使用CRISPR/Cas9系统阻断人乳头瘤病毒18型（HPV18）在宫颈癌细胞中的活性。
Blocking activity of the HPV18 virus in cervical cancer cells using the CRISPR/Cas9 system.
作者信息
Wang Jing, Guo Meng, Wang Quanxing, Dang Jianhong, Liu Xiaojun, Jin Zhijun
机构信息
Changzheng Hospital, Second Military Medical University Shanghai, China.
Department of Immunology, Second Military Medical University Shanghai, China.
出版信息
Int J Clin Exp Pathol. 2018 Aug 1;11(8):4230-4235. eCollection 2018.
OBJECTIVE
Specific sgRNA-sequences targeting oncogenes E6 and E7 in HPV18 were designed using the CRISPR/Cas9 system. These sgRNAs knocked out E6 and E7 expressions and were used to study their effects on the proliferation and cell cycle of the cervical cancer HeLa cell line.
METHODS
Lentivirus vectors targeting E6 and E7 oncogenes were constructed and transfected into HeLa cells. mRNA and protein expression levels of E6 and E7 were measured by RT-PCR and Western blot, respectively. The cell cycle was detected by flow cytometry. A colony formation assay was applied to evaluate the proliferation capacity of the HeLa cells.
RESULTS
Three E6 Cas9-sgRNA vectors targeting E6 and three E7 Cas9-sgRNA vectors targeting E7 genes were constructed and transfected into HeLa cells, respectively. RT-PCR results showed that all three E6 and E7 sgRNAs inhibited the expressions of E6 or E7 mRNA, respectively, when compared with the control groups. The inhibition ratios of the three groups of E6-sgRNAs were 28%, 85%, and 19%; the E7-sgRNAs were 86%, 25%, and 27%, respectively (P<0.05), with E6-sgRNA2 and E7-sgRNA1 having the greatest inhibitory effects. Western blot results showed that, compared with the control group, the protein expressions of E6 and E7 in the sgRNAs transfected group were also decreased, and E6-sgRNA2 and E7-sgRNA1 had the most inhibitory effects on E6 and E7 proteins. Flow cytometry results showed that the number of cells in G1/G0 was increased by 14.2% in the E6-sgRNA2 transfection group, and by 7.1% in the E7-sgRNA1 transfection group. Colony formation assay results showed that after transfection of E6 or E7 sgRNA plasmids, the HeLa cell colony was reduced significantly compared with the control group.
CONCLUSIONS
The CRISPR/Cas9 system targeting HPV18 E6 or E7 genes effectively blocked the transcription and expression of oncogenes E6 or E7 in HeLa cells, which resulted in cell cycle arrest and reduced cell proliferation.
目的
利用CRISPR/Cas9系统设计靶向人乳头瘤病毒18型（HPV18）致癌基因E6和E7的特异性sgRNA序列。这些sgRNA敲除E6和E7的表达，并用于研究它们对宫颈癌HeLa细胞系增殖和细胞周期的影响。
方法
构建靶向E6和E7致癌基因的慢病毒载体，并转染到HeLa细胞中。分别通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法（Western blot）检测E6和E7的mRNA和蛋白表达水平。通过流式细胞术检测细胞周期。采用集落形成试验评估HeLa细胞的增殖能力。
结果
分别构建了三种靶向E6的E6 Cas9-sgRNA载体和三种靶向E7基因的E7 Cas9-sgRNA载体，并分别转染到HeLa细胞中。RT-PCR结果显示，与对照组相比，所有三种E6和E7 sgRNA分别抑制了E6或E7 mRNA的表达。三组E6-sgRNA的抑制率分别为28%、85%和19%；E7-sgRNA的抑制率分别为86%、25%和27%（P<0.05），其中E6-sgRNA2和E7-sgRNA1的抑制作用最大。Western blot结果显示，与对照组相比，sgRNAs转染组中E6和E7的蛋白表达也降低，且E6-sgRNA2和E7-sgRNA1对E6和E7蛋白的抑制作用最强。流式细胞术结果显示,E6-sgRNA2转染组G1/G0期细胞数量增加了14.2%，E7-sgRNA1转染组增加了7.1%。集落形成试验结果显示，转染E6或E7 sgRNA质粒后，HeLa细胞集落与对照组相比显著减少。
结论
靶向HPV18 E6或E7基因的CRISPR/Cas9系统有效阻断了HeLa细胞中致癌基因E6或E7的转录和表达，导致细胞周期停滞并减少细胞增殖。
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