Mechanisms of the suppression of proliferation and invasion ability mediated by microRNA-147b in esophageal squamous cell carcinoma

Abstract

Objective: To explore the expression of microRNA(miR)-147b in esophageal squamous cell carcinoma (ESCC) and its regulatory roles in cell proliferation, cell cycle and invasion as well as its molecular mechanisms. Methods: Real-time quantitative PCR (qPCR) was used to investigate the expression of miR-147b in ESCC tissues and cells. Negative control (NC) and miR-147b inhibitor were transfected into ESCC EC1 and EC9706 cells, which were divided into two groups: NC group and miR-147b inhibitor group, and qPCR was employed to detect the miR-147 level and CCK-8. Flow cytometry and Transwell chamber were utilized to investigate the effects of miR-147b downregulation on cell proliferation, cell cycle and invasion in ESCC cells. Besides, target genes of miR-147b was confirmed by double luciferase reporter assay. Subsequently, qPCR and Western blot were used to examine the effects of miR-147b downregulation on NDUFA4 expression, and NDUFA4 expression and its correlation with miR-147b were investigated in ESCC tissues. Results: Relative level of miR-147b in ESCC tissues (3.03±0.27) and cells were markedly higher than that in para-carcinoma tissues (1.00±0.01) and normal esophageal epithelial cell, and the differences had statistical significance (P<0.01), and its high expression was closely associated with clinical staging, invasion depth, histological grading and lymph node metastasis(P<0.05). Importantly, clinical staging, lymph node metastasis and miR-147b may be an independent prognostic factor in ESCC. Moreover, miR-147b downregulation dramatically suppressed cell proliferation, arrested cell cycle in G0/G1 phase and reduced invasion ability in ESCC cells. Most importantly, NDUFA4 was a direct target gene of miR-147b, and miR-147b inhibitor evidently upregulated the expression of NDUFA4. Furthermore, NDUFA4 displayed low expression in ESCC tissues and its expression exhibited negative correlation with miR-147b expression. Conclusions: The downregulation of miR-147b expression significantly suppresses the proliferation and invasion abilities as well as alters cell cycle distribution in ESCC.