Study On The Association Between HCV F Protein And APC Gene Methylation And Chronicity Of HCV Infection

As with high chronicity,incidence rate of liver cirrhosis and hepatocellular carcinoma,hepatitis C virus(HCV)infection has become one of the most popular global public health problems.There is still no prophylactic vaccine available and researches on it seem to be hard to be achieved in a short term.Screening HCV high-risk and susceptible populations,and then make them easy to early detection and therapy,and further study the mechanisms of HCV chronicity and related therapeutic regimens has been national major infectious disease prevention and control content.HCV encodes 10 viral proteins(core,El,E2,P7,NS2,NS3,NS4A,NS4B,NS5A,and,NS5B),HCV core protein(HCV Core,Core protein,C protein)is the main pathogen to participate in the assembly of viruses and a series of biochemical processes in the organism,which is closely related to chronic HCV infection and the occurrence and development of HCC.HCV F protein is produced by thift of nucleotide codon encoding HCV Core gene during HCV natural infection.Its expression may be closely related to the expression of Core protein,and may play a similar regulatory mechanism with Core protein.DNA methylation is an important pathway for epigenetic modification by catalyzing the activity of S-adenosyl methionine(SAM)by DNA methyl-transferase(DNMT)as a methyl donor,which converts cytosine(C)in CpG islands into 5-methylcytosine.After DNA methylation,the basic genetic information does not change,which is still involved in transcription inhibition,gene expression and regulation.In recent years,in a number of epigenetic studies associated with hepatitis B virus(HBV),HCV infection,a series of diseases associated with significant DNA methylation abnormalities have been found,which provides a strong reference for the diagnosis and prognosis of disease progression.Studies have found that compared with liver biopsy of normal liver and HCV-related liver cancer liver biopsy,using methylation fluorescence detection to analyze a variety of gene methylation,the methylation rate of adenomatous polyposis coli(APC)gene increased significantly in malignant liver tissue than normal liver tissue.A number of studies have found that HCV Core protein can affect the expression of methyltransferase and participate in the regulation of gene methylation,which affects the change of methylation level and participates in the occurrence and development of disease.F protein is produced by the mutation of the Core gene,so it is assumed that it may also have the same methylation regulation.In order to investigate the relationship between HCV F protein and APC gene methylation in chronic HCV infection,this study was to collect blood samples from patients with chronic HCV1b and detect the positive rate of F antibody by ELIS A,and according to the detection results divided into two groups[F-Ab(+)CHP group,F-Ab(-)CHP group],collected healthy subjects as the control group.Peripheral blood mononuclear cells(PBMCs)were isolated and stimulated with HCV Core and F protein respectively.After 72 hours,DNA was extracted and the methylation level of APC gene was detected by the second generation.The APC gene Differences in the degree of differentiation were analyzed.The preliminary results showed that the methylation degree of APC gene was statistically significant in F-Ab(+)CHP group,F-Ab(-)CHP group and healthy control group.The methylation degree of APC gene in F-Ab(+)CHP group was higher than F-Ab(-)CHP group,which was lowest in the healthy control group.After PBMCs of three groups were stimulated by Core/F protein,the difference of APC gene methylation was significantly different(P<0.05).Compared with the blank control,the methylation degree of APC gene in the co-stimulated group of Core protein and F protein group was higher than the F protein stimulation group,which was lowest in the Core protein stimulation group.At the same time,the serum of each subject was collected and the quantitative detection of cytokines IFN-? and IL-5 was carried out.The culture supernatants of PBMCs stimulated by different antigens for 72 hours were collected for quantitative detection of cytokines IFN-? and IL-5.The results showed that serum levels of IFN-?in HCV were significantly lower than those in healthy controls,and IL-5 concentrations were significantly higher in HCV patients than in healthy controls.PBMCs cells were cultured in vitro,The concentration of IFN-? in vitro cultured PBMCs was significantly different from that in stimulated antigens.Core+F protein was the most potent stimulated antigen,and the concenteations were significantly lower in stimulated PBMCs than those without stimulation.The healthy control group did not show significant differences.Both core and F proteins inhibited the expression of IFN-?,and the inhibitory effect of F protein was weaker than that of Core.The concentration of IL-5 was also different due to the different antigens.Core+F protein was still the strongest stimulated antigen,and the concenteations were significantly higher in stimulated PBMCs than those without stimulation.The healthy control group did not show significant differences.In HCV patients,both Core and F protein increased IL-5 expression,but there was no significant difference.In healthy controls,Core and F protein had little effect on IL-5 expression.The results showed that both the stimulation of Core and F could inhibit the expression of IFN-? and promote the expression of IL-5 in HCV patients.In healthy control group,the effects of Core and F on IFN-? and IL-5 were not obvious.In order to further explore the association of changes in methylation level of APC gene single locus with the outcome of HCV infection,the molecular epidemiological study of case-control and case-case was adopted in this study.Fresh EDTA anticoagulant whole blood of 156 chronic HCV patients,55 HCV self limiting patients and 66 healthy controls were extracted,and DNA was quantified.The levels of methylation of APC gene were detected by two-generation sequencing.Carry out CpG island search of APC gene by software to select the detection fragment we need.The difference of methylation level of single locus of APC gene in each group was analyzed to identify the specific sites associated with the outcome of chronic HCV infection.The results showed that the difference of methylation level of single CG site in the two target fragments of the APC gene,the rs112073406 site on the target fragment APC_M1 and the rs 112043397 site on the target fragment APC M2,was statistically significant in the healthy control,self-limiting,sustained infection.Methylation level of rs112073406 locus on fragment APC_M1 and rs112043397 locus on fragment APC M2 was significantly different between chronic HCV patients and healthy controls,between persistent infection group and healthy control group,self-limiting group and healthy control group,but there was no significant difference between the continuous infection group and the self-limiting group.The results indicated that the methylation level of rs112073406 and rs112043397 in the APC gene was significantly associated with HCV susceptibility,but was not associated with HCV infection outcome.The study was carried out in Chinese Han population,and the associations of HCV F protein and APC gene methylation in chronic HCV infection were investigated.All these results will contribute to the personalized prophylactic and therapeutic regimens and means a lot to Chinese HCV patients.