fmnl2基因沉默抑制SW620体外侵袭能力的机制  

Mechanism of fmnl2 gene silencing for inhibiting in vitro invasion ability of SW620

作　　者：朱曦龄[1] 黎功[1] 李黛[1] 

机构地区：[1]中国武警总医院肿瘤二科,北京100039

出　　处：《检验医学与临床》2017年第22期3279-3281,3286,共4页Laboratory Medicine and Clinic

基　　金：国家自然科学基金资助项目(30400206)

摘　　要：目的研究fmnl2基因沉默抑制SW620体外侵袭能力的机制。方法筛选并建立fmnl2基因沉默的SW620细胞;反转录(RT)-聚合酶链反应(PCR)和蛋白质印迹(Western blot)检测干扰效率;Boyden试验检测fmnl2基因对SW620侵袭能力的影响。利用Active Rho pull-down试验和Western blot检测fmnl2基因沉默后,细胞中RhoA、RhoB、RhoC活性和蛋白水平的变化;利用Western blot检测RhoA/Rock信号转导通路中3种效应蛋白[Phospho-肌球蛋白轻链(MLC)、Phospho-LIM激酶(LIMK)、Phospho-Cofilin]及细胞内肌动蛋白纤维(F-actin)、核内MAL的蛋白变化。检测fmnl2基因沉默前后,磷脂酸(LPA)对细胞体外侵袭能力、RhoA的活性和蛋白水平、Phospho-MLC及MLC的影响。结果 fmnl2基因沉默后明显抑制SW620体外侵袭能力。fmnl2表达沉默后,细胞中RhoA活性降低,但蛋白水平没有明显变化;细胞中Phospho-Cofilin、Phospho-LIMK、Phospho-MLC、F-actin和核内MAL的蛋白水平均减少。fmnl2表达沉默后,LPA对细胞体外侵袭能力,RhoA活性及Phospho-MLC均无影响。结论 fmnl2表达沉默后,通过抑制RhoA/Rock信号转导通路,会影响肿瘤细胞的侵袭运动能力。ObjectiveTo explore the mechanism of fmnl2 gene silencing for inhibiting the in vitro invasion ability of SW620.MethodsSW620 cells with fmnl2 gene silencing were screened and established.specific shRNA plasmid vectors as well as controls.fmnl2 silenced cells were screened by G418.The real time fluorescence quantitative RT PCR and Western blot analysis were used to detect the interference efficiency;the influence of fmnl on SW620 invasion ability was detected by the Boyden test.The active Rho pull down test and Western blot were used to detect the Rho A,Rho B,Rho C activity and protein level change after fmnl2 gene silencing;the 3 kinds of effector proteins Phospho MLC,Phospho LIMK Phospho Cofilin,and F actin,intranuclear MAL protein changes.The in vitro invasion ability of LPA on cells,RhoA activity and protein level,Phospho MLC and MLE influence were detected before and after fmnl2 gene silencing.Resultsfmnl2 gene silencing significantly inhibited the SW620 cell invasion ability in vitro.After fmnl2 expression silencing,the intracellular RhoA activity was decreased,but the protein level had no obvious change;the protein levels of Phospho Cofilin,Phospho LIMK,Phospho MLC,F actin and intranuclear MAL were decreased.After fmnl2 expression silencing,LPA had no influence on the cell in vitro invasion ability,PhoA activity and Phospho MLC.Conclusionfmnl2 silencing could influence the invasion and motor ability of tumor cells by inhibiting the RhoA/Rock signaling transduction pathway.

关 键 词：大肠癌 fmnl2基因 DRFs家族 侵袭 RHO蛋白 

分 类 号：R282.71[医药卫生—中药学]