沉默RhoA通过调控Wnt/β-catein信号通路对甲状腺癌TPC-1细胞周期、侵袭及迁移能力的影响及机制研究  

RhoA Works on the Cell Cycle, Invasion and Migration of Thyroid Cancer Cells TPC-1 via Regulating Wnt/β-catein Signal Pathway

作　　者：马建仓[1] 李宗禹 徐金错 赖媾胡 关昊[1] MA Jiancang;LI Zongyu;XU Jinkai;LAI Jinvue;GUAN Hao(The Second Affiliated Hospital of Xi'an Jiaotong University General surgery , Xi' an , 710004, China)

机构地区：[1]西安交通大学第二附属医院普外科,西安市710004

出　　处：《医学分子生物学杂志》2019年第2期150-156,共7页Journal of Medical Molecular Biology

基　　金：陕西省社会发展科技攻关基金(No.2016SF-137).

摘　　要：目的探究Ras同源物基因家族成员A (ras homolog family member A, RhoA)对甲状腺癌TPC-1细胞周期和侵袭迁移能力的影响及作用机制方法RT-PCR检测RhoA在甲状腺癌组织、癌旁组织、甲状腺癌细胞株及甲状腺细胞株中的inRNA水平将TPC-1细胞分为Control, siRNA Control, si-RhoA-1及si-RlioA-2组siRNA Control, si-RhoA-1及si-RhoA-2分别转染细胞后,MTT检测细胞增殖情况;流式检测细胞周期分布;Transwell检测细胞侵袭能力;划痕实验检测细胞迁移能力;Western印迹检测细胞周期蛋白Bl (cyclinBl)、周期蛋白依赖性激酶 I (cyclin-dependent kinases, Cdkl )、Ki67、基质金属蛋白酶-2 (matrix metalloproteinase-2 , MMP-2)、MMP-9、Wntl、-连环素(P-catenin)及钙依赖性黏附蛋白 E (calciumdependent adhesion protein E, E-ea<lherin)的表达水平.结果与癌旁组织及甲状腺细胞株Nthy-ori 3-1比较,RhoA在甲状腺癌组织及细胞株KTC1、KTC1-VA7, TPC-1和B-CPAP中的mRNA水平显著升高,选取TPC-1细胞进行后续研究与Control组比较,siRNA Control组无显著差异;si-RhoA-1及si-RhoA-2组TPC-1细胞增殖速率及Ki67表达显著降低同时,与Control组比较,siRNA Control组无显著差异;si-RlwA-1及si-RhoA-2组TPC-1细胞G2/M期阻滞比例显.著增加.G1/G1期细胞比例及Cyelin Bl、Cdkl表达显著减少。此外,与Control组比较,siRNA Control组无显著差异;si-RhoA-1及si-RhoA-2组TPC-1细胞划痕闭合率、侵袭细胞数及MMP-2、MMP-9表达显著降低si-RhoA-1及si-RhoA-2可显著降低TPC-1细胞中Wntl及0-catenin的表达水平,升高E-cadherin的表达水平、结论沉默RhoA可通过下调Wnt/β-catein信号通路诱导TPC-1细胞G2/M期阻滞并抑制细胞的侵袭及迁移。Objective To investigate the effect of Ras Homolog Family Member A ( RhoA ) on thyroid cancer TPC-1 cell cycle, invasion and migration. Methods RT-PCR was performed to evaluate the mRNA level of RhoA in thyroid cancer tissue, normal tissue, thyroid cancer cell line and thyroid cell line. TPC-1 cells were divided into control, siRNA control, si-RhoA-1 and si-RhoA-2 groups after transfection with siRNA control, si-RhoA-1 and si-RhoA-2 respectively. MTT was performed to evaluate cell proliferation;flow cytometry was clone to assess cell cycle distribution;Transwell was used to delect cell invasion;wound healing assay was employed to observe cell migration;Western blot were utilized to examine the expression level of Cyelin Bl , cyclin-dependent kinases ( Cdkl ), Ki67 , Matrix met ci 1 loprotei nase -2 (MMP-2), MMP-9 , Wntl ,β-catenin and Calciuin-dependent adhesion protein E ( E-cadherin). Results Compared with normal tissue and thyroid cells Nthy-ori 3-1 , the mRNA level of RhoA was increased in thyroid cancer tissue and cell lines KTC1 , KTC1-VA7, TPC-1 and B-CPAP. TPC-1 was used for subsequent studies. There was no significant difference between the control group and siRNA control group in the rate of TPC-1 cell proliferation and expression of Ki67. The rate of TPC-1 cell proliferation and expression of Ki67 were decreased in si-RhoA-1 and si-RhoA-2 group Meanwhile, there was no significant difference between the control group and siRNA control group in the ratio of TPC-1 cell G2/M phase arrest.The ratio of TPC-1 cell G2/M phase arrest was increased in si-RhoA-1 and si-RhoA-2 group,but the ratio of TPC-1 cell at G0/G1 phase and expression of Cyclin Bl and Cdkl were decreased significantly. Additionally, the was not significant difference between then control group and siRNA control group in the wound closure rate of TPC-1 cell, invasive cells and expression of MMP-2 and MMP-9. The wound closure rate of TPC-1 cells, invasive cells and expression of MMP-2 and MMP-9 dropped remarkably. Si-RhoA-1 and si-RhoA-2 could

关 键 词：怙同源物基因家族成员A 甲状腺癌 W nt/fJ-eatein信号通路 细胞侵袭 细胞迁移 

分 类 号：R736.1[医药卫生—肿瘤]