RNAi沉默STAT3对结直肠癌SW480细胞的抑制作用及其机制

Growth inhibition and underlying mechanisms following siRNA silencing of STAT3 in colorectal cancer SW480 cells

目的:慢病毒介导siRNA沉默结直肠癌SW480细胞内信号转导和转录活化因子3(signal transducer and activators of transcription 3,STAT3)的表达,观察其对SW480细胞凋亡、侵袭、集落形成及下游信号分子Mcl-1、caspase3表达的影响。方法:用携带针对STAT3的siRNA的慢病毒Lenti-STAT3-siRNA感染SW480细胞,Real-time PCR和Western blotting分别检测Lenti-STAT3-siRNA感染对SW480细胞内STAT3、Mcl-1及caspase3 mRNA和蛋白表达的影响,流式细胞术检测下调STAT3表达对SW480细胞凋亡的影响。Transwell实验检测下调STAT3表达对SW480细胞侵袭能力的影响。结果:Lenti-STAT3-siRNA组SW480细胞STAT3 mRNA和蛋白的相对表达量较Lenti-GFP组和对照组显著降低(均P<0.05)。Lenti-STAT3-siRNA组SW480细胞集落形成能力受到抑制,对照组、Lenti-GFP组和Lenti-STAT3-siRNA组SW480细胞凋亡率分别为1.32%、4.92%及11.9%,Lenti-STAT3-siRNA组SW480细胞穿膜细胞数较对照组和Lenti-GFP组显著下降[(178.49±15.42)vs(340.20±41.31)、(320.61±13.30)个,均P<0.05]。Lenti-STAT3-siRNA组Mcl-1 mRNA和蛋白的相对表达量显著降低(均P<0.05),caspase3 mRNA和蛋白的相对表达量显著增加(均P<0.05)。结论:慢病毒Lenti-STAT3-siRNA感染能够有效下调结直肠癌细胞SW480内STAT3基因的表达,促进其凋亡并抑制其侵袭、集落形成能力,其机制可能与降低Mcl-1、提高caspase3的表达有关。

Objective：To determine the effect of siRNA silencing of signal transducer and activators of transcription 3 （ STAT3 ） gene on proliferation/apoptosis, invasion, colony formation, and Mcl-1 and caspase3 expression of colorectal cancer SW480 cells in vitro . Methods： SW480 cells were infected by a GFP-STAT3-siRNA-carrying lentivirus vector or a GFP-carrying control vector. At 72 h after infection, mRNA and protein levels of STAT3, Mcl-1, and caspase3 were analyzed by Real-time PCR and Western blotting respectively, apoptosis by flow cytometry, the invasive activity by transwell assays in the infected SW480 cells. Results： The colony forming ability of SW480 cells was significantly suppressed after infection with the lentiviral vector carrying GFP-STAT3-siRNA as compared to the GFP-carrying control vector （ P 〈005）. Infection with the lentirival vector carrying GFP-STAT3-siRNA significantly decreased mRNA and protein levels of STAT3 and Mc1-1 （ P 〈0.05）, significantly increased mRNA and protein levels of caspase3 （ P 〈0.05）, significantly increased the percentage of apoptotic cells （11.9% vs 4.92%, P 〈0.05）, and significantly reduced the invasive activity （178.49±15.42 vs 320.61±13.30, P 〈0.05） in SW480 cells as compared with the control vector infection. Conclusion： Silencing of the STAT3 gene in colorectal cancer cells promotes apoptosis and inhibits invasion and colony formation, possibly through modulating the expression of Mc1-1 and caspase3.