Hsa-mir-634对Vero细胞增殖和凋亡的影响

Effects of microRNA hsa-mir-634 on the proliferation and apoptosis of Vero cells

目的探讨hsa—mir-634在Vero细胞中的功能与作用机制。方法运用生物信息学方法在线预测hsa—mir-634的潜在靶基因细胞周期蛋白Dl（CyclinDl，CCNDl）的结合位点，将含有hsa—mir-634结合位点的CCNDl的3’UTR片段连人psi—CHECK2载体，构建双荧光报告基因载体。通过定点突变的方法突变hsa—mir-634和CCNDl的结合位点，构建双荧光报告突变基因载体。将构建的重组质粒转染293T细胞，进行双荧光检测；将化学合成的hsa—mir-634模拟物转染Vero细胞，荧光定量PCR和Western印迹法检测hsa—mir-634对内源性CCNDlmRNA水平和蛋白水平表达的影响，MTS法检测hsa—mir-634对Veto细胞增殖能力的影响，流式细胞仪检测hsa—mir-634对Vero细胞凋亡的影响。结果用生物信息学方法成功预测到hsa—mir-634与CCNDl的结合位点；测序结果显示，野生型及突变型CCND13’UTR序列成功连接到psi—CHECK2报告基因载体；双荧光检测结果显示，过表达hsa—mir-634可以抑制荧光素酶的表达。过表达hsa—mir-634后，其靶基因CCNDl在mRNA水平上无明显变化，但可以显著影响CCNDl蛋白的表达。过表达hsa—mir-634可抑制Vero细胞增殖，这种抑制作用在转染后第4天最为明显。另外，过表达hsa—mir-634可诱导Veto细胞的凋亡，空白细胞组、阴性对照组及hsa—mir-634过表达组晚期凋亡的比例分别为8．03％、7．96％和17．33％。结论Hsa—mir-634通过影响CCND1的表达影响Vero细胞的增殖和凋亡。

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 DI （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 duaMuciferase reporter vectors, including CHECK2-CCND1 wild, CHECK2-CCND1 mut 1, CHECK2- CCND1 mut 2 and CHECK2-CCND1 mut 3. Then, 293T ceils were transfected with the four constructed plasmids, and luciferase activity was measured 48 hours after the transfection. Vero cells were transfected with hsa-mir-63d 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 ~ero 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.