Abstract: WANG Ying, FAN Jian-yong, YANG Hui-lan. Department of Dermatology, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China 【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 hsa-mir-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 hsa-mir-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. 【Key words】 Cyclin D1; Vero cells; Apoptosis; Cell proliferation; Herpesvirus 2, human; Hsa-mir-634

Key words: Vero cells