The Mechanism Study Of Interferon Regulatory Factor 7 And Serotonergic Genes On Antiviral Immunity And Nicotine Addiction

Since cigarette smoking is a driver of a lot of diseases,it has attracted a lot of attention.Smoking increases the mortality rate of smokers and also increases the risk of multiple diseases,which may adversely affect many tissues and organs of the body.Therefore,it is urgent to conduct researches on smoking to better understand the mechanism of smoking,which is the foundation for developing drugs of smoking cessation.（1）Biological mechanism of smoking on antiviral immunity:Background:Previous studies have shown that smoking suppresses the antiviral innate immune response,which leads to increased susceptibility of smokers to virus and also increased risk of infection.Interferon regulatory factor 7（IRF7）plays an important role in the type Ⅰ interferon dependent antiviral immune response.However,whether IRF7 also plays any role in the inhibition of smoking on antiviral immune response is still unclear.Method:In this study,firstly,we edited IRF7 by using the CRISPR/Cas9 gene editing technique,and obtained the IRF7 stable edited HEK293FT cell line.Since polyinosinic-polycytidylic acid（poly I:C）is a synthetic viral double-stranded RNA analog,it was used to treat both wild-type and IRF7-edited cells to mimic viral infections causing antiviral immune responses.Given that nicotine is one of the key active ingredients in tobacco,the poly I:C-treated cells were then exposed to nicotine to investigate whether IRF7 is involved in this process.Results:The results showed that in wild-type cells,the stimulation of poly I:C increased the expression of type Ⅰ interferon,and nicotine treatment significantly inhibited the increased expression of type Ⅰ interferon and nicotine also suppressed the expression of genes related to the production of interferon,including interferon-stimulated genes,interferon-stimulated gene factor 3（ISGF3）complexes.However,in the IRF7-mutant cells,the stimulation responses by poly I:C was weakened,and the inhibitory effect of nicotine on the expression of genes involved in interferon production were no longer significantly changed.Conclusion:It suggests that IRF7 plays a key role in the inhibition of smoking on antiviral innate immune responses.（2）Genetic mechanism of cigarette smoking addiction:Background:Although smoking causes many diseases and inhibits the process of antiviral immune response,epidemiological data showed that there are still a lot of smokers.Especially in China,the smoking rate of men is as high as 66.1%,in which a large number of them are highly nicotine dependent,that is to say nicotine addiction or smoking addiction.Thus,the differences between smokers and non-smokers are worthy to be further studied.Smoking is a complex behavior that is influenced by both environmental and genetic factors.Previous studies have found some genetic variations in smokers,but most of them were couducted in the European or American populations.The study on Chinese population is still largely unknown.Method:In this study,we investigated the mechanism of single-nucleotide polymorphisms（SNPs）of serotoninergic genes in nicotine addiction by candidate gene association analysis.Totally,we collected 2,616 Chinese Han male smokers.The basic information and smoking-related information were collected through questionnaires.We also collected peripheral blood,which is used for DNA isolation and genotyping.The selected SNPs were genotyped by using Taqman Openarray.Then,genetic association analyses were conducted to determine the correlations between genetic variations of serotonergic genes and FTND（Fagerstrom Test for Nicotine Dependence）,an indicator for evaluating the degree of nicotine addiction.Further,expression and methylation quantitative trait loci（cis-meQTL）analysis was employed to determine the association of individual SNPs with the extent of methylation of each CpG locus or the expression level of located genes.Results:The results showed that,from individual SNP-based association analysis,it revealed that rs1176744 in HTR3B was significantly associated with FTND;from haplotype-based association analysis,we found that one major haplotype,T-T-A-G,formed by SNPs rs3758987-rs4938056-rs1176744-rs2276305,located in the 5’ region of HTR3B,showed a significant association with FTND.Further,a significant genetic interactive effect affecting nicotine dependent was detected among SNPs rs10160548 in HTR3A,and rs3758987,rs2276305,and rs1672717 in HTR3B.Finally,we found four CpG sites（CpG₄543549,CpG₄543464,CpG₄543682,and CpG₄546888）to be significantly associated with three cis-meQTL SNPs（i.e.,rs3758987,rs4938056,and rs1176744）located in our detected haplotype within HTR3B.Conclusion:From individual level,haplotype level and gene-gene interaction level,combined with DNA methylation and mRNA expression to conduct cis-mQTL and cis-eQTL analyses,we found the important genetic role of HTR3B involved in nicotine dependence.Rs1176744,rs1176744,rs3758987 and rs4938056 in HTR3B may be the key loci playing important role in Chinese smokers.These findings will enlarge our knowledge of 5-HT in nicotine addiction which will help us to look for genetic loci associated with smoking cessation in Chinese smokers.In summary,biological studies demonstrate that interferon regulatory factor 7 is involved in the inhibition of smoking on antiviral immune responses,and genetic analyses indicate that the genetic variation of the serotoninergic system plays an important role in the smokers of Chinese Han population.These findings can explain,at least partly,the genetic mechanisms of smokers different from non-smokers and the reasons of smokers are more susceptible to the viral infection,which will extend our knowledge and help researchers look for drug targets of smoking cessation and treatments for smoking addiction.