Identification And Functional Studies Of Development Related Non-coding RNAs In Shaziling Pig Testicular Tissue

The significance of boars in swine production is mainly reflected by the key influences of semen quality on the pregnancy rate and litter size of sows,as well as the offspring production performance.Besides,as an important reproductive organ in boars,the normal growth and development processes of testicular tissue are closely related to ensure the orderly processes of spermatogenesis.Recently,studies have shown that non-coding RNAs are widely expressed in testicular tissues of male animals and they play key regulatory roles in spermatogenesis.However,limited number of lncRNAs related to boar testicular tissue development or spermatogenesis were identified,and their function roles have still not be achieved.Based on our previous studies,the objective of this dissertation is to identify non-coding RNAs(lncRNA,circRNA,miRNA)from pig testicular tissues at different developmental stages through constructing cDNA and small RNA RNA-seq libraries,and further study their biological roles at cellular level.The main results are as follows:(1)In the present study,Shaziling pig testicular tissues were used as the investigated object.Four treatments were set in different days of age,which including 60,90,120,and 150 days of age.Twelve cDNA sequencing libraries were constructed using total RNA from each testes sample.Three independent biological replicates were performed in this experiment.The libraries were then sequenced on an Illumina Hiseq 4000 platform.In total,we obtained 164.26 Gb clean reads and assembled 207,110 transcripts.Using a series of bioinformatics analysis methods,we identified 25,491 protein coding transcripts,5,032 pig known lncRNAs,10,496 pig novel lncRNAs,7,232 TUCPs,and 5,380 pig novel circRNAs.In addition,pairwise comparisons identified 733 lncRNAs that showed differential expression patterns.GO enrichment analyses results demonstrated that their targeted genes were significantly enriched in 64 GO terms.We also validated the expression patterns of nine differential expressed lncRNAs and other six lncRNAs in developing porcine testicular tissues using quantitative real-time PCR technology.In addition,the splice site of eight circRNAs were also validated using PCR technology.(2)In the present study,Shaziling pig testicular tissues were used as the investigated object.Four treatments were set in different days of age,which including 60,90,120,and 150 days of age.Equal amounts of total RNA of three pig testis tissues at the same developmental stage was pooled to construct each of four small RNA libraries.The library preparations were sequenced on an Illumina HiSeq 2500 platform.In total,we obtained 53,309,184 clean reads from these above mentioned small RNA libraries.The bioinformatics analysis results demonstrated that 293 pig known mi RNAs,36 pig novel miRNAs,and 86 differential expressed miRNAs were identified in the present study.The target genes of mi RNAs were predicted using the integrated analysis of mi RNA and mRNA expression profiles based on our previous studies.GO enrichment analysis results showed that target genes of miRNAs were significantly enriched in 81 GO terms.The expression patterns of 12 differentially expressed mi RNAs and other 4 miRNAs were validated in developing porcine testicular tissues using quantitative real-time PCR technology.(3)In order to confirm the regulatory roles of miR-10 b in porcine immature Sertoli cell growth and development,Flow cytometry,CCK8,EdU,Annexin V-FITC/PI staining,or quantitative real-time PCR assay was used to measure the cell proliferation or apoptosis.The results indicated that over-expression of miR-10 b or knockdown of DAZAP1 gene significant increased the percentage of cells in G2 phase(P<0.05),decreased the percentage of cells in S phase(P<0.05),and enhanced the cell proliferation activity compared with the NC group(P<0.05).However,over-expression of mi R-10 b or knockdown of DAZAP1 gene had no effect on cell apoptosis(P>0.05).In addition,the results from co-transfection treatments showed that mi R-10 b promoted porcine immature Sertoli cell proliferation by targeting DAZAP1 gene.(4)To confirm the regulatory roles of miR-362 in porcine immature Sertoli cell growth and development,we over-expressed miR-362 in porcine immature Sertoli cells.Flow cytometry results showed that more cells were checked in G1 phase(P<0.05),and fewer cells in S phase compared with the NC group(P<0.05).Results from CCK8 and EdU assays indicated that cell proliferation activity was significantly decreased compared with the NC group(P < 0.05).Annexin V-FITC/PI staining results demonstrated that cell apoptosis rate was significantly increased compared with the NC group(P < 0.05).Quantitative real-time PCR assay results indicated that the Bcl2 expression level was significantly decreased(P < 0.05),while BAX and Caspase-3 expression level were significantly increased compared with the NC group(P < 0.05).In addition,these phenomena were similar to the siRNA-mediated knockdown of the RMI1 gene.The luciferase reporter assay results demonstrated that miR-362 directly target the RMI1 gene and regulated its expression in porcine immature Sertoli cell.In conclusion,the present study identified lncRNAs,circRNA,and mi RNA from pig testes at different developmental stages,and their functions were also predicted and annotated using bioinformatic methods.In addition,we also studied the regulatory roles of mi R-10 b and miR-362 on the growth and development of porcine immature Sertoli cells.These results will offer new theoretical basis into the molecular mechanisms of non-coding RNAs in regulating the porcine spermatogenesis,thus to provide new insights for understanding the molecular mechanisms of porcine spermatogenesis.