Effects of miR-760 targeting c-Myc on biological characteristics of human esophageal squamous cell carcinoma TE-10 cells / 上海交通大学学报（医学版）

ABSTRACT

Objective • To detect the effects of miR-760 on proliferation, apoptosis, migration and invasion of human esophageal squamous cell carcinoma (ESCC) TE-10 cells and to analyse the underlying mechanism. Methods • The mRNA and protein expression levels of miR-760 and c-Myc in five ESCC cell lines (normal esophageal epithelial cells as control) and 14 pieces of ESCC tissue specimens (paracancerous tissues as control) were detected by using reverse transcription quantitative PCR (RT-qPCR) and Western blotting. TE-10 cells were transfected with miR-760 mimics, miR-760 inhibitor (miR-mimics/miR-inhibitor group) and corresponding negative controls (mimics-NC/inhibitor-NC group), in which the overexpression and inhibition efficiency of miR-760 and the expression of c-Myc were verified by RT-qPCR. The effect of miR-760 on the proliferation of TE-10 cells was assessed by CCK-8 and colony formation assay. Changes of cell cycle distribution and proportion of apoptotic cells were measured by flow cytometry. Expression levels of cell cycle-, apoptosis-, migration-, and invasion-associated proteins as well as c-Myc were analyzed by Western blotting. The targeting relationship between miR-760 and c-Myc was verified by using the dual luciferase reporter assay. Results • The expressions of miR-760 were down-regulated and the expressions of c-Myc mRNA and protein were up-regulated in five ESCC cell lines compared with those in the normal esophageal epithelial cells. In 14 cases of ESCC tissue specimens, the expressions of miR-760 were down-regulated but the expressions of c-Myc were up-regulated compared with those in the cancer-adjacent tissues. The proliferation ability of TE-10 cells in the miR-mimics group was markedly attenuated, and colony numbers were also decreased. Flow cytometry assay showed that the proportion of cells in G1 phase was notably augmented, and the proportion of apoptotic cells was also increased. The miR-mimics group cells had weaker migration and invasion potential compared with mimics-NC group. Western blotting analysis conformed that expression levels of cyclin D1, B cell lymphoma 2, matrix metalloproteinase 2 and vimentin were decreased, but expression levels of cleaved-caspase3, E-cadherin and β-catenin were elevated. The miR-inhibitor group showed opposite results compared with the miR-mimics group. The dual luciferase reporter assay validated the direct targeted binding of miR-760 to the 3'UTR of c-Myc. Conclusion • miR-760 can suppress proliferation, migration and invasion, and induce apoptosis of TE-10 cells by directly targeting c-Myc.