稳定表达人MCPH1基因shRNA的宫颈癌Caski细胞系的建立

Establishment of Cervical Cancer Caski Cell Line for Stable Expression of shRNA Targeting Human MCPH1 Gene

目的建立稳定表达人MCPH1基因shRNA的宫颈癌Caski细胞系,并观察shRNA对Caski细胞中MCPH1基因表达的影响。方法将人MCPH1基因RNA干扰双链DNA片段重组到逆转录病毒质粒pSUPERRetro中,构建携带人MCPH1基因RNA干扰的逆转录病毒载体pSUPER-shRNA-MCPH1,经PT67细胞包装后,产生重组逆转录病毒,感染宫颈癌细胞株Caski,经puromycin筛选稳定的细胞克隆,实时荧光定量PCR和Westernblot法检测细胞中MCPH1基因mRNA的转录水平和蛋白的表达水平。结果重组逆转录病毒质粒经测序鉴定正确;逆转录病毒感染Caski细胞后可筛选出稳定的细胞克隆;pSUPER-shRNA-MCPH1组细胞中MCPH1基因mRNA的转录水平及蛋白的表达水平明显低于阴性对照组及正常对照组。结论已成功建立了稳定表达人MCPH1基因shRNA的Caski细胞系,shRNA能明显抑制MCPH1基因的表达,为进一步研究MCPH1在宫颈癌中的作用奠定了基础。

Objective To establish the cervical cancer Caski cell line for stable expression of shRNA targeting human MCPHI gene and observe the effect of shRNA on expression of MCPHI gene. Methods A double stranded DNA fragment for RNA interference on human MCPH1 gene was cloned into retrovirus vector pSUPER Retro, and the constructed recombinant retrovirus vector pSUPER-shRNA-MCPH1 was packaged in PT67 cells. The obtained recombinant retrovirus was infected to Caski cells, and the stable clones were screened with puromycin, in which the transcription level of MCPH1 mRNA and expression level of MCPH1 protein were determined by real-time fluorescent quantitative PCR and Western blot. Results Sequencing result proved that recombinant retrovirus vector pSUPER-shRNA-MCPH1 was constructed correctly. Stable clones were screened from Caski cells infected with recombinant retrovirus. Both the transcription level of MCPH 1 mRNA and expression level of MCPH 1 protein in Caski cells infected with pSUPER-shRNA-MCPH1 were significantly lower than those in negative control and normal control cells. Conclusion The Caski cell line for stable expression of shRNA targeting human MCPH1 gene was successfully established, and shRNA inhibited the expression of MCPH 1 gcne significantly, which laid a foundation of further study on the role of MCPH 1 in cervical cancer.