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目的:分别构建信号转导与转录活化因子6(signal transducer and activator of transcription6,STAT6)过表达及RNA干扰(RNA interference,RNAi)慢病毒载体,研究STAT6基因表达对人结肠癌RKO细胞增殖和迁移的影响。方法:以人胚肾HEK293FT细胞cDNA为模板,PCR扩增STAT6基因的完整开放阅读框(open reading frame,ORF)片段,化学合成针对STAT6基因的RNAi靶向单链片段,通过pLVTHm慢病毒载体三质粒包装系统对以上2种片段进行慢病毒颗粒包装;再用病毒感染结肠癌RKO细胞,获得稳定表达STAT6-ORF和STAT6-RNAi的细胞株。RT-PCR和蛋白质印迹法检测稳定感染细胞株中STAT6基因的表达情况,CCK8法检测细胞活性的变化,Transwell实验检测细胞迁移能力的变化。结果:成功构建STAT6-ORF和STAT6-RNAi慢病毒表达载体,并获得稳定表达STAT6的相应RKO细胞株。与对照组细胞相比,STAT6-ORF转染组细胞的增殖速度加快不明显(P=0.609),细胞迁移能力显著增强(P<0.01);而STAT6-RNAi转染组的细胞活性显著降低(P<0.001),迁移能力显著下降(P<0.01)。结论:STAT6可能是促进结肠癌细胞增殖和迁移的重要转录因子。
OBJECTIVE: To construct the lentiviral vector with signal transducer and activator of transcription6 (STAT6) overexpression and RNA interference (RNAi) to study the effect of STAT6 gene on the proliferation and migration of human colon cancer RKO cells Impact. Methods: The open reading frame (ORF) fragment of STAT6 gene was amplified by PCR using human embryonic kidney HEK293FT cDNA as a template. RNAi targeted single-stranded fragment of STAT6 gene was chemically synthesized and cloned by pLVTHm lentiviral vector three Plasmid packaging system for these two fragments of lentivirus particles packaging; and then infected with colon cancer RKO cells to obtain stably expressing STAT6-ORF and STAT6-RNAi cell lines. The expression of STAT6 gene in stable infected cell lines was detected by RT-PCR and Western blotting. The changes of cell viability were detected by CCK8 assay. The changes of cell migration were detected by Transwell assay. Results: STAT6-ORF and STAT6-RNAi lentiviral vector was successfully constructed and the corresponding RKO cell line stably expressing STAT6 was obtained. Compared with control cells, the proliferation of STAT6-ORF transfected cells did not accelerate significantly (P = 0.609), and the migration ability of STAT6-ORF transfected cells was significantly increased (P <0.01) P <0.001), migration ability decreased significantly (P <0.01). Conclusion: STAT6 may be an important transcription factor to promote the proliferation and migration of colon cancer cells.