Tumor-suppressor microRNA-139-5p restrains bladder cancer cell line ECV-304 properties via targeting Connexin 43

Abstract

Background: In our previous paper, we demonstrated that Connexin 43 (CX43) was highly expressed in bladder cancer (BC) tissues. But the molecular mechanism about microRNAs (miRNAs) regulation upstream of CX43 in BC has not been well elucidated and remains to be further studied. MicroRNA-139-5p (miR-139-5p) is a tumor suppressor in progression of multifarious cancers including BC. Nevertheless, the underlying mechanisms of CX43/miR-139-5p in tumorigenesis of BC are still not well illustrated. The specific objective of our study was to inquiry the effect of CX43/miR-139-5p on BC progression and its underlying mechanism.

Methods: The bioinformatics analysis softwares were applied to predict the miRNAs in the upstream of CX43. First, the expression levels of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal) were investigated using the data from The Cancer Genome Atlas database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of miR-139-5p in three human BC cell lines 5637, T24, ECV-304 and a human bladder epithelial immortalized cell line SV-HUC-1 (normal control). Then si-CX43, si-control, miR-139-5p mimic, and its negative control (NC) were transfected into BC cell line ECV-304. The relationship of miR-139-5p and CX43 was analyzed by dual-luciferase reporter assay. The qRT-PCR and Western blotting were used to test the mRNA and protein expression level of CX43. The proliferation of ECV-304 and T24 cells were examined by cell counting kit-8. The migration and invasion of ECV-304 cells were tested by transwell assay. To determine whether miR-139-5p would affect cell proliferation, migration and invasion by targeting CX43, we executed the rescue assay. The comparison between two groups was analyzed by Student's t test, and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.

Results: The expression of miR-139-5p was remarkably down-regulated in BC tissues (tumor vs. normal, 2.286 ± 0.017 vs. 3.211 ± 0.034, t = 11.540, P < 0.0001) and cell lines (P < 0.01 in all BC cell lines). Besides, we also indicated that over-expression of miR-139-5p reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005). Moreover, miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. Furthermore, the relative luciferase activity of CX43-wild type 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), nevertheless the activity of CX43-mutant type construct was untouched (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). Finally, the rescue assay revealed that CX43 deletion enhanced the depressor effect of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014).

Conclusion: MiR-139-5p, as a tumor-suppressor, repressed cell proliferation, invasion, and migration in BC, which might be achieved by regulating CX43.

Figure 1

MiR-139-5p was preliminary predicted as a candidate upstream miRNA of CX43 and it is lowly expressed in BC tissues. (A) Venn diagram for common miRs in miRs predict websites miRanda, miRWalk, TargetScan, miRDB (the purple round on the left) and TCGA database (the yellow round on the right). (B) The mRNA expression level of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal). ^∗P < 0.0001 compared with Normal group. BC: Bladder cancer; CX43: Connexin 43; MiR-139-5p: MicroRNA-139-5p; TCGA: The Cancer Genome Atlas.

Figure 2

MiR-139-5p was lowly expressed in BC cell lines. (A) The relative expressions of miR-139-5p in normal cell line SV-HUC-1 and BC cell lines 5637, T24, and ECV-304 were detected by qRT-PCR. (B) The expressions of miR-139-5p in BC cells under miR-139-5p mimic transfection were tested by qRT-PCR. ^∗P < 0.01 compared with SV-HUC-1 group. ^†P < 0.05 compared with mimic NC group. BC: Bladder cancer; MiR-139-5p: MicroRNA-139-5p; NC: Negative control; qRT-PCR: Quantitative reverse transcription-polymerase chain reaction.

Figure 3

Over-expression of miR-139-5p restrained the proliferation, invasion, and migration potentials of BC cells. (A) CCK8 assay determined the role of miR-139-5p on the proliferation of ECV-304 cells. (B) CCK8 assay determined the role of miR-139-5p on the proliferation of T24 cells. (C) Transwell assay (bar = 200 μm) was used to detect ECV-304 cell migration and invasion. Representative images of crystal violet staining were shown on the upper panel (original magnification ×200). (D) Quantification of (C). ^∗P < 0.01 compared with mimic NC group. BC: Bladder cancer; CCK-8: Cell counting kit-8; MiR-139-5p: MicroRNA-139-5p; NC: Negative control.

Figure 4

CX43 was a direct target of miR-139-5p, and was negatively modulated by miR-139-5p. (A) The binding sites between CX43 and miR-139-5p were predicted by Targetscan. (B) The relative luciferase activity of CX43-wt and CX43-mut. (C) The relative mRNA expression level of CX43 in ECV-304 cells. (D) The protein expression of CX43 in ECV-304 cells. (E) Quantification of (D). ^∗P < 0.01 compared with mimic NC group. CX43: Connexin 43; MiR-139-5p: MicroRNA-139-5p; mut: Mutant type; NC: Negative control; wt: Wild type.

Figure 5

CX43 deletion strengthened the inhibitory effect of miR-139-5p in bladder cancer cells. (A) The proliferation ability of ECV-304 cells. (B) The numbers of invaded and migrated ECV-304 cells (bar = 200 μm). Representative images of crystal violet staining were shown on the upper panel (original magnification ×200). (C) Quantification of (B). ^∗P < 0.01 compared with control group. ^†P < 0.01 compared with mimic + si-con group. ^‡P < 0.05 compared with mimic + si-con group. CX43: Connexin 43; MiR-139-5p: MicroRNA-139-5p; Si-con: Si-control.