The Establish And Screening Of RNAi Library Of Acetyltransferase Genes And Its Impact On HBV Replication

Background and aims:cccDNA is the transcription template of pgRNA/mRN A in liver cell, and is import ant marker to chronic HBV infection. In the host cell, the transcription activity of ccc DNA is closely related to chromosome structure. Epigenetic modification directly is involved in the formation of chromosome structure. Studies have shown that histone acetylation is associated with HBV replication. In this thesis, we investigatedthe role of human acetuyltransferase genes in the transcription of HBV cccDNA and HBV replication,and identifiedthe key genes in HBV replication,.These provide new potential drug targets for CHB antiviral treatment. Methods:1. Construction of lentiviral RNAi library targeting human acetyltransferase genes. Eight targeting sequences were designed based on mRN A sequence and used for construction of lentivirus mediated shRNA library.2. A-D and E-H lentiviral plasmids were mixed respectively. Each gene had two lentivirus mixtures and each mixture contained four shRNA. We used shRNA lentivirus mixture to infect Hep G2.2.15 cells. After 72 hours of infection, qPC R was used to detect HBV pgRNA. Then, we collected Hep G2.2.15 cells supernatant after 96 hours infection, and used ELISA to detect HBsAg level of the cell supernatant. According to the result of qPCR and ELISA/MTS, we identified acetyltrasferase genesthat affected HBV replication.3. Each shRNA lentivirus targeting positive genes was produced for identification of effective shRNA inhibiting HBV replication.4. After silencing the positive gene, we used qPCR to detect the expression level of pgRNA, detected the content of HBs Ag and HBeAg in HepG2.2.15 cells supernatant through CMIA after silence 72 hours and 96 hours, and also used qPCR to detect the level of HBV DNA. Results:1. We constructed RNAi library targeting sixteen human acetyltransferase genes which totally contained one hundred and twenty-eight lentiviral vectors. Based on the results of qPCR and ELISA/MTS, we identified eight potential positive genes and their efficient shRNA sequences: CREBBP, EP300, EPC1, HAT1, KAT5, ESCO1, CSRP2 BP and KAT8.2. Individual lentivirus harboring single shRNA sequence for eight geneswas packed. These shRN A lentivirus were used to infect cells respectively. qPC R and ELISA/MTS results revealed that HAT1, CSRP2 BP and KAT8 had more than two effective shRNA fragments, which significantly inhibited HBV replication. For efficient shRNA gene fragments, we used quantitative PCR to determinetheir interference efficiency, and found that other shRNAs can silence target genes except EPC1.3. After silencing HAT1, CSRP2 BP and KAT8 genes, pgRNA e xpression level decreased obviously, HBsAg, HBe Ag and HBV DN A expression decreased with the increase of time, and the suppression effect of KAT8 repression on HBV repliacation is the most significant. Conclusions:After RNAi screening of sixteen acetyltrasnsferase genesand analysis of the function and interference efficiency of the effective shRNA fragments, we identifiedthree positive genes, which had at least two shRN A fragments that can effectively repressthe target gene expression and inhibit the content of HBs Ag. The HBsAg, HBe Ag and HBV DNA were significantly reduecdafter silencing positive genes, suggesting that HAT1, CSRP2 BP and KAT8 have important roles on HBV replication.