Original Research: miR-194 inhibits proliferation and invasion and promotes apoptosis by targeting KDM5B in esophageal squamous cell carcinoma cells

Abstract

Increasing evidence suggests that miR-194 is down-regulated in esophageal squamous cell carcinoma tumor tissue. However, the role and underlying mechanism of miR-194 in esophageal squamous cell carcinoma have not been well defined. We used DIANA, TargetScan and miRanda to perform target prediction analysis and found KDM5B is a potential target of miR-194. Based on these findings, we speculated that miR-194 might play a role in esophageal squamous cell carcinoma development and progression by regulation the expression of KDM5B. We detected the expression of miR-194 and KDM5B by quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot assays, respectively, and found down-regulation of miR-194 and up-regulation of KDM5B existed in esophageal squamous cell carcinoma cell lines. By detecting proliferation, invasion and apoptosis of TE6 and TE14 cells transfected with miR-194 mimics or mimic control, miR-194 was found to inhibit proliferation and invasion and promote apoptosis of esophageal squamous cell carcinoma cells. miR-194 was further verified to regulate proliferation, apoptosis and invasion of esophageal squamous cell carcinoma cells by directly targeting KDM5B. Furthermore, animal studies were performed and showed that overexpression of miR-194 inhibited the growth of esophageal squamous cell carcinoma tumors in vivo. These results confirmed our speculation that miR-194 targets KDM5B to inhibit esophageal squamous cell carcinoma development and progression. These findings offer new clues for esophageal squamous cell carcinoma development and progression and novel potential therapeutic targets for esophageal squamous cell carcinoma.

Keywords: Esophageal squamous cell carcinoma; apoptosis; histone demethylase lysine demethylase 5b; miR-194; proliferation.

Figure 1

miR-194 expression is obviously low and KDM5B protein expression is significantly high in ESCC cell lines compared with those in human esophageal epithelial cells (HEEpiC). (a) qRT-PCR shows that miR-194 is down-regulated in ESCC cell lines compared with that in HEEpiC. (b) The expression level of KDM5B protein is significantly increased in ESCC cell lines compared with that in HEEpiC. *P < 0.05 and **P < 0.01

Figure 2

miR-194 inhibits proliferation and invasion and promotes apoptosis in ESCC cells. (a) qRT-PCR confirmed successful transfection TE6 and TE14 cells with miR-194 mimics. (b and c) CCK-8 assay shows that TE6 and TE14 cells transfected with miR-194 mimics both have a significantly lower proliferation rate than controls. (d and e) Flow cytometer assay shows that TE6 and TE14 cells transfected with miR-194 mimics both have an obviously high apoptosis compared with controls. (f and g) Transwell chamber shows that the invasion capacity of TE6 and TE14 cells transfected with miR-194 mimics is significantly reduced compared with the control. (h) TE6 and TE14 cells transfected with miR-194 mimics both have obvious decreases in Ki67, Bcl-2, MMP-2 and MMP-9 protein expression, and significant increases in Bax protein expression compared with controls. *P < 0.05, **P < 0.01 and ***P < 0.001

Figure 3

KDM5B is a direct target of miR-194. (a) Bioinformatics-based target prediction analysis shows that KDM5B is a potential target of miR-194. (b) Luciferase reporter assay shows that in the miR-194 mimic group, the luciferase activity driven by 3′UTR of KDM5B is obviously decreased compared with that in the negative control and 3′UTR-MUT group. (c) qRT-PCR shows that miR-194 level is lower in HEEpiC transfected with miR-194 inhibitors than in control cells. (d) Down-regulation of miR-194 in HEEpiC cells increased KDM5B protein expression, and up-regulation of miR-194 in TE6 cells significantly decreased KDM5B protein expression. (e) CCK-8 assay shows that the siKDM5B group has a lower cell proliferation rate than si-control group. (f) Flow cytometer assay shows that the siKDM5B group has a significantly higher apoptosis rate than si-control group. (g) Transwell chamber shows that the siKDM5B group has a markedly weaker invasion capacity than si-control group. (h) Western blot assay shows that the siKDM5B group has obvious decreases in Ki67, Bcl-2, MMP-2 and MMP-9 protein expression, and significant increase in Bax protein expression compared with si-control group. *P < 0.05, **P < 0.01 and ***P < 0.001

Figure 4

The antitumor effect of miR-194 in vivo. TE6 cells are used in this study. (a) In the miR-194 mimic group, the tumor volume is significantly lower than that in control. (b) After 30 days, in the miR-194 mimic group, the tumor weight is significantly lighter than that in control. (c) qRT-PCR shows that after 30 days, miR-194 level in the miR-194 mimic group was not significantly different from than in control. (d) Western blot assay shows that compared with control, the miR-194 mimic group has marked decreases in KDM5B, Ki67, Bcl-2, MMP-2 and MMP-9 protein expression, and significant increase in Bax protein expression. *P < 0.05, **P < 0.01 and ***P < 0.001, n = 6