中华皮肤科杂志 ›› 2013, Vol. 46 ›› Issue (11): 795-799.

• 论著 • 上一篇    下一篇

Hsa-mir-634对Vero细胞增殖和凋亡的影响

王颖1,樊建勇2,杨慧兰2   

  1. 1. 广州军区广州总医院皮肤科
    2. 广州军区总医院皮肤科
  • 收稿日期:2012-12-03 修回日期:2013-07-09 出版日期:2013-11-15 发布日期:2013-11-01
  • 通讯作者: 王颖 E-mail:wangying301@hotmail.com
  • 基金资助:
    microRNA-LAT在生殖器疱疹潜伏-复发中的应用研究

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

  • Received:2012-12-03 Revised:2013-07-09 Online:2013-11-15 Published:2013-11-01

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摘要： 【摘要】 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D1（CyclinD1，CCND1）的结合位点，将含有hsa-mir-634结合位点的CCND1的3′UTR片段连入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CCND1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CCND1 mRNA水平和蛋白水平表达的影响，MTS法检测hsa-mir-634对Vero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CCND1的结合位点；测序结果显示，野生型及突变型CCND1 3′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CCND1在mRNA水平上无明显变化，但可以显著影响CCND1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Vero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。 【关键词】 细胞周期蛋白D1； Vero细胞； 细胞凋亡； 细胞增殖； 疱疹病毒2型，人； Hsa-mir-634

关键词: 细胞周期蛋白D1, Hsa-mir-634, 细胞凋亡, 细胞增殖, Vero细胞, 疱疹病毒2型，人

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