The Molecular Mechanism Of Suv39h1 Gene Expression Regulated By Hbv In Human Sperm

【Backgroud and Objectives】 Hepatitis B is a global disease that seriously threatens human health and life.About 400 million people in all countries of the world are infected by hepatitis B virus(HBV),and the number of new infected persons increased at an annual rate of 50 million.The number of people infected with HBV in China is about 93 million,accounting for about 7% of the country’s population.Hepatitis B plays an important role in the occurrence of liver cancer.Chronic active hepatitis and cirrhosis are also one of the major causes of human death.Because the disease has a wide wide spread and great harm,therefore the research on its transmission routes,pathogenic mechanisms,and diagnosis and treatment measures has always been a highly valued topic to governments and scientists of different countries.Some studies have confirmed that HBV can not only integrate into the genome of male germ cells through the blood testosterone barrier to increase the instability of sperm chromosome and induce the aberrations of multiple chromosome;but also it can destroy the function of the sperm mitochondria,and lead to the loss of mitochondrial membrane potential,so as to result in the damage of sperm vitality and the decrease of fertility rate and fertility index.However,there are few reports about the pathogenic mechanisms of HBV affecting human sperm quality and function.The purpose of this research was to reveal the pathomechanism of HBV affecting the quality and function of spermatozoa by the research on the molecular mechanism of HBV regulating Suv39h1 gene expression in human spermatozoa and the characteristics of LncRNAs expression in sperm of HBV infected people.【Materials and Methods】 Materials The sperm samples used in this experiment were from the Reproductive Medicine Center of the Third Affiliated Hospital of Guangzhou Medical University.All participants had signed their informed consent.The spermatogonia of the GC-1 spg mouse were purchased from Guangzhou Ginio Biotechnology Co.,Ltd.Methods In this study,spermatozoa from HBV infected person and normal healthy males were used as research samples.Spermatozoa were collected by density gradient centrifugation method.RT-qPCR and Western-blot were used to verify the histone methyltransferase Suv39h1 gene of the significant differential expression in the expression profiles of human sperm mRNA.BSP cloning and sequencing method was used to detect the methylation level of the promoter region of the Suv39h1 gene in human spermatozoa.Proteins interacting with HBx protein in human spermatozoa were analyzed by co-immunoprecipitation combined with mass spectrometry technology to analyze its function by bioinformatics;lentivirus transfection technology was used to construct HBx stably expressed murine spermatogonia cell line(GC-1 spg),Annexin V-FITC and PI staining for detecting spermatogonia apoptosis and cell cycle,siRNA interference and other experiments were used to explore the effect of HBx on the function of spermatogonia;through the screening of differentially expressed LncRNAs in sperm by gene chip technology,RT-qPCR technology was used to verify the accuracy of the chip results,and it conducted the screening for differentially expressed genes and bioinformatics analysis.【Result】 1.Compared with normal healthy males,the expression level of mRNA and protein in histone methyltransferase Suv39h1 gene of HBV infected persons’ spermatozoa were significantly down-regulated(P<0.05).2.There was no significant difference in the methylation level of the Suv39h1 gene promoter in the spermatozoa of HBV infected persons and normal males(P>0.05).3.The expression level of mRNA and protein in Suv39h1 of the spermatogonia that stably expressed by HBx was significantly down-regulated(P<0.05).4.After that murine spermatogonia was transfected by lentivirus pGFP-HBx,the expression level of Suv39h1 gene was significantly decreased.5.After that murine spermatogonia was transfected by lentivirus pGFP-HBx,the apoptotic rate of murine spermatogonia was increased and proliferation delayed.6.After it interfered the expression of transcription factor UBP1,the expression level of was HBx was significantly down-regulated and the expression level of Suv39h1 was significantly up-regulated.7.A total of 300 LncRNAs were significantly differentially expressed in the spermatozoa of HBV infection group(214 up-regulated and 86 down-regulated).【Conclusion】 HBV infection can cause significant down-regulation of Suv39h1 gene expression in human spermatozoa;HBV X gene may regulate the expression of Suv39h1 gene through long chromatin;HBV infection can alter the expression of LncRNAs in sperm and thus affect sperm function.