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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (6): 24-31    DOI: 10.13523/j.cb.20160604
研究报告     
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
秦海霞1, 崔红凯2, 潘莹3, 户瑞丽1, 朱利红1, 王世进1
1. 新乡医学院第一附属医院妇产科 卫辉 453100;
2. 新乡医学院第一附属医院介入科 卫辉 453100;
3. 新乡医学院第三附属医院 新乡 453000
miR-335 Regulate Cell Proliferation by Targeting Rho Associated Coiled-coil Forming Protein Kinase 1 in Ovarian Cancer Cells SKOV3
QIN Hai-xia1, CUI Hong-kai2, PAN Ying3, HU Rui-li1, ZHU Li-hong1, WANG Shi-jin1
1. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, China;
2. Department of Interventional Radiology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, China;
3. Department of Obstetrics and Gynecology, The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453000, China
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摘要：

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。
关键词： miR-335;  靶向;  ROCK1;  细胞增殖;  卵巢癌    
Abstract:

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.
Key words: miR-335    Target    ROCK1    Cell proliferation    Ovarian cancer
收稿日期: 2015-12-14 出版日期: 2016-03-16
ZTFLH:  Q819  
基金资助:

河南省卫生厅科技攻关项目(200802009)、河南省教育厅自然科学研究计划(2008A320011)资助项目
通讯作者: 王世进     E-mail: 13569858153@163.com
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秦海霞
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引用本文:

秦海霞, 崔红凯, 潘莹, 户瑞丽, 朱利红, 王世进. 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[J]. 中国生物工程杂志, 2016, 36(6): 24-31.

QIN Hai-xia, CUI Hong-kai, PAN Ying, HU Rui-li, ZHU Li-hong, WANG Shi-jin. miR-335 Regulate Cell Proliferation by Targeting Rho Associated Coiled-coil Forming Protein Kinase 1 in Ovarian Cancer Cells SKOV3. China Biotechnology, 2016, 36(6): 24-31.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160604    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I6/24

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