稳定沉默STAT3的HPV16阳性宫颈癌细胞株的建立

Establishment of HPV16 Positive Cervical Cancer Cell Lines with Stable Silence of STAT3

目的:建立针对人信号传导与转录激活因子3(STAT3)的小发夹RNA(shRNA)逆转录病毒表达载体,建立稳定感染的人乳头瘤病毒16(HPV16)阳性宫颈癌细胞系,以进一步探讨STAT3在HPV16阳性宫颈癌发生和发展中的作用。方法:设计合成3条靶向STAT3基因的小干扰RNA(siRNA),从中选择1条具有最佳干扰效果的siRNA,构建STAT3-shRNA载体pSUPERretro-puro-STAT3,用293FT细胞包装质粒产生STAT3 shRNA逆转录病毒,感染HPV16阳性的宫颈癌细胞株SiHa和CaSki细胞后,通过嘌呤霉素筛选2周,约3 d传代1次,建立稳定细胞株,稳定沉默STAT3的细胞株命名为SiHa/STAT3-sh和CaSki/STAT3-sh。通过实时定量逆转录聚合酶链反应(qRT-PCR)和蛋白质印迹(Westernblotting)分别从mRNA和蛋白水平检测STAT3的表达量。结果:经RT-PCR和Western blotting实验证实,在SiHa/STAT3-sh和CaSki/STAT3-sh细胞中,STAT3 mRNA的抑制率达99%以上(P<0.01),STAT3总蛋白抑制率达70%以上(P<0.01),磷酸化STAT3蛋白的抑制率达78%以上(P<0.01)。结论:稳定沉默STAT3的HPV16阳性宫颈癌细胞株建立成功。

Objective：Constructing retroviral vector of small hairpin RNA （ shRNA ） targeting human transducer and activator of transcription 3（STAT3）,establishing stable infected HPV16 positive cervical cancer cell lines,to further explore the role of STAT3 in the development of HPV16 positive cervical cancer. Methods：Design and synthetise three siRNA sequences targeting STAT3,then select one with best gene interference effect,and construct STAT3-shRNA vector,pSUPERretro-puro-STAT3. The recombinant plasmid was transfected into packaging cell line 293FT and then infected into HPV16 positive cervical cancer cell line SiHa and CaSki cells. Cells were selected with puromycin for 2 weeks,passaged around every 3 days,and named with SiHa/STAT3-sh and CaSki/STAT3-sh. Expression of STAT3 was analyzed by quantitative reverse transcription polymerase chain reaction （qRT-PCR） and western blotting. Results：The knockdown of the expression of STAT3 mRNA was over 99%（ P〈0 . 01 ） , that of the expression of STAT3 total protein was over 70% （ P〈0 . 01 ） and that of the expression of STAT3 phosphorylated protein was over 78% （P〈0.01） in both SiHa/STAT3-sh and CaSki/STAT3-sh cells. Conclusions：HPV16 positive cervical cancer cell lines with stable silence of STAT3 were successfully established.