MiR-382 Regulates Proliferation Of Spermatogonia Via Targeting Kmt5a Gene

MicroRNA is a class of non-coding small RNA with regulation function by binding to the 3' UTR region of the target mRNA.Previous studies have shown that miR-382 is involved in the regulation of proliferation in multiple cell types and inhibits the invasion of tumor cells.In order to investigate the effect of miR-382 on the proliferation of spermatogonia in mice,the target gene of miR-382 was predicted using bioinformatics software.The dual-luciferase reporter gene assay system was used to confirm the target gene.miR-382-5p mimics and inhibitor were transfected into the GC-1 spg cells,the effects of miR-382-5p on cell viability and proliferation of spermatogonia were examined by CCK-8 and EdU assays.The expression level of Kmt5 a mRNA in different developmental stages and different cell types in pig testes was detected by RT-qPCR.Molecular cloning,DNA sequencing and bioinformatics analysis were used to investigate the molecular structure differences of Kmt5 a splice variants.Main results were as follows:The predicted target genes of miR-382-5p were Kmt5 a and Top1.The dual-luciferase reporter gene assay system confirmed that Kmt5 a was a target gene of mi R-382-5p,but Top1 was not.The results of CCK-8 and EdU assay showed that after the transfection of 120 nM of miR-382-5p mimics,the cell viability of GC-1 spg cells was decreased by 13.4%(p<0.05)and the cell proliferation ability of GC-1 spg cells was decreased by 8.6%(p<0.05)compared to the control group.miR-382-5p inhibitor did not increase the viability and proliferation of GC-1 spg cells(p>0.05).The results of RT-qPCR and western blotting showed that after the transfection of120 nM of miR-382-5p mimics,the relative mRNA abundance of Kmt5 a in GC-1 spg cells decreased by 39.8%(p<0.05)and the KMT5 A protein content decreased by 36.5%.The relative mRNA abundance of Kmt5 a in 5-month-old pig testis tissue was significantly higher than those in 7-day-old and 2-month-old pig testes(p<0.05).The relative m RNA abundance of Kmt5 a in Sertoli cells of 7-day-old pig testes was significantly higher than that in interstitial cells and gonocytes(p<0.05).DNA sequencing showed that the Kmt5 a gene had two splice variants in pig testes.The analysis of protein structure showed that protein KMT5A-X1 was lack of PIP(PCNA-interacting motifs)box1 in the N-terminal compared with KMT5A-X2.In conclusion,this study found that Kmt5 a gene was a target gene of miR-382-5p,and miR-382-5p regulated the proliferation of GC-1 spg cells.The expression and distribution of Kmt5 a and the molecular structure of two splice variants were studied in pig testes.The results enriched the regulatory network of miRNA in the process of spermatogonia,and provided a preliminary experimental basis for further analysis of spermatogenesis in male mammalian animals.