利用基因芯片分析RNA干扰PNAS-2后凋亡相关基因表达的变化

目的:采用基因芯片技术,比较RNA干扰法封闭U937细胞中PNAS-2基因前后,与凋亡有关基因的变化情况,从而了解PNAS-2在凋亡通路中的可能定位。方法:将急性单核细胞白血病细胞株U937分别转染已构建的靶向封闭PNAS-2的干扰质粒组和对照质粒组,合并使用流式细胞仪(FCM)筛选出GFP阳性细胞,并运用半定量PCR及实时PCR方法验证RNAi效果。采用affymetrix u133A 2.0基因芯片技术比较2组细胞间基因表达的差异。进一步通过半定量RT-PCR对其中的2条上调基因(IER3、CCL2)和6条下调基因(CASP8、CD74、VEGF、DNASE2、PRTN3、TERT)的表达情况进行验证。结果:半定量及实时PCR检测结果表明,合并使用G418及FCM筛选后得到的转染细胞在90%以上,干扰质粒组对PNAS-2的抑制率达到70%以上;基因芯片筛选结果提示共有1651条出现上调,1404条出现下调。其中比值≥2倍的基因共有336条,上调114条,下调222条。根据功能分类初步筛选出与凋亡相关基因共22条,上调基因11条,下调基因11条。RT-PCR检测证实基因芯片结果可靠。结论:抑制PNAS-2表达后促进U937细胞凋亡的机制可能与c-Jun过表达、JNK通路的激活及VEGF基因下调有关。

A study on apoptosis-related gene changes using genechip after the suppression of PNAS-2 expression by RNA interference

Objective To study the apoptosis-related gene changes using genechip after the suppression of PNAS-2 gene expression by RNA interference in U937 leukemia cell line in order to understand the role of PNAS-2 in apoptosis pathway. Methods After transfecting with interference and control recombinant plasmids into U937 cells, G418 with GFP cell subpopulation sorted by FCM was selected to purify the tansfected cells. Both semiquantitative PCR and realtime PCR (RT-PCR) were used to evaluate the RNAi efficiency. The affymetrix u133A 2.0 genechip was used to study the gene changes between the interference group and the control transfected group. And then 2 up-regulated and 6 down-regulated genes were selected and the results of genechip were confirmed through RT-PCT. Results Confocal microscopy comfirmed that almost pure tranfected U937 cells, were gotten and both semiquantitative PCR and RT-PCR confirmed that the inhibition rate was over 70%. The result of microarray indicated 1651 genes were up-regulated and 1404 downregulated in the PNAS-2-shRNA transfected group in comparison with the control group, among which 22 apoptosis relate genes' change rates were above 2 times including 11up-regulated and 11 downregulated. The results were confirmed reliable by RT-PCR. Conclusions The mechanism of U937 cell apoptosis induced by the inhibition of PNAS-2 may be concerned with over-expression of c-jun and activation of JNK pathway, and also may be concerned with the down-regulation of VEGF gene.