Tumor-suppressor microRNA-139-5p Restrains Bladder Cancer Cell Line ECV-304 Properties via Targeting Connexin 43

Tumor-suppressor microRNA-139-5p Restrains Bladder Cancer Cell Line ECV-304 Properties via Targeting Connexin 43

Bladder cancer (BC) is one of the most common malignant tumors in the urinary system, with 549,393 new cases reported in 2018. Despite advancements in diagnostic approaches, imaging, surgical techniques, chemotherapy, and urinary tumor markers, the 5-year survival rate for BC has seen minimal improvement. This underscores the need for further research into the underlying mechanisms of BC to develop more effective treatments. Connexins (CX), particularly Connexin 43 (CX43), have been found to be highly expressed in various cancers, including BC. However, the molecular mechanisms involving microRNAs (miRNAs) upstream of CX43 in BC remain poorly understood. This study focuses on the role of microRNA-139-5p (miR-139-5p) as a tumor suppressor in BC and its interaction with CX43.

The study begins by identifying potential upstream regulatory miRNAs for CX43 using bioinformatics analysis software such as miRanda, miRWalk, TargetScan, and miRDB. These tools predicted 120 miRNAs upstream of CX43. Additionally, data from The Cancer Genome Atlas (TCGA) database revealed 22 miRNAs with low expression in BC. The intersection of these two sets identified miR-139-5p as a candidate regulatory miRNA for CX43. The expression levels of miR-139-5p were then investigated in BC tissues and paracancer tissues using TCGA data, revealing significantly lower expression in BC tissues compared to normal tissues (tumor vs. normal, 2.286 ± 0.017 vs. 3.211 ± 0.034, t = 11.540, P < 0.0001).

To further validate these findings, the expression levels of miR-139-5p were examined in three human BC cell lines (5637, T24, ECV-304) and a human bladder epithelial immortalized cell line (SV-HUC-1) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that miR-139-5p was significantly down-regulated in all BC cell lines compared to the normal cell line, with the lowest expression observed in the ECV-304 cell line. Consequently, ECV-304 was chosen for subsequent experiments.

The study then explored the functional role of miR-139-5p in BC by transfecting ECV-304 cells with miR-139-5p mimic and its negative control (NC). Over-expression of miR-139-5p significantly reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005), as determined by the cell counting kit-8 (CCK-8) assay. Additionally, transwell assays revealed that miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. These results indicate that miR-139-5p acts as a tumor suppressor by inhibiting the proliferation, invasion, and migration of BC cells.

To investigate the relationship between miR-139-5p and CX43, a dual-luciferase reporter assay was conducted. The results showed that the relative luciferase activity of the CX43-wild type (wt) construct was distinctly lessened by up-regulation of miR-139-5p (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.916 ± 0.063 vs. 0.356 ± 0.048, t = 7.085, P = 0.002), while the activity of the CX43-mutant type (mut) construct remained unchanged (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.918 ± 0.057 vs. 0.878 ± 0.039, t = 0.577, P = 0.595). This confirms that CX43 is a direct target of miR-139-5p, which negatively regulates its expression.

Further validation was provided by qRT-PCR and Western blotting, which demonstrated that the mRNA (P = 0.005) and protein (P = 0.002) expression levels of CX43 in ECV-304 cells were significantly reduced in the miR-139-5p mimic group compared to the mimic NC group. These findings reinforce the notion that miR-139-5p directly targets and down-regulates CX43.

To determine whether miR-139-5p exerts its tumor-suppressive effects through CX43, a rescue assay was performed. Co-transfection of CX43 knockdown (si-CX43) with miR-139-5p mimic revealed that CX43 deletion enhanced the inhibitory effects of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014). This suggests that miR-139-5p mediates its tumor-suppressive functions, at least in part, by targeting CX43.

The study also discusses the broader implications of these findings. Connexins, including CX43, form gap junction channels that facilitate direct intercellular communication. Aberrant regulation of connexins has been implicated in various cancers, with CX43 showing reduced expression in solid tumors such as breast cancer, ovarian cancer, and lung cancer. However, previous research by the authors has shown that CX43 is highly expressed in BC tissues, suggesting a potential role in BC progression.

MicroRNAs, such as miR-139-5p, are short non-coding RNAs that regulate gene expression by binding to the 3′ untranslated region (3’UTR) of target mRNAs. MiR-139-5p has been identified as a tumor suppressor in several cancers, including non-small cell lung cancer, endometrial cancer, colorectal cancer, and BC. This study adds to the growing body of evidence supporting the tumor-suppressive role of miR-139-5p in BC and highlights its potential as a therapeutic target.

In conclusion, this study demonstrates that miR-139-5p is down-regulated in BC tissues and cell lines, and that its over-expression inhibits the proliferation, invasion, and migration of BC cells. CX43 was identified as a direct target of miR-139-5p, and its deletion enhanced the tumor-suppressive effects of miR-139-5p. These findings suggest that miR-139-5p and CX43 hold significant potential as therapeutic targets for BC. However, further research, including in vivo studies and experiments in additional cell lines, is needed to fully validate these results and explore their clinical applications.

doi.org/10.1097/CM9.0000000000000455

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