Ssc-miR-155 Targeting Hsd17b3 Regulates The Function Of Leydig Cells

Leydig cells(LCs)are the major cell types for steroid hormone synthesis.17β-hydroxysteroid dehydrogenase 3(HSD17B3)can catalyze androstenedione directly conversing to testosterone.The mutation of genes related to steroid synthesis pathway,LCs apoptosis,inflammation and other factors can cause abnormal synthesis of testosterone,which affects spermatogenesis and male reproduction.In this study,pigs were used as research objects.By using population genetics,Hsd17b3 was selected as the important candidate genes that affected pig male reproduction.Hsd17b3 gene expression,functions and regulatory mechanisms were further studied.The results would provide experimental evidence for further exploring the post-transcriptional regulatory mechanism of pig Hsd17b3 genes and would provide references for researching testosterone synthesis-related genes.miR-155(miR-155-5p)acts as an inflammatory miRNA(Micro RNA)molecule,might play roles in the testicular inflammation and male reproduction.Research about the function of porcine miR-155(sscmiR-155)is still scarce in male reproduction.Thus,investigating the regulation mechanism of porcine Hsd17b3 by miRNA has important reference significance on understanding the role of Hsd17b3 in testosterone synthesis and steroid metabolism in testicular cells.By using molecular and cellular biology techniques,the important miRNAs associated with porcine reproduction in testicular cells,Hsd17b3 gene expression patterns and molecular mechanisms were explored;the role of ssc-miR-155 and Hsd17b3 genes in regulating male reproduction were clarified;the functions of ssc-miR-155 targeted to Hsd17b3 in Leydig cells were analysis,which finally provide molecular basis for androgen synthesis and metabolism.1.Effect of Hsd17b3 gene on testicular morphological traits of pig testisIn this part of the experiment,q RT-PCR experiments were used to determine the expression of Hsd17b3 gene in different tissues and cell samples.Results revealed that Hsd17b3 expression was most abundant in testes.The main transcript sequence information of Hsd17b3 gene in testes was identified by cloning and sequencing.Using population genetics analysis method,it revealed that a segment of 11 bp indel in the promoter region was strongly linked to the 15 bp indel of the intron region,which affected the expression of Hsd17b3 in pigs and was significantly correlated with porcine testis weight.These two key indels can be used as potential molecular markers in animal genetic breeding.2.Study on the targeting relationship between ssc-miR-155 and Hsd17b3 geneBioinformatic analysis showed that ssc-miR-155 was predicted to target Hsd17b3 and was conserved among different species.The expression of ssc-miR-155 in various tissues of commercial pigs was detected by q RT-PCR experiments.Results revealed that ssc-miR-155 was expressed in a variety of tissues,among which ssc-miR-155 had a higher expression in the epididymis.The dual luciferase reporter system demonstrated that ssc-miR-155 can target the pig Hsd17b3 gene and regulate the expression of endogenous Hsd17b3 gene in pig testis cell line(ST).3.The function of ssc-miR-155 in Leydig cellsThrough q RT-PCR,Western blotting,TUNEL,Ed U,and flow cytometry experiments,it was found that EDS treatment of mesenchymal cells can induce cellular inflammation,cause apoptosis and induce the upregulation of ssc-miR-155;overexpression of ssc-miR-155 can induce inflammation and apoptosis of TM3 cell,but has no effect on cell proliferation;overexpression of ssc-miR-155 can activate the NF-κB / p-NF-κB signaling pathway of TM3 cells and cause cellular inflammatory responses.In summary,this study found that ssc-miR-155 can target the Hsd17b3 gene and induce TM3 cell inflammation and apoptosis.It can provide scientific data for the study of the key gene for testosterone synthesis Hsd17b3.At the same time,it lays the foundation for the analysis of the molecular mechanism of ssc-miR-155 regulating LCs.