miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1抑制人骨肉瘤细胞MG-63侵袭转移的实验研究

doi:10.3969/j.issn.1007-3969.2014.11.001

中国癌症杂志 ›› 2014, Vol. 24 ›› Issue (11): 801-807.doi: 10.3969/j.issn.1007-3969.2014.11.001

miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1抑制人骨肉瘤细胞MG-63侵袭转移的实验研究

王勇¹,王科峰²,赵伟¹

1.沈阳医学院附属中心医院骨四科，辽宁沈阳 110024；
2.中国医科大学附属盛京医院泌尿外一科，辽宁沈阳110004

出版日期:2014-11-30 发布日期:2015-05-05
通信作者: 赵伟　E-mail:zhaowei3332@163.com
基金资助:
辽宁省教育厅科学研究一般项目(No: L2014428)；辽宁省社会发展公关计划项目（No: 2013225086）；
沈阳医学院青年科学基金(No: 20132041)。

miR-335 inhibits migration and invasion of human osteosarcoma cell line MG-63 by targeting ROCK1

WANG Yong¹, WANG Ke-feng², ZHAO Wei¹

1.The 4th Department of Orthopaedics, the Central Hospital Affiliated to Shenyang Medical College, Shenyang Liaoning 110024, China; 2. The 1st Department of Urinary Surgery, Shengjing Hospital Affiliated to China Medical University, Shenyang Liaoning 110004, China

Published:2014-11-30 Online:2015-05-05
Contact: ZHAO Wei E-mail: zhaowei3332@163.com

摘要/Abstract

摘要：

背景与目的：微小RNA(microRNA，miRNA)是一类小分子内源性RNA，主要在转录后水平调节靶基因的表达。microRNA-335(miR-335)作为一种肿瘤抑制因子，参与了多种人类肿瘤的发生、发展过程。本研究旨在探讨miR-335是否靶向抑制Rho相关卷曲螺旋形成蛋白激酶1(Rho associated coiled-coil formingprotein kinase，ROCK1)基因的表达，并以此调控人骨肉瘤细胞MG-63侵袭及转移。方法：理论预测并通过荧光素酶基因报告验证miR-335与ROCK1基因的3'-非翻译区(untranslated region，UTR)的特异性结合作用；real-time PCR和蛋白质印迹法(Western blot)分别从基因和蛋白水平检测miR-335对ROCK1表达的负性调控作用；Transwell小室法检测miR-335过表达及下调ROCK1表达后MG-63侵袭及转移能力的变化。结果：Targetscan预测显示，miR-335与ROCK1 3'-UTR存在结合位点。荧光素酶基因报告实验结果显示，miR-335 mimic和ROCK1 3'-UTR能够靶向结合；miR-335在MG-63细胞中低表达，ROCK1则呈高表达。Western blot检测结果显示，转染miR-335 mimic或转染ROCK1 siRNA后ROCK1的蛋白表达减少。Transwell小室法检测结果显示，过表达miR-335或下调ROCK1后穿过基膜的细胞数目明显下降。结论：miR-335能特异性结合于ROCK1基因的3'-UTR并下调ROCK1的表达，抑制人骨肉瘤细胞MG-63侵袭转移。

关键词:

margin: 0cm 0cm 0pt, text-autospace: , mso-layout-grid-align: none" align="left">font-family: 宋体, mso-bidi-font-family: 宋体, mso-font-kerning: 0pt, mso-hansi-font-family: Calibri, mso-hansi-theme-font: minor-latin">微小RNA-335, Rho相关卷曲螺旋形成蛋白液酶1, 人骨肉瘤细胞MG-63, 侵袭及转移

Abstract:

Background and purpose: MicroRNAs are endogenous small RNAs and involved in target gene regulation in post-transcription level. As a tumor suppressor, microRNA-335 (miR-335) was participated in the occurrence and progression in many types of human tumors. This study aimed at investigating whether miR-335 can regulate cell migration and invasion by negative targeting Rho associated coiled-coil forming protein kinase (ROCK1) in human osteosarcoma cell line MG-63. Methods: The specific binding ability of miR-335 to ROCK1 3’-untranslated region (UTR) was theoretically predicted and detected by the luciferase reporter gene assay. Western blot and realtime PCR were used to evaluate the effect of miR-335 on the expression of ROCK1 at protein and mRNA levels. Cell migration and invasion assays were performed by transwell chambers in MG-63 cells intervened by over-expression of miR-335 and knockdown of ROCK1. Results: Luciferase reporter gene assay showed that miR-335 could target ROCK1 3’-UTR. Lower miR-335 but higher ROCK1 was expressed in MG-63 cells. The results of Western blot showed that ROCK1 protein expression was decreased by transfection of miR-335 mimic or siROCK1. Transwell chambers results showed decreased cells invading through the substrate membrane after over-expression of miR-335 or knockdown of ROCK1 gene. Conclusion: miR-335 inhibits the migration and invasion ability of MG-63 human osteosarcoma cell line, which may through targeting ROCK1 3’-UTR and subsequent down-regulating ROCK1 expression.

Key words:

font-family: "TimesNewRomanPSMT",serif, mso-bidi-font-family: TimesNewRomanPSMT, mso-font-kerning: 0pt, mso-hansi-font-family: Calibri, mso-hansi-theme-font: minor-latin">miR-335, Rho associated coiled-coil forming protein kinase, MG-63, Migration and Invasion

王勇,王科峰,赵伟. miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1抑制人骨肉瘤细胞MG-63侵袭转移的实验研究[J]. 中国癌症杂志, 2014, 24(11): 801-807.

WANG Yong, WANG Ke-feng, ZHAO Wei. miR-335 inhibits migration and invasion of human osteosarcoma cell line MG-63 by targeting ROCK1[J]. China Oncology, 2014, 24(11): 801-807.

miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1抑制人骨肉瘤细胞MG-63侵袭转移的实验研究

miR-335 inhibits migration and invasion of human osteosarcoma cell line MG-63 by targeting ROCK1

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