Study On The Effect Of CRISPR/Cas9 System Insilencing The Activity Of HPV18-Virus In Cervical Cancer Cell

Part One: The effect of CRISPR/Cas9 system on the Expression of HPV18 E6 and E7Objective:We designed a specific sg RNA-sequence by using the CRISPR/Cas9 system,which can knockout the oncogenes E6 and E7 of HPV18 site-directly,and then study its effect on the expression of the oncogenes in cervical cancer Hela cell.Methods: They would be transfected into Hela cells after the specific lentivirus had been packaged based on the three sequences constructed targeted the oncogenes E6 and E7 of HPV18's DNA by CRIPSR/Cas9 system.The effect of different types of sg RNA sequences on the transcription of the oncogenes E6 or E7 m RNA were detected by RT-PCR.Similarly,the effect of different types of sg RNA sequences on the expression of the oncogenes E6 or E7 protein were detected by Western Blot.Results: After the targeted HPV18E6 and E7 Cas9-sg RNA vectors were transfected into Hela cells for 48 hours respectively,RT-PCR results showed that,both types of sg RNAs inhibited the expression of E6 and E7 m RNAs when compared with the control groups.And the inhibition ratio of the groups of E6-sg RNAs were 28%,85% and 19%,the groups of E7-sg RNAs were 86%,25% and 27%,respectively,all showing significant difference(P<0.05).The results also showed that the inhibitive effect of the two groups,the E6-sg RNA2 and the E7-sg RNA1,was more obvious.Western Blot results showed that,compared with CT group,the amount of protein expression of the E6 sg RNAs' group were decreased,and the E6-sg RNA2 was more obvious.While the amount of protein expression of the E7-sg RNA1's group were lower than other three groups.The performances were consistent with the results of PCR.Conclusion: After CRISPR/Cas9 system blocked HPV18 E6 or E7 gene,the levels of the transcription of m RNAs and the expression of proteins of the oncogenes E6 or E7 in Hela cells were decreased.Part Two: The effect of CRISPR/Cas9 system on the Viability of Hela cellObjective : Here We would further explore the effects of the above effective sequences on the cell cycle and independent survival ability of cervical cancer Hela cell.Methods: According to the above experiment,we acquired two sequences which can effectively inhibit the expression of the oncogenes,namely the groups of E6-sg RNA2 and E7-sg RNA1 sequences.Then they would be transfected into Hela cells.PI staining flow cytometry tested the effects of different types of plasmids on the Hela cell cycle arrest.Flat plate clone formation assay were applied to evaluated the effects of different types of plasmids on the group dependence and the proliferation capacity of Hela cells.Results: After the targeted groups of E6-sg RNA2 and E7-sg RNA1 vectors were transfected into Hela cells respectively,Flow cytometry instrument cycle analysis results showed that,the number of E6 group cells in G1/G0 increased by 14.2%,while the number of E7 group cells increased by 7.1%,both of which showed cell cycle arrest obviously;Flat plate clone formation assay results showed that after the transfection of E6 or E7 sg RNA plasmid,the number of Hela cells colony was reduced significantly compared with the control group,which meant the clone formation ability decreased obviously.Conclusion: CRISPR/Cas9 system could cause the Hela cell cycle arrest after blocking HPV18 E6 or E7 gene,and the cellular group dependence and proliferation ability were also affected.