Study On Abnormal Expression Of Host Cell DNA Repair System After HBV Stable Transfection

Hepatitis B virus(Hepatitis B virus,HBV)infection can cause chronic hepatitis,liver cirrhosis,hepatocellular carcinoma etc.About 1 million people worldwide die from liver diseases caused by HBV each year.Currently,there is no effective cure,which is a serious threat to the public health in the world.The process of liver cancer caused by HBV infection is very complicated,at present,the recognized HBV carcinogenic mechanisms mainly include chronic inflammation caused by the persistence of hepatitis B surface antigen(HBs Ag),hepatitis B X protein(HBx)as a proto-oncogene activator to activate the expression of proto-oncogenes in host cells,and the integration of hepatitis B virus DNA(HBV DNA)into the host cell genome to cause chromosome instability.In recent years,researchers,including our group,have found that the abnormality of the host cell DNA repair system after HBV infection may be related to the subsequent accumulation of genome mutations,viral genome integration,chromosomal instability,and the occurrence of liver cancer.In order to further explore the effects of HBV infection on DNA repair system of host cells,we used HBV stably transfected cell lines Hep G2.A64,Hep G2.2.15 and their background cell line Hep G2 as research objects,and RNA-seq sequencing technology was used to analyze the transcriptome of host cells before and after HBV stable transfection.The results showed that a large number of differentially expressed genes appeared in host cell transcriptional group after HBV stable transfection.Using RT-q PCR and Western Blot technology for further testing,we found that the m RNA and protein expression of RAD52,a gene related to DNA homologous recombination repair(HRR),were significantly down-regulated in HBV stable transfected cell lines.Then,we used the CRISPR/Cas9 gene editing system and recombinant luciferase expression vector to construct a CRISPR-SSA report system for detecting cell DNA HRR ability.Using this system,we confirmed that the host cell DNA HRR ability decreased significantly after stable HBV transfection.Finally,in order to verify the role of RAD52 gene in host cell DNA repair,we targeted mutated RAD52 gene in HEK 293 T cells by CRISPR-Cas9 gene editing technology.The results showed that several DNA repair related genes were down-regulated in HEK 293 T cell lines after RAD52 gene mutation,and the host cell DNA homologous recombination repair ability decreased significantly.It is suggested that RAD52 gene may play a key role in the inhibition of DNA repair caused by HBV infection.1.Analysis of host cells transcriptome before and after stable HBV transfection.In order to explore the changes of transcriptome level of host cells before and after HBV stable transfection,we used RNA-seq sequencing technology to analyze the cells Hep G2,Hep G2.A64 and Hep G2.2.15 before and after HBV stable transfection.Results showed that compared with Hep G2 cells,a total of 613 genes were differentially expressed in the HBV stable transfected cell line Hep G2.A64,and a total of 6085 genes were differentially expressed in another HBV stable transfected cell line Hep G2.2.15.Through Reactome enrichment analysis,it was found that these differentially expressed genes(DEGs)were enriched in various metabolic pathways of the cells.It is worth noting that some of the down-regulated genes were enriched in the pathways related to DNA double-strand breaks and DNA repair.Using RT-q PCR method,we verified the key genes in DNA repair pathway.The results showed that compared with Hep G2 cells,the expression of DNA repair related genes such as RAD51,ERCC2,PARP1,ATM,DDB1 and RPA1 in HBV stably transfected cell lines Hep G2.A64 and Hep G2.2.15 decreased.The expression of RAD52 and XRCC2 genes decreased significantly in Hep G2.A64 and Hep G2.2.15 cell lines(In Hep G2.A64 and Hep G2.2.15 cell lines,the expression of RAD52 decreased by 68.96 ±7.59% and 60.63% ±5.04% and the expression of XRCC2 decreased by 69.58% ±6.32% and 58.87% ±9.34%,respectively).Using Western blot method,we verified the protein expression level of key genes in DNA repair pathway.The results showed that compared with Hep G2 cells,the expression of RAD52 in HBV stably transfected cell lines Hep G2.A64 and Hep G2.2.15 decreased by 69.93% ±3.88% and 85.74% ±2.1%,respectively.The above results showed that a large number of genes were differentially expressed in HBV stable transfected cell lines,and the expression of DNA homologous recombination related gene RAD52 was significantly down-regulated.2.Evaluation of host cells DNA homologous recombination repair(HRR)ability before and after stable HBV transfection.To further explore the changes of DNA HRR ability in HBV stable transfected cell lines,we independently established a set of CRISPR-SSA report system to detect the DNA HRR ability of cells by using CRISPR/Cas9 system and recombinant luciferase expression vector.In this system,an insertion sequence with homologous arm is introduced into the p GL3 luciferase plasmid,so that luciferase cannot be expressed normally.The luciferase plasmid is cut by CRISPR/Cas9 system with targeted insertion sequence,and luciferase emits fluorescence after repair with the help of host cell DNA homologous recombination system,which reflects the host cell DNA HRR repair ability.Using the CRISPR-SSA report system,we tested the HRR ability of cell lines Hep G2,Hep G2.A64 and Hep G2.2.15 before and after stable transfection of HBV.Results show that HRR ability of Hep G2.A64 cell,48 hours after transfection of SSA,is only10.96%±0.39% of that of Hep G2 cell,and HRR ability of Hep G2.2.15 cell is only28.44%±1.82% of that of Hep G2 cell.The results indicate that the HRR ability in HBV stably transfected cell lines was significantly reduced.3.Study on the relationship between RAD52 gene and host cell DNA repair system.To further explore the role of RAD52 gene in host cell DNA HRR and its relationship with other DNA repair genes,we used CRISPR-Cas9 gene editing technique to construct RAD52 gene mutant cell line based on the HEK 293 T cell line.Compared with the wild HEK 293 T cell line,the expression of RAD52 in this cell line decreased by 97.98%±1.19%.Subsequently,we used the CRISPR-SSA report system to evaluate the HRR ability in HEK 293 T cells before and after RAD52 mutation.The results showed that compared with the wild HEK 293 T cell line,the DNA HRR ability in the cell line with RAD52 gene mutation decreased by 31.18% ±2.5%.At the same time,we used RT-q PCR technology to detect other DNA repair related genes in the cell lines with RAD52 gene mutation.The results showed that compared with the wild HEK293 T cell line,the expression of ERCC2 in the cell line with RAD52 gene mutation decreased by 49.07% ±0.49%,the expression of XRCC2 decreased by 49.35% ±0.08%and the expression of DDB1 decreased by 43.93% ±3.57%.The results suggest that the down-regulated expression of RAD52 gene can significantly decrease the host cells DNA HRR ability,and at the same time can inhibit the expression of other genes related to DNA repair in host cells.This gene may play a key role in host cell DNA repair.In summary,this study found that a large number of genes were differentially expressed in the cell lines stably transfected with HBV,and the DNA repair related gene RAD52 was down-regulated.At the same time,the DNA HRR ability in HBV stably transfected cell lines were significantly inhibited.Subsequently,it was confirmed by experiments that the down-regulation of RAD52 gene expression in host cells can significantly decrease the DNA HRR ability and inhibit the expression of other genes related to DNA repair in host cells.These results suggest that HBV may inhibit the ability of host cells to repair DNA homologous recombination by down-regulating the RAD52 gene in the homologous recombination repair pathway,which in turn leads to a large number of differential expressions of host genes due to the inability of normal repair.This study explored the relationship between HBV and host cell DNA HRR system,and provided new ideas for the subsequent exploration of HBV and host cell genome interaction and carcinogenic mechanism.