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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.
P130Cas and paxillin molecules are important adapter molecules downstream of the integrin family.In order to explore the role of these two molecules in the anti-anoikis of tumor cells, RNAi technology was used to inhibit the expression of paxillin in the anti-anoikis cell line BGC82 3 and p130cas gene and observe their effects on the growth of cells.According to the principle of siRNA design, two sequences targeting p130cas and paxillin were designed respectively, and the vectors pWH1 p130cas and pWH1 paxillin were successfully constructed to block these two molecules. The constructed vector was transiently transfected into adherent culture and anaerobic culture BGC82 3, RT-PCR and Western blot detection of paxillin and p130Cas expression of mRNA and protein levels were significantly reduced; inverted microscope observation found that adherent culture Of the gastric cancer cell shrinkage, shedding; the phenomenon of agglomeration of cells cultured in vitro was significantly inhibited, the cell mass smaller than the control group, and more loose; MTT results showed that in vitro cultured BGC82 3pWH1 paxillin group cell survival rate (32 19 ± 6 11%) and BGC82 3pWH1 p13 0cas group (28 52% ± 502%) were significantly decreased compared with the control group (P <0 0 1vscontrol). Compared with control group, BGC82 3pWH1 paxillin and BGC82 3pWH1 p13 0cas group showed elevated G1 phase and apoptotic peak in FCM assay compared with that of control group. The RNAi technique inhibited the proliferation of BGC82 3 cells The expression of paxillin and p130Cas, preliminary evidence that paxillin and p130Cas is an important signal molecule of cell survival, plays an important role in anti-anoikis in tumor cells.