miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1对卵巢癌细胞增殖的影响

目的: 探讨miR-335 靶向Rho相关卷曲螺旋形成蛋白激酶1(rho associated coiled-coil forming protein kinase 1,ROCK1)对卵巢癌细胞系SKOV3增殖的调控作用。方法:(1)选取卵巢癌细胞系SKOV3及人正常卵巢上皮细胞系IOSE80,采用RT-PCR检测各组细胞中miR-335表达;采用Western blot检测各组细胞中ROCK1蛋白表达;(2)选取卵巢癌细胞系SKOV3,分别转染miR-335 mimic及mimic control,采用RT-PCR检测细胞中miR-335表达;(3)选取卵巢癌细胞系SKOV3,将SKOV3荧光素酶报告载体与miR-335 mimic共转染,采用荧光素酶活性实验验证miR-335对SKOV3的靶向作用;(4)选取卵巢癌细胞系SKOV3,分为3组,即SKOV3组(转染mimic control)、miR-335 mimic组(转染miR-335 mimic)及miR-335 mimic+ROCK1组(共转染miR-335 mimic+ROCK1),采用MTT法检测各组细胞增殖活性,采用Western blot检测各组细胞中ROCK1蛋白表达,采用RT-PCR检测细胞中Cyclin D1表达。结果: (1)RT-PCR结果显示,卵巢癌细胞SKOV3中miR-335表达显著低于人正常卵巢上皮细胞IOSE80(P < 0.05);Western blot结果显示,卵巢癌细胞SKOV3中ROCK1蛋白表达显著高于人正常卵巢上皮细胞IOSE80(P < 0.05);(2)RT-PCR结果显示,转染miR-335 mimic可使卵巢癌细胞SKOV3中miR-335表达上调,与转染mimic control相比较差异具有统计学意义(P < 0.05);(3)双荧光素酶活性检测结果显示,miR-335 mimic可显著抑制野生型ROCK1-Wt报告载体的荧光素酶活性,但对突变型ROCK1-Mut报告载体的荧光素酶活性并无显著抑制作用;(4)转染miR-335mimic后,卵巢癌细胞SKOV3增殖活性及Cyclin D1表达较阴性对照组显著降低(P < 0.05);而转染miR-335 mimic+ROCK1后,卵巢癌细胞SKOV3增殖活性及Cyclin D1表达较单纯转染miR-335 mimic组显著提高(P < 0.05),但仍显著低于阴性对照组(P < 0.05)。Western blot检测结果显示,转染miR-335mimic后,卵巢癌细胞SKOV3中ROCK1蛋白表达较阴性对照组显著降低(P < 0.05);而转染miR-335 mimic+ROCK1后,ROCK1蛋白表达较单纯转染miR-335mimic组显著增高(P < 0.05),且显著高于阴性对照组(P < 0.05)。结论: miR-335可通过靶向ROCK1抑制卵巢癌细胞系SKOV3增殖。

miR-335 Regulate Cell Proliferation by Targeting Rho Associated Coiled-coil Forming Protein Kinase 1 in Ovarian Cancer Cells SKOV3

Objective: To evaluate the effect of miR-335 target regulating rho associated coiled-coil forming protein kinase 1(ROCK1)on the cell proliferation of ovarian cancer cells SKOV3. Methods: (1)Chose ovarian cancer cells SKOV3 and human normal ovarian epithelial cells IOSE80 as research object. The miR-335 expression was detected by RT-PCR. The ROCK1 protein expression was detected by Western blot.(2)Chose ovarian cancer cells SKOV3 as research object, transfected miR-335 mimic and mimic control respectively. The miR-335 expression was detected by RT-PCR. (3)Chose ovarian cancer cells SKOV3 as research object, the SKOV3 luciferase report carrier and miR-335 mimic were co-transfection to SKOV3, and the targeting effect of miR-335 to SKOV3 was verified by luciferase activity experiment. (4)Chose ovarian cancer cells SKOV3 as research object, which is divided into three groups: SKOV3 group(transfection mimic control), miR-335 mimic group(transfection miR-335 mimic)and miR-335 mimic+ROCK1 group(co-transfection miR-335 mimic+ROCK1). The cell proliferation activity of each group were detected by MTT method. The ROCK1 protein expression was detected by Western blot. The Cyclin D1 expression was detected by RT-PCR. Results: (1)RT-PCR result showed that, the miR-335 expression of ovarian cancer cells SKOV3 significantly lower than human normal ovarian epithelial cells IOSE80(P < 0.05). Western blot result showed that, the ROCK1 protein expression of ovarian cancer cells SKOV3 significantly higher than human normal ovarian epithelial cells IOSE80(P < 0.05). (2)RT-PCR result showed that, transfection miR-335 mimic made miR-335 expression of ovarian cancer cells SKOV3 significantly increased, and significantly higher than transfection mimic control(P < 0.05).(3)Luciferase activity experiment result showed that, transfection miR-335 mimic made the luciferase activity of ROCK1-Wt significantly decreased, but not inhibited the luciferase activity of ROCK1-Mut.(4)After transfection miR-335 mimic, the proliferative activity of ovarian cancer cells SKOV3 and the expression of Cyclin D1 significantly lower than negative control(P < 0.05). After transfection miR-335 mimic+ROCK1, the proliferative activity of ovarian cancer cells SKOV3 and the expression of Cyclin D1 significantly higher than only transfection miR-335 mimic(P < 0.05), but still significantly lower than negative control(P < 0.05). Western blot result showed that, after transfection miR-335 mimic, the ROCK1 protein expression of ovarian cancer cells SKOV3 significantly lower than negative control(P < 0.05). After transfection miR-335 mimic+ROCK1, the ROCK1 protein expression of ovarian cancer cells SKOV3 significantly higher than only transfection miR-335 mimic(P < 0.05), and still significantly higher than negative control(P < 0.05). Conclusion: miR-335 can target ROCK1 to inhibit the proliferation activity of ovarian cancer cells SKOV3.