Identification And Functional Studies Of Differentially Expressed Genes And MicroRNAs During Development Of Porcine Testicular Tissue

Pig(Sus Scrofa)is an important economic animal and one of ideal model animals for human biology because of the similarity physiology,organ development and disease progression.Testicular development and spermatogenesis are complex and strictly regulated processes as which are related to the complex interactions between several cell types in testis,such as Sertoli cells,Leydig cells and germ cells.Elucidating the potential key regulators and signaling pathways in testis development and spermatogenesis will provide us more clues to understand the normal germ cell development or generate novel hypotheses.In the present study,RNA-seq technology was used to identifiy and select the differential expressed protein coding genes and miRNAs in Shaziling pig testes at different development stages,and the main results were showed as follows:(1)Four cDNA libraries named E60,E90,D30 and DM were constructed and sequenced using Illumina HiSeq 2500 platform,and 56296294,49177458,48837300,41545444 clean reads were generated and used further analysis,respectively.1007(646 up and 361 down),1929(911 up and 1018 down),7420(3998 up and 3422 down)differential expressed genes were identified from E90 vs.E60,D30 vs.E90 and D180 vs.D30,respectively,which were enriched 37,20,77 GO terms and 11,23,9 KEGG signaling pathways.In addition,the results showed that 92738 alternative splicing events were observed from Sus Scrofa genome corresponding to 20398 genes(80.6%).Alternative 5? first exon(TSS),alternative 3? last exon(TTS),skipped exon(SKIP)and alternative exon ends(AE)were the highest frequency events.(2)Four small libraries named E60,E90,D30 and DM were constructed and sequenced using Illumina HiSeq 2000 platform,and 6347414,6287768,6148364,6333541 clean reads were generated and used for further analysis,respectively.A total of 304 mature and 50 novel miRNAs were identified in this study.93(48 up and 45 down),104(49 up and 55 down),122(49 up and 73down)differential expressed miRNAs,as well as 1007(646 up and 361 down),1929(911 up and 1018down),7420(3998 up and 3422 down)differential expressed genes were identified in E90 vs.E60,D30 vs.E90 and D180 vs.D30,respectively.Integrating analysis of miRNA and mRNA expression profiles predicted 55339 mi RNA-mRNA interaction sites.GO enrichment and KEGG pathway analysis of the predicted target genes illustrated the likely roles of differentially expressed miRNAs in testis development and spermatogenesis.For example,PI3K-Akt signaling pathway and Hippo signaling pathway related development,and carbon metabolism,fatty acid metabolism,protein digestion and absorption,were involved in metabolite synthesis.(3)miR-10b-DAZAP1/KLF11 and miR-26a-ULK2 were selected from the results of integrating analysis and validated in swine testicular(ST)cells.The expression levels of miR-10 b,mi R-26 a,DAZAP1,KLF11 and ULK2 genes were checked in pig testes at 1-day-old,30-day-old,60-day-old,90-day-old,120-day-old,150-day-old and 180-day-old using quantitative real-time PCR.The expression level results showed that miR-10 b and DAZAP1 gene were negative correlation(Pearson value=-0.6,P=0.002),as well as miR-26 a and ULK2 gene were negative correlation(Pearson value=-0.771,P=0.015).In addition,western blot results indicated that the protein expression levels of DAZAP1 and ULK2 genes were consensus with their mRNA expression level in each testis sample.The wild type and mutant type 3?-UTRs of DAZAP1 and ULK2 genes were constructed and transfected in Swine Testicular cells with miRNA mimics and mimic NCs,and the dual luciferase report results confirmed that DAZAP1 and ULK2 genes were the target genes of miR-10 b and mi R-26 a,respectively.Then,we transfected miR-10b/26 a mimic,mimic NC,inhibitor,inhibitor NC in ST cell,and the quantitative real-time PCR and western blot results showed that miR-10 b can repress the DAZAP1 gene at mRNA and protein level,as well as miR-26 a can repress the ULK2 gene at mRNA and protein level.In conclusion,the present study provided a comprehensive view of differences in pig testes with different development stages using miRNAome and transcriptome,and the function roles of these different expressed mi RNAs and genes were predicted using GO and KEGG databases.In addition,the target genes of mi R-10 b and miR-26 a were validated on tissue and cell levels.These results not only contribute to the understanding of the pig testes mi RNA transcriptome profiles and transcriptome but also provide a valuable resource for further studying the regulation mechanisms in testis development and spermatogenesis.