell Signaling Technology, Danvers, MA, USA), P-EGFR (#2234, Cell Signaling Technology), MMP2 (#40994, Cell Signaling Technology), MMP9 (PA5-13199, Invitrogen), and VEGF-A (ab46154, abcam, Cambridge, UK) were detected using enhanced chemiluminescence (ECL). The loading control in this experiment was vinculin (PA5-29688, Invitrogen).
Transwell invasion assay. TSGH8301 and T24 cells were either transfected with the miR-124-3p mimic or treated with erlotinib for 48 hours. For the invasion assay, the upper chamber was pre-coated with Matrigel. Following treatment, 5×105 cells were seeded into the upper chamber of transwell inserts and allowed to invade for 24 hours. After incubation, the transwell membranes were fixed with a methanol:acetic acid solution (3:1) and stained with 0.5% crystal violet. Invaded cells on the lower surface of the membrane were visualized and imaged using a microscope. The number of cells was quantified using ImageJ software (ImageJ Version 1.54, NIH) (21).
Immunofluorescence (IF) staining assay. TSGH8301 and T24 cells were transfected with the miR-124-3p mimic for 48 h and seeded at a density of 5×105 cells per well. Following transfection, cells were washed with 1× PBS and fixed with 4% paraformaldehyde (Alfa Aesar, Ward Hill, MA, USA) for 20 min. They were then permeabilized with 0.1% Triton X-100 (Bio Basic, Markham, ON, Canada) for 10 min, washed three times with 1×PBS, and blocked with 1% BSA for 1 hour at room temperature. Subsequently, cells on slides were incubated overnight at 4°C with an unconjugated rabbit anti-EGFR primary antibody (1:300 in 1% BSA; #5605, Cell Signaling Technology). The next day, after washing, a FITC-labeled goat anti-rabbit IgG (H&L) secondary antibody (1:300 in 1% BSA; ab6702, abcam, Cambridge, MA, USA) was applied for 1 hour at room temperature in the dark. Finally, 50 μl of DAPI mounting medium (Leica, Wetzlar, Germany) was added, and EGFR fluorescence was captured using the EVOS M5000 imaging system (22).
Statistical analysis. For comparisons involving more than two groups, a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparisons post hoc test to identify specific group differences. For comparisons between two groups, an unpaired two-tailed Student’s t-test was applied. A p-value <0.05 was considered statistically significant.
Results
miR-124-3p suppresses bladder cancer cell progression via EGFR inactivation. To initially evaluate the effect of erlotinib on TSGH8301 and T24 bladder cancer cells, an MTT assay was conducted. As shown in Figure 1A, cell viability decreased in a dose-dependent manner following erlotinib treatment, indicating its cytotoxic effects. To further investigate the anti-proliferative activity of erlotinib, a colony formation assay was performed. As demonstrated in Figure 1B, erlotinib significantly inhibited colony formation in both TSGH8301 and T24 cells, confirming its suppressive effect on cell proliferation. Next, the potential binding sites of miR-124-3p on the EGFR gene were predicted using the miRDB database (Figure 1C). Notably, erlotinib treatment was found to upregulate the expression of miR-124-3p in both cell lines, as shown in Figure 1D. Furthermore, transfection with the miR-124-3p mimic resulted in reduced proliferation of TSGH8301 and T24 cells, further supporting its tumor-suppressive role. In summary, our findings suggest that miR-124-3p suppresses the progression of bladder cancer cells, likely through the downregulation of EGFR signaling, which is also targeted by erlotinib.
miR-124-3p inhibits bladder cancer cell progression through EGFR inactivation. (A) Cell viability of TSGH8301 and T24 cells treated with increasing concentrations of erlotinib, assessed by MTT assay. (B) Colony formation assay showing a significant reduction in clonogenic growth of TSGH8301 and T24 cells following erlotinib treatment. (C) Predicted binding sites of miR-124-3p on the 3″ UTR of EGFR mRNA, as identified using the miRDB database. (D) Relative expression of miR-124-3p in TSGH8301 and T24 cells after erlotinib treatment, measured by qPCR. (E) Effects of miR-124-3p mimic on cell proliferation in TSGH8301 and T24 cells, evaluated via colony formation assay. Data are presented as mean ± standard error (SE) from at least three independent experiments (n=5). *p<0.05, **p<0.01 compared to control using one-way ANOVA followed by Tukey’s post hoc test.
Confirming the targeting effect of miR-124-3p on EGFR. To validate the regulatory relationship between miR-124-3p and EGFR, we first assessed EGFR protein levels by Western blotting. As shown in Figure 2A, transfection with the miR-124-3p mimic led to a marked reduction in phosphorylated EGFR (p-EGFR) and EGFR levels in both TSGH8301 and T24 cells. Quantification revealed that phosphorylation at the Tyr1068 site was suppressed by over 70%, the EGFR expression levels were suppressed by 20-40% in response to miR-124-3p overexpression (Figure 2B), indicating effective inactivation of EGFR signaling. To further visualize this effect, we performed immunofluorescence staining for EGFR expression. As illustrated in Figure 2C and D, both miR-124-3p and the EGFR inhibitor erlotinib substantially reduced EGFR fluorescence signals in bladder cancer cells, supporting the inhibitory role of miR-124-3p on EGFR expression. To determine whether this regulation is mediated through direct binding to the EGFR 3″UTR, we introduced three point mutations within the predicted miR-124-3p binding site to create a mutant EGFR construct (EGFR Mut; Figure 2E, F). Luciferase reporter assays showed that miR-124-3p significantly reduced luciferase activity in cells transfected with the wild-type EGFR 3′ UTR (EGFR WT), but not with the mutant construct, indicating that the direct binding of miR-124-3p to EGFR is essential for its regulatory effect. Together, these findings confirm that miR-124-3p suppresses EGFR expression in bladder cancer cells by directly targeting its 3′ UTR.
miR-124-3p directly targets and inhibits EGFR expression in bladder cancer cells. (A) Western blot analysis showing reduced phosphorylation of EGFR (Tyr1068) in TSGH8301 and T24 cells transfected with the miR-124-3p mimic. (B) Quantification of EGFR levels showing over 20% inhibition by miR-124-3p, and p-EGFR (Tyr1068) levels showing over 70% inhibition by miR-124-3p. (C-D) Immunofluorescence staining for EGFR expression following treatment with miR-124-3p or erlotinib. Both treatments significantly reduced EGFR signal intensity. (E) Schematic representation of the wild-type (WT) and mutant (Mut) EGFR 3″ UTR sequences used in the luciferase reporter assay. (F) Luciferase activity in cells co-transfected with miR-124-3p and either EGFR WT or EGFR Mut constructs. miR-124-3p suppressed luciferase activity in the WT group but not in the mutant group. Data are presented as mean ± standard error (SE) from at least three independent experiments (n=3). *p<0.05, **p<0.01 versus control using Student’s t test. Scale bar=100 μm.
miR-124-3p inhibits bladder cancer cell invasion and migration via EGFR-associated pathways. To further explore the functional role of miR-124-3p in bladder cancer progression, we evaluated it impact on cell invasion using transwell assays. As shown in Figure 3A and B, transfection with the miR-124-3p mimic significantly inhibited the invasive capacities of TSGH8301 and T24 cells compared to the control group. To elucidate the underlying mechanism, we examined the expression of EGFR downstream effectors involved in cell motility and metastasis, including matrix metalloproteinases (MMP2 and MMP9) and vascular endothelial growth factor A (VEGF-A) (23-25). Western blot analysis revealed a substantial downregulation of MMP2, MMP9, and VEGF-A protein levels following miR-124-3p transfection in both cell lines (Figure 3C). Densitometric analysis showed that miR-124-3p reduced the expression of these proteins by approximately 80–90% (Figure 3D-F). These results suggest that miR-124-3p effectively suppresses bladder cancer cell invasion, likely through the inactivation of EGFR and its downstream signaling pathways associated with metastatic behavior.
miR-124-3p suppresses invasion of bladder cancer cells through EGFR-mediated pathways. (A-B) Transwell invasion assay with Matrigel-coated chambers indicating suppressed invasive ability in both cell lines following miR-124-3p overexpression. (C) Western blot analysis and quantification of MMP2 (D), MMP9 (E), and VEGF-A (F) expression in TSGH8301 and T24 cells transfected with miR-124-3p. All three proteins, known to promote invasion and metastasis, were significantly downregulated by miR-124-3p. Data are presented as mean ± standard error (SE) (n=3). *p<0.05, **p<0.01 versus control using Student’s t test.
Discussion
This study aimed to investigate whether miR-124-3p is involved in the regulation of EGFR signaling expression in bladder cancer cells. MicroRNAs typically exert their gene-silencing effects by partially or fully pairing with the 3′ untranslated region (3′ UTR) of target mRNAs, thereby promoting mRNA degradation or inhibiting translation (26, 27). According to the miRDB database prediction, EGFR is a potential target gene of miR-124-3p (Figure 1C). To validate this direct regulatory relationship, EGFR mutation luciferase assay was conducted, and the results showed that miR-124-3p significantly suppresses EGFR expression (Figure 2F). Furthermore, immunofluorescence staining and Western blot analysis revealed that overexpression of miR-124-3p effectively downregulated EGFR activity and protein levels (Figure 2A-D), further supporting the negative regulatory role of miR-124-3p in the EGFR signaling axis in bladder cancer cells.
EGFR is a key upstream activator of multiple oncogenic kinase signaling pathways, such as PI3K/AKT and RAS/RAF/MEK/ERK. Activation of EGFR signaling promotes the downstream pathways, thereby driving cancer cell proliferation, enhancing invasiveness, and inducing tumor angiogenesis. Therefore, inhibition of EGFR signaling is considered an effective strategy for suppressing tumor progression (28-30). In this study, we evaluated the effects of Erlotinib and miR-124-3p on the growth of bladder cancer cells using a colony formation assay. The results demonstrated that both agents significantly inhibited cancer cell proliferation (Figure 1B and E). Notably, qPCR analysis revealed that erlotinib treatment significantly upregulated the expression of miR-124-3p (Figure 1D), suggesting a potential regulatory interaction between EGFR signaling inhibition and miR-124-3p expression.
Metastasis is a major cause of poor prognosis in cancer patients, including those with bladder cancer (31). Cell invasion is a critical step in the metastatic process. Previous studies have demonstrated that upregulation of miR-124-3p suppresses the invasive capacity of bladder cancer cells (12, 13). These findings are consistent with our results (Figure 3A and B). VEGF, MMP-2, and MMP-9 are downstream effector proteins of the EGFR signaling pathway. They promote tumor metastasis by enhancing angiogenesis and increasing the invasive capacity of cancer cells (32-34). In this study, we further investigated the relationship between miR-124-3p and these proteins. Our results revealed that miR-124-3p significantly downregulated the expression of VEGF, MMP-2, and MMP-9 in bladder cancer cells (Figure 3C-F). Consistent with recent evidence that miR-124-5p modulates PI3K signaling in lung cancer patients (35), our findings suggest that the miR-124 family may broadly regulate oncogenic pathways, including the EGFR axis in bladder cancer.
In addition to miRNA-mediated regulation, emerging evidence suggests that circular RNAs (circRNAs) also play crucial roles in bladder cancer oncogenesis and progression. CircRNAs may function as competing endogenous RNAs (ceRNAs) that sponge miR-124-3p, thereby indirectly modulating EGFR expression and activity. Future work should focus on characterizing these circRNA-miRNA-mRNA regulatory networks, as they may represent novel biomarkers and therapeutic targets for precision treatment of bladder cancer (36).
Conclusion
In conclusion, our findings demonstrate that miR-124-3p plays a critical tumor-suppressive role in bladder cancer cells by directly targeting the EGFR signaling pathway. miR-124-3p not only downregulates EGFR expression and activity, but also effectively inhibits cell proliferation, reduces invasiveness, and diminishes EGFR downstream effector proteins, such as VEGF, MMP-2, and MMP-9. We suggest that miR-124-3p could be a promising therapeutic target to inhibit EGFR-mediated growth and metastatic potential in bladder cancer cells.
Acknowledgements
Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China Medical University, Taichung, Taiwan, R.O.C.
Footnotes
Authors’ Contributions
KP Chen, TL Liao and FT Hsu performed experiments, analyzed data and wrote the draft of manuscript. GH Chen, CH Yang and JD Yang conceived the ideas, oversaw the research and wrote the final version of the manuscript.
Conflicts of Interest
The Authors have no potential competing interests.
Funding
This study was supported by the Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: BRD-112010), China Medical University Hsinchu Hospital, Hsinchu, Taiwan (CMUHCH-DMR-112-020), and National Yang-Ming Chiao Tung University Hospital, Yilan, Taiwan (ID: RD2025-014).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT-5, OpenAI) was used solely for language editing and stylistic refinement in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced using generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.
- Received August 12, 2025.
- Revision received September 10, 2025.
- Accepted September 16, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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