The Regulatory Study Of Steroidogenic Genes Expressision In Leydig Cells And Somatic Fibroblast Cells Based On The Intergrated Analysis Of Genomic DNA Methylation And Transcriptome

Leydig Cell?LC?is the main source of androgen in male individuals.Androgen plays a decisive role in the development of male reproductive system and the maintenance of male reproductive function.DNA methylation is one of the main manifestations of epigenetic modification of nucleic acids,which can regulate the expression of target genes at the transcriptional level.So far,there have been no reports on epigenetic expression of key enzymes in androgen synthesis pathway and regulation of androgen secretion in LC.In this study,Progenitor Leydig Cell?PLC?,Immature Leydig Cell?ILC?,and Adult Leydig Cell?ALC?,witch were at different developmental stages of LC were isolated,and rat tail fibroblasts?TTF?were used as a control.Using transcriptome sequencing RNA-seq technology and genomic methylation sequencing RRBS technology to analyze the changes on genes transcription level and genomic DNA methylation map of LC at different developmental stages,exploring the epigenetic regulation mechanism of genomic DNA methylation on the expression of key enzymes in androgen synthesis pathway during LC development,provides a scientific basis for understanding the development of LC lineage,the regulation mechanism of androgen synthesis,and the future reprogramming of somatic cells into functional leydig cells.The main results of this study are as follows:1.Transcriptome sequencing of LC?PLC,ILC,ALC?at different developmental stages,and TTF 8 samples?2 replicates?,and finally 6.60 Gb data were obtained for each sample.A total of 15852 genes expressed in were detected in LC,of which 10636genes were co-expressed at three developmental stages,and 710 genes were differentially expressed at each developmental stage?|log₂FC|?1,FDR<0.01?.Gene ontology analysis,and the significantly enriched biologic processes associated with steroid synthesis?P<0.05?were:lipid transport?GO:0006869?,hormone metabolism?GO:0044445?,steroid metabolism?GO:0008202?,cholesterol transport?GO:0030301?,testosterone response?GO:0033574?,lipid catabolism?GO:0016042?.The genes involved in the regulation of steroidogenesis were Cyp11a1,Hsd11b1 and Tspo.KEGG pathway enrichment analysis,and the significantly enriched the pathways involved in steroid synthesis?P<0.05?were:steroid hormone biosynthesis?rno00140?;cholesterol metabolism?rno04979?;cytochrome P450 metabolism of xenobiotics?rno00980?.The genes involved in the regulation of steroidogenesis were Hsd11b1,Cyp11a1,and Tspo.A total of 28,365 genes were detected in LC and TTF cells,and a total of 13446genes were co-expressed.There were 7707,9850 and 8659 differentially expressed genes in TTF vs PLC,TTF vs ILC and TTF vs ALC,among which 4913 genes were differentially expressed?|log₂FC|?1,FDR<0.01?in the three groups.Gene ontology analysis,and the significant enrichment biological processes associated with steroid hormone regulation?P<0.05?were:development of major male sexual characteristics?GO:0046546?,gonadal development?GO:0008406?,sex differentiation?GO:0007548?,Leydig cell differentiation?GO:0033327?,steroid biosynthesis process?GO:0006694?,response to gonadotropin?GO:0034698?,steroid metabolism process?GO:0008202?,and so on.The genes involved in steroid synthesis regulatory such as Nr0b1,Ar,Cyp11a1,Cyp17a1,Gata4,Nr5a1,Wt1,Star,Hsd17b3,Hsd3b1,Lhcgr,and so on.KEGG pathway enrichment analysis,and the significantly enriched pathways involved in steroid synthesis?P<0.05?were:cortisol synthesis and secretion?rno04927?,cytokine-cytokine receptor interaction?rno04060?,MAPK signaling pathway?Rno04010?,cytochrome P450 metabolism of xenobiotics?rno00980?,AGE-RAGE signaling pathway?rno04933?in diabetic complications.The genes involved in steroid synthesis regulatory were Nr5a1,Cyp11a1,Hsd3b1,Star,Nr0b1.2.Using RRBS technology to construct the genomic DNA methylation map of LC and TTF,revealing the DNA methylation characteristics of genomic DNA:mCpG is the main genomic DNA methylation pattern of LC and TTF;methylation level is significant decreased near TSS,but significantly increased in the gene body region;methylation level analysis of differential functional elements,the methylation level of promoter region and 5'UTR are the lowest.The comparison of LC methylation groups at different developmental stages?PLC vs ILC,ILC vs ALC,PLC vs ALC?detected 5749,5710,and 6317 differential methylation regions?DMRs?,associated with 3936,3914,and 4286 differential methylation genes,in which 512,638 and 654 genes mRNA level were negatively correlated with changes in methylation level;DMRs of the three comparison groups were distributed on chromosomes 1 to 10 and X chromosome,and rarely distributed on the Y chromosome;DMRs were concentrated in the intergenic region,partially distributed in the intron region of the genes,and a few were distributed in the exon region,the promoter region and the 3'UTR,and a few were distributed in the 5'UTR of gene.Three LCs were compared with TTF?TTF vs PLC,TTF vs ILC,TTF vs ALC?,and 20254,23275,and 22169 DMRs were detected,associated with 8640,9445,and9167 genes,in witch 2160,2570,and 2509 genes mRNA level were negatively correlated with changes in methylation level.Differential methylation of one gene occurs in the promoter region.The DMRs of three comparison groups were distributed on chromosomes 1 to 10 and X chromosome,and rarely distributed on the Y chromosome;DMRs were concentrated in the intergenic region,partially distributed in the intron region of the gene,and a small part was distributed in the exon region of the gene.The promoter region and the 3'UTR are rarely distributed in the 5'UTR of the gene.3.Combined analysis of methylation and transcriptome,obtained a negative correlation between changes in transcription levels and in methylation levels.For the differental genes of three LCs,gene ontology analysis,and the significant enriched biological processes associated with steroid synthesis GO term?P<0.05?were:genitourinary system development?GO:0001655?,response to gonadotropin?GO:0034698?,gonadal development?GO:0008406?,sex differentiation?GO:0007548?and the like,the genes involved in steroid synthesis regulatory were:Nr2f2,Nr0b1,Pdgfra,Wt1,Cyp11a1.TTF was compared with PLC,ILC and ALC respectively to obtain genes with negative correlation between changes in transcription level and chang in methylation level,gene ontology analysis,and the significant enrichment biological processes GO term related to steroid synthesis?P<0.05?were:male gonad development?GO:0008584?,male sex differentiation?GO:0046661?,stress-activated MAPK cascade?GO:0043410?,genitourinary system development?GO:0001655?,Gonadal development?GO:0008406?,transcriptional activator activity,RNA polymerase II core promoter proximal region sequence-specific binding?GO:0001077?and the like.Genes involved in androgen synthesis were:Nr5a1,Hsd17b3,Lhcgr,Cyp11a1,Nr0b1,which are initially thought to be differentially expressed candidate genes mediated by methylation.KEGG pathway enrichment analysis of differential genes,significant differentially enriched pathways for steroid synthesis?P<0.05?:regulation of steroid biosynthesis processes?GO:0050810?,stress-activated MAPK cascade?GO:0051403?,response to gonadotropin?GO:0034698?,response to cAMP?GO:0051591?,male gonadal development?GO:0008584?,gonadal development?GO:0008406?,sex differentiation?GO:0007548?,C21-steroid hormone metabolism process?GO:0008207?and the like.The genes involved in steroid synthesis regulatory were:Nr5a1,Cyp11a1,Cyp17a1,Cyp21a1,Lhcgr,Dhh.It is initially thought to be differentially expressed candidate genes mediated by methylation.Among them,the differential methylation region of Nr5a1 and Hsd17b3 occurs in the promoter region,which are presumed to be differentially methylated genes with androgen synthesis functional mediated by methylation of the promoter region.