RNAi引起的Paxillin和p130Cas下调抑制胃癌细胞失巢性生长

Silence of Paxillin and P130Cas by RNAi Inhibited Growth of Gastric Cancer Cells

P130Cas和paxillin分子是整合素家族下游重要的衔接分子.为了探索这两个分子在肿瘤细胞抗失巢凋亡中的作用,应用RNAi技术分别抑制抗失巢凋亡的胃癌细胞BGC82 3中paxillin和p130cas基因的表达,观察它们对细胞失巢性生长的影响.依据siRNA设计原则,分别设计针对p130cas和paxillin的两条序列;成功的构建了特异性封闭上述两分子的载体pWH1 p130cas和pWH1 paxillin .构建的载体瞬时转染贴壁培养和失巢培养的BGC82 3后,RT PCR和Western印迹检测发现paxillin和p130Cas分子在mRNA及蛋白水平的表达量均明显降低;倒置显微镜下观察发现,贴壁培养的胃癌细胞发生皱缩、脱落;失巢培养的细胞聚集成团的现象受到明显抑制,细胞团比对照组小,且较松散;MTT实验结果表明,失巢培养的BGC82 3pWH1 paxillin 组细胞存活率(32 19%±6 11% )和BGC82 3pWH1 p13 0cas组细胞存活率(2 8 5 2 %±5 0 2 % )与对照组相比显著下降(P <0 0 1vscontrol) ;FCM实验结果发现与失巢培养的对照组相比,BGC82 3pWH1 paxillin和BGC82 3pWH1 p13 0cas组细胞G1期抬高,并出现了凋亡峰.运用RNAi技术分别抑制了BGC82 3细胞中paxillin和p130Cas分子的表达,初步证明paxillin和p130Cas是细胞存活的重要信号分子,在肿瘤细胞抗失巢凋亡过程中具有重要的作用.

Focal adhension kinase (FKA) is a key component of the signal transduction pathways triggered by integrins and activation of FAK lead to acts on potential substrates paxillin and p130Cas,which causes increases cell migration and potentially regulates cell proliferation.To investigate the effects of the two signal molecules on anoikis (apoptosis that occurs in cells that are detached from matrix). RNA interference technolgy was applied to silence the genes of the two proteins in gastric cancer cell line BGC823 and observed the the effects on cell anchorage independent growth.According to the mechanism of RNAi technology,two target sequences of paxillin and p130Cas gene were designed and subsequently,two vectors (pWH1-p130cas,pWH1-paxilin) were constructed.Following trasient transfected into BGC823 cell line cultured both in adhence and suspension,the mRNA and protein expression level of the two genes were significantly decreased by RT-PCR and Western blot assay.Furthermore,the inverted phase contrast microscope demonstrated that the attached BGC823 cell shrank and detached from the matrix and that clustering phenomenon of suspended cells were inhibited following the two molecules were being silenced,respectively.MTT assay also showed that the survival rate of the detached BGC823 pWH1-paxillin and GGC823 pWH1-p130Cas were markedly decrease to 32.19% and 28,52% as compared with control group (P<0.01).This was mainly due to the enhanced G1 phase as demonstrated by FCM assay.The above results suggested that p130Cas and paxillin have played an important role in the gastric cancer cell resistant to anoilks.