The functions of microRNA-124 on bladder cancer

Abstract

Background: To detect the expression of miR-124 in bladder cancer (BC) cell lines and tissue specimens and to analyze its association with the growth of the BC cells. Methods: Quantitative real-time polymerase chain reaction (qPCR) was applied to examine the expression of miR-124 in BC cell lines and tissues. The function of miR-124 in modulating cell proliferation was assessed in BC cells with miRNA-124 overexpression; the cell viability was identified by Cell Count Kit-8; flow cytometry was employed to detect the cell cycle; the expressions of E2F3, cyclin-dependent kinase 4 (CDK4), Ki-67 and vascular endothelial growth factor (VEGF) were tested by qPCR and Western blot; angiogenesis experiment was performed to analysis changes in angiogenesis rate; and bioinformatics prediction and dual luciferase reporter system were employed to identify the target of miR-124. Results: Survival curve data showed that the expression of MicroRNA-124 was positively correlated with survival. MicroRNA-124 expression was significantly decreased in BC cell lines and tissues. Bioinformatics prediction and dual luciferase reporter system verified CDK4 as a direct target of miR-124, which regulated the proliferation of BC cells by directly inhibiting CDK4. BC cells over-expressing miR-124 showed significantly inhibited cell viability, decreased angiogenesis rate, prevented cell proliferation and diminished the expression of E2F3, CDK4, Ki-67 and VEGF. All of these changes were reversed by over-expressing CDK4. Conclusion: MicroRNA-124 suppressed the proliferation of CRC cells by directly targeting CDK4, which provides a target for improving the therapeutic effect of BC.

Keywords: CDK4; VEGF; bladder cancer; miR-124; proliferation.

Figure 1

MicroRNA-124 was downregulated inbladder cancer ( BC) tissues. (A) Reverse transcription-quantitative polymerase chain reaction was conducted to test the miR-124 expression in BC tissues and matched adjacent non-tumor tissues. (B) BC patients with low miR-124 expression presented a shorter survival time when compared with patients with a high expression of miR-124. P<0.01 vs normal.

Figure 2

MicroRNA-124 was obviously decreased in bladder cancer ( BC) cell lines. Reverse transcription-quantitative polymerase chain reaction was conducted to examine the miR-124 expression in four BC cell lines and normal bladder mucosa epithelial cells. **P<0.01 vs SV-HUC-1.

Figure 3

Overexpression of miR-124 suppressed cells viability and E2F3 and CDK4 was predicted to be target genes of miR-124. T24 (A) cells and 5637 (B) cells transfected with miR-124 mimic or miR-NC. Cell Count Kit-8 (CCK-8) was used to detect the viability of T24 (C) cells and 5637 (D) cells. (E) The potential targets of miR-124 were analyzed by bioinformatics prediction. Reverse transcription-quantitative polymerase chain reaction was conducted to confirm the potential target of miR-124 in T24 (F) cells and 5637 (G) cells. ^P<0.05, ^^P<0.01 vs control, ^#P<0.05, ^##P<0.01 vs NC.

Figure 4

(A) TargetScan website was used to find target sites. (B) The luciferase activity was detected by dual-luciferase reporter gene assay. **P<0.01 vs 1.

Figure 5

MicroRNA-124 inhibits the malignant phenotypes of T24 cells. (A) Transfection efficiency of CDK4 was tested by reverse transcription-quantitative polymerase chain reaction. (B) Cell Count Kit-8 (CCK-8) was used to detect the viability of cells. (C) Angiogenesis rate was identified by in vitro angiogenesis experiment. (D) Cell cycle was described by flow cytometry. The images of angiogenesis rate and cell cycle were presented in (E) and (F), respectively. ^P<0.05, ^^P<0.01 vs control, ^#P<0.05, ^##P<0.01 vs NC. ^&P<0.05, ^&&P<0.01 vs mimics.

Figure 6

MicroRNA-124 inhibits the malignant phenotypes of 5637 cells. (A) Transfection efficiency of CDK4 was tested by reverse transcription-quantitative polymerase chain reaction. (B) Cell Count Kit-8 (CCK-8) was used to detect the viability of cells. (C) Angiogenesis rate was identified by in vitro angiogenesis experiment. (D) Cell cycle was described by flow cytometry. The images of angiogenesis rate and cell cycle were presented in (E) and (F), respectively. ^P<0.05, ^^P<0.01 vs control, ^#P<0.05, ^##P<0.01 vs NC. ^&P<0.05, ^&&P<0.01 vs mimics.

Figure 7

Overexpression of miRNA-124 inhibited the expression of E2F3, CDK4, Ki-67 and VEGF, which were reversed by CDK4. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the expression levels of E2F3, CDK4, Ki-67 and VEGF in T24 (A) cells and 5637 (B) cells. Western blot was used to determine the expression levels of E2F3, CDK4, Ki-67 and VEGF in T24 (C) cells and 5637 (D) cells. The relative levels of proteins were counted by β-actin as normalization in T24 (E) cells and 5637 (F) cells). ^P<0.05, ^^P<0.01 vs control, ^#P<0.05, ^##P<0.01 vs NC, ^&&P<0.01 vs mimics.