RNA干扰STAT3基因对结肠癌细胞sw620凋亡的影响

Effects of interfering STAT3 gene by RNAi on apoptosis of colon cancer cell sw620

摘要:

目的：构建针对信号转导和转录活化因子3（signal transducers and activators of transcription 3，STAT3）的小RNA干扰表达质粒，利用该质粒特异性抑制人结肠癌sw620细胞中STAT3基因的表达，观察RNA干扰STAT3基因对人结肠癌细胞sw620凋亡的影响。方法：将针对STAT3基因的干扰短发卡RNA（short hairpin RNA，shRNA）克隆到pGenesil-1质粒表达载体中，命名为p-shSTAT3。质粒转染sw620细胞48 h后，利用RT-PCR（reverse transcription PCR）和Western blot技术检测STAT3基因mRNA和蛋白水平上的表达情况。利用Hochest 33258染色对转染后sw620细胞的凋亡情况进行检测；利用Western blot技术对STAT3基因调控相关的凋亡信号通路中的p53、Caspase 3、Bax、Bcl-XL和Bcl-2蛋白的表达情况进行检测。结果：转染STAT3干扰质粒能在细胞水平上有效抑制sw620细胞中STAT3基因mRNA和蛋白水平上的表达（P=0.000）。Hochest 33258染色表明在sw620细胞中干扰掉STAT3基因的表达能促进细胞凋亡的发生（P=0.000），乱序对照对细胞无明显的凋亡促进作用（P=0.218）。对作用机理进行研究后发现，在sw620细胞中干扰掉STAT3基因能促进p53、Caspase 3和Bax 3个凋亡诱导蛋白的表达；同时下调Bcl-XL和Bcl-2 2个凋亡抑制蛋白的表达。结论：利用RNA干扰技术干扰掉sw620细胞中STAT3基因的表达能促进sw620细胞凋亡，为结肠癌治疗提供新的研究思路和作用靶点。

Abstract:

Objective：To construct SiRNA-signal transducers and activators of transcription 3（STAT3） expression vector，to silence protein of STAT3 expression in human colon cancer cell sw620 and to study apoptosis of sw620 after STAT3 interference in vitro. Methods：In this study，short hairpin RNA targeting STAT3 was cloned into pGenesil-1 plasmid vector，named as p-shSTAT3. Forty-eight hours after transfection with p-shSTAT3，the expression level of STAT3 mRNA and protein were measured using RT-PCR and Western blot. Moreover，Hochest 33258 staining was used to measure the apoptosis of sw620 cell after transfection. Furthermore，downstream apoptosis-related targets of STAT3，p53，Caspase 3，Bax，Bcl-XL and Bcl-2 in sw620 cell was investigated by Western blot. Results：Our results indicated that p-shSTAT3 could significantly silence STAT3 mRNA and protein expression in sw620 cancer cells（P=0.000）. Hochest 33258 staining indicated that p-shSTAT3 induced sw620 cancer cell apoptosis（P=0.000） whereas controls had no effects on cell apoptosis（P=0.218）. Knockdown of STAT3 expression induce p53，Caspase 3 and Bax protein expression，inhibit Bcl-XL and Bcl-2 protein expression in sw620 cells. Conclusions：Our data showed that interference of STAT3 expression promote sw620 cell apoptosis，which provide new ideas and targets for the treatment of colon cancer.