摘要
目的:探讨RNAi技术沉默Tiam1对大肠癌LoVo细胞上皮-间质转化(EMT)的影响。方法:应用RNAi技术将Tiam1的特异性干扰片段转染处理LoVo细胞,采用Real time-RT PCR方法检测Tiam1表达水平和沉默前后LoVo细胞E-cadherin、Vimentin、RhoA、RhoC、Rac1的表达水平;扫描电镜、透射电镜,HE染色观察细胞结构及形态变化;考马斯亮蓝染色观察细胞骨架变化。结果:与对照组比较,RNAi组的Tiam1mRNA表达水平明显降低;RNAi组的E-cadherin mRNA表达水平比对照组上调,差异具有统计学意义(P<0.05),RNAi组Vimentin、RhoA、RhoC、Rac1的mRNA表达水平与对照组比较差异均无显著性(全部P>0.05);扫描电镜显示RNAi组细胞胞体比对照组变圆、细胞突起明显变短或消失,丝状微绒毛减少,变为扁平盘状突起;透射电镜显示RNAi组细胞表面丝状微绒毛比对照组有较明显减少;HE染色显示对照组细胞散在生长,呈细长梭形,RNAi组细胞生长较集中,呈圆形或类圆形;RNAi组细胞质中丝网状的骨架蛋白结构及点状肌动蛋白小体比对照组减少。结论:沉默Tiam1能在一定程度上使已发生EMT的大肠癌细胞出现上皮表型及降低其迁移运动能力,对EMT有一定程度的逆转作用,推测Tiam1通过诱导EMT来促进大肠癌侵袭、转移,至于其确切机制尚待进一步研究。
Objective To investigate the effects and mechanism of RNA interference-induced gene silence of Tiaml on epithelial-mesenchymal transition（EMT） of human colon cancer cell line LoVo. Methods Colon cancer cell line LoVo was transfected by Tiaml small interfering RNA, and the mRNA levels of Tiaml, E-cadherin, Vimentin, RhoA, RhoC, and Racl were determined by real-time reverse transcription-polymerase chain reaction （RT-PCR）. The change of microstructure and morphology were observed by scanning electron microscope （SEM）, transmission electron microscope （TEM） and HE staining. The cytoskeleton of LoVo was observed by coomassie brilliant blue staining. Results In comparison with that in the control group, the expression of Tiara1 mRNA was down-regulated remarkably in the RNAi group. The expression of E-cadherin mRNAs between control group and RNAi group were significantly different （P 〈 0.05）, but there were no significant differences in expressions of Vimentin,RhoA, RhoC, and Racl mRNA （P 〉 0.05）. SEM showed after Tiaml gene silenced, LoVo cells became round, the processes of both ends significantly became thin even disappeared, the surface microvilli reduced and was replaced by disciform processes. TEM showed the surface microvilli of LoVo cells in RNAi group were less than the control group obviously. Cells in RNAi group had less surface projections, cytoskeletal structures and spot-like actin bodies than those in control group cells. Conclusions Silence Tiaml gene can induce an epithelial phenotype, and reduce motility and migration capacity of colon cancer cells with EMT. Tiaml promote invasion and metastasis of colorectal cancer involved in EMT, the regulatory mechanism of Tiaml in EMT needs further study.
出处
《实用医学杂志》
CAS
北大核心
2011年第13期2321-2324,共4页
The Journal of Practical Medicine
作者简介
通信作者：韩慧霞E—mail：hhxia@fimmu．com
