Effects of microRNA hsa-mir-634 on the proliferation and apoptosis of Vero cells / 中华皮肤科杂志

ABSTRACT

Objective To investigate the function and possible action mechanisms of microRNA hsa-mir-634 in Vero cells.Methods The binding sites for hsa-mir-634 in the 3' UTR of cyclin D1 (CCND1) were predicated by bioinformatics methods.Then,the 3'UTR sequence of CCND1 containing the binding sites for hsamir-634 was amplified by PCR.Site-directed mutagenesis was used to create mutations in the binding sites.The wild and mutant 3' UTR sequences of the CCND1 gene were ligated into the psi-CHECK2 vector separately to construct dual-luciferase reporter vectors,including CHECK2-CCND1 wild,CHECK2-CCND1 mut 1,CHECK2-CCND1 mut 2 and CHECK2-CCND1 mut 3.Then,293T cells were transfected with the four constructed plasmids,and luciferase activity was measured 48 hours after the transfection.Vero cells were transfected with hsa-mir-634 mimics and negative control separately,and harvested after additional culture for different durations; the Vero cells remaining untreated served as the blank control.Subsequently,fluorescence-based quantitative PCR and Western blot were performed to detect the mRNA and protein expressions of CCND1 respectively in,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay to evaluate the proliferation of,and flow cytometry to detect the apoptosis in,Vero cells.Results The binding sites for hsa-mir-634 in the 3'UTR of CCND1 were successfully predicated.Sequencing results showed the successful construction of dual-luciferase reporter vectors.As the luciferase assay revealed,the overexpression of hsa-mir-634 could significantly inhibit the CCND1 3'UTR-mediated luciferase activity.Compared with the negative control,the hsamir-634 mimics markedly decreased the protein expression of CCND1,but had no obvious effect on the mRNA expression of CCND1 in Vero cells.The proliferation of Vero cells transfected with hsa-mir-634 mimics was significantly restrained compared with those transfected with the negative control,and the strongest restraining effect was observed on day 4 after the transfection.In addition,the overexpression of hsa-mir-634 also induced the apoptosis of Vero cells,with the apoptosis rate being 8.03％,7.96％ and 17.33％ in the blank control group,negative control group and mimics group respectively.Conclusion Hsa-mir-634 may regulate the proliferation and apoptosis of Vero cells via influencing the expression of CCND1.