慢病毒介导shRNA沉默STAT3表达对大肠癌HT-29细胞生长的影响

目的观察慢病毒介导shRNA沉默STAT3表达对大肠癌HT-29细胞生长的影响。方法将pRNAT-shSTAT3重组慢病毒质粒进行包装产生病毒液,测定其滴度,感染大肠癌HT-29细胞,筛选获得阳性细胞株,实时定量PCR和Westernblot检测shRNA对STAT3基因的沉默效率,MTT法检测细胞生长情况,流式细胞仪检测细胞周期变化。结果成功包装慢病毒,其病毒悬液的滴度为2×107TU/mL。病毒液感染大肠癌HT-29细胞,经G418筛选获得稳定细胞株,实时定量PCR和Western blot结果分别显示HT-29细胞STAT3mRNA表达和蛋白明显减弱,表达量分别为(16.9±2.1)%、(18.8±2.4)%(P0.01)。MTT结果显示STAT3基因沉默后的大肠癌HT-29细胞生长明显减慢,G0/G1期细胞占(68.73±2.88)%,S期细胞占22.93±1.10%,与对照组相比差异显著(P0.01)。结论慢病毒介导shRNA沉默STAT3表达对大肠癌HT-29细胞生长有明显的影响,细胞生长速度明显减慢,细胞阻滞于G0/G1期。

Effects of STAT3 gene silencing by shRNA mediated with letiviral vector on human colorectal cancer cell line HT-29

Objective To observe the interference effects of STAT3 gene silencing by shRNA mediated with letiviral vector on human carcinoma HT-29 cells growth.Methods The re-combinant vector pRNAT-U6.2/Lenti was packaged in 293T cells.The virus in the supernatant was collected and the titer was measured.HT-29 cells transfected with the virus were selected by G418 and transfected cells were gained and harvested.Real time quantitative PCR and Western blot were used to detect the interference effects.The cell growth was assessed by MTT assay and the cell cycle distribution was detected by flow cytometry.Results Virus particles were packaged in 293T cell line,and the titer of virus was 2×107TU/ml.In HT-29 cells transfected with virus,the ex-pressing level of STAT3 was down-regulated as Real time quantitative PCR and Western blot analy-ses demonstrated.In transfected group,MTT assay showed the growth of HT-29 cells were sup-pressed and FCM assay indicated the proportion of cells at G0/G1and S phase was 68.73±2.88% and 22.93±1.10% respectively,which was obviously different from that of the control group(P< 0.01).Conclusion STAT3 targeting shRNA can silence STAT3 gene remarkably,induce HT-29 cell growth inhibition and block cell cycle at G0/G1phase.