Effects of HPV Pseudotype Virus in Cutting E6 Gene Selectively in SiHa Cells
| Autor: | Xiao Yang, Yanxiang Cheng, Gantao Chen, Li Hong, Yanqing Wang |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Carcinogenesis viruses Mice Nude Biology Biochemistry Virus 03 medical and health sciences 0302 clinical medicine Plasmid Cell Line Tumor Gene expression Genetics CRISPR Animals Humans Neoplasm Invasiveness RNA Messenger Cell Proliferation Human papillomavirus 16 Mice Inbred BALB C Cell migration Transfection Oncogene Proteins Viral Molecular biology female genital diseases and pregnancy complications Blot Repressor Proteins Titer 030104 developmental biology CRISPR-Cas Systems 030217 neurology & neurosurgery Plasmids RNA Guide Kinetoplastida |
| Zdroj: | Current medical science. 38(2) |
| ISSN: | 2523-899X |
| Popis: | The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method. We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells. Experimental subjects were divided into control group, empty virus group, E6-gRNA transfected group, Cas9 transfected group and Cas9+E6-gRNA transfected group. The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis, and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups; the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups. Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6. The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells, and there were two cutting zones in the Cas9+E6-gRNA transfected group. However, the empty virus group, E6-gRNA transfected group and Cas9 transfected group had no corresponding zone. Compared with those in the control group, the empty virus group, E6-gRNA transfected group and Cas9 transfected group, the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P |
| Databáze: | OpenAIRE |
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