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目的:利用RNAi技术研究STAT3对肺腺癌细胞A549的生长抑制作用,同时研究STAT3对CyclinD1、Bcl-2、BAX、Caspase-9的影响及其意义。方法:利用荧光定量PCR法检测后STAT3mRNA的表达,选取1条最能有效抑制STAT3mRNA的siRNA进行后续实验;利用AM-BLUE法检测STAT3siRNA的抑制率;利用流式细胞术(FCM)检测STAT3沉默前后肺腺癌A549细胞周期分布状况和凋亡情况;利用Westernblot检测RNA干扰前后STAT3与CyclinD1、Bcl-2、BAX、Caspase-9的蛋白表达。结果:STAT3被沉默后,肺腺癌细胞A549生长被抑制(P<0.05);细胞处于G0/G1期(P<0.05),而S,G2/M期的细胞相对减少(P<0.05);凋亡细胞明显增多(P<0.05);STAT3基因被沉默后,CyclinD1、Bcl-2蛋白的表达明显降低(P<0.05),BAX、Caspase-9蛋白的表达增高(P<0.05)。Person相关性分析显示,RNAi后人肺腺癌细胞A549中STAT3与CyclinD1、Bcl-2蛋白表达呈正相关(P<0.05),STAT3与BAX蛋白表达呈负相关(P<0.05),STAT3与Caspase-9蛋白表达无相关性(P>0.05)。结论:RNA干扰技术抑制A549细胞STAT3基因后,可阻断A549的细胞周期,使细胞阻滞在G0/G1期,无法进入到S、M期。同时可诱导肺腺癌A549细胞的凋亡。其机制可能为STAT3siRNA抑制了STAT3基因的表达,从而使CyclinD1、Bcl-2蛋白表达降低,BAX蛋白表达增高。
OBJECTIVE: To study the effect of STAT3 on the growth of human lung adenocarcinoma A549 cells by RNA interference (RNAi), and to study the effect of STAT3 on the expression of CyclinD1, Bcl-2, BAX and Caspase-9. Methods: The expression of STAT3 mRNA was detected by real-time PCR. One of the most effective siRNAs for STAT3 mRNA suppression was selected for follow-up experiments. The inhibition rate of STAT3 siRNA was detected by AM-BLUE assay. Flow cytometry (FCM) The cell cycle distribution and apoptosis of lung adenocarcinoma A549 cells were detected by Western blot. The protein expressions of STAT3, CyclinD1, Bcl-2, BAX and Caspase-9 were detected by Western blot. Results: The growth of lung adenocarcinoma A549 cells was inhibited after STAT3 was silenced (P <0.05). The cells in G0 / G1 phase (P <0.05), while the cells in S and G2 / M phase decreased (P <0.05). (P <0.05). After STAT3 gene was silenced, the expression of CyclinD1 and Bcl-2 protein was significantly decreased (P <0.05), and the expression of BAX and Caspase-9 protein was increased (P <0.05). Person correlation analysis showed that there was a positive correlation between the expression of STAT3 and CyclinD1 and Bcl-2 in human lung adenocarcinoma A549 cells (P <0.05) and the expression of STAT3 and BAX protein (P <0.05). STAT3 and Caspase- 9 protein expression no correlation (P> 0.05). CONCLUSION: RNAi can inhibit the cell cycle of A549 cells after inhibiting the STAT3 gene in A549 cells. The cell cycle arrest in G0 / G1 phase can not reach the S phase and M phase. At the same time, apoptosis of lung adenocarcinoma A549 cells can be induced. The mechanism may be that STAT3 siRNA inhibits the expression of STAT3 gene, thereby decreasing the expressions of CyclinD1 and Bcl-2 proteins and increasing the expression of BAX protein.