Mechanism Of MiR-let-7c Rugulates Cyclin-depedent Kinase Inhibitors 1A In Mouse Spermatogonial Cell Line GC-1spg

Objective:The World Health Organization predicts that infertility will become the third largest disease after cancer and cardiovascular disease.Male factors account for about 50%of infertility,and there is an increasing tendency.Asthenozoospermia is the most common type of male infertility,and about 70%of infertile men suffer from Asthenozoospermia.So far,the pathogenesis of Asthenozoospermia has not been clarified.It is generally believed that abnormal gene regulation is the mainly cause.In recent years,microRNA(miRNA)function is continuously deepening,and in the field of male infertility,the researchers agree that miRNA affects spermatogenesis by regulating the transcription and translation of its specific target genes.MiR-let7family is known to regulate the proliferation and development of spermatogonial stem cells and play an important role in sperm motility,capacitation,fertilization and embryonic development.Cyclin-depedent kinase inhibitors 1A(CDKN1A)is one of the most important cell cycle regulators.This study takes miR-let-7c as the research contents,to establish miR-let-7c overexpressing and silencing lentivirus vector to infect GC-1spg cells,in vitro to find the effect of miR-let-7c on the proliferation of spermatogonia,the expression of CDKN1A mRNA and protein,to explore the mechanism of mi R-let-7c on spermatogenesis and its role in the pathogenesis of Asthenozoospermia.Methods:1.To construct Lentivirus-vectors to transfected GC-1spg cells including miR-let-7c overexpressing lentivirus,silencing lentivirus and no vector lentivirus.GC-1spg cells were divided into four groups:negative control group transfected with no lentivirus,miR-let7c upregulated group transfected with mi R-let-7c overexpression lentivirus,downregulated miR-let-7c group transfected with miR-let-7c silencing and blank control group without lentivirus transfection.The transfected cells were collected 72 hours after transfection and the transfection efficiency was observed by fluorescence microscope.2.Experimental verification:The proliferation of GC-1spg cells was detected by CCK8,qTR-PCR and Western Blotting were used to detect CDKN1A mRNA and protein levels among the four groups.Results:1.The recombinant plasmids of miR-let-7c lentivirus were sequenced,and the results were all consistent with the target sequences,and the lentivirus titer were 3×10~8TU/ml and 6×10~8 TU/ml after the packaging.2.With the condition of DMEM and Polybrene(5?g/mL),MoI value was 80,GC-1spg cells were infected lentivirus for 8 hours,then replaced the fresh medium,after72 hours culture,the infection efficiency was to be more than 90%estimated by fluorescence microscope.3.The results of CCK8 showed that the growth rate of GC-1spg cells in the miR-let-7c upregulated group was slower than that in the control group,and the cell proliferation was significantly inhibited(P<0.01).4.qRT-PCR and Western Blotting showed that miR-let-7c up-regulated the expression of CDKN1A mRNA and protein in the control group,but the expression of CDKN1A mRNA and protein in the down-regulated miR-let-7c group was significantly higher than that in the control group(P<0.01).Conclusion:1.Successfullydesignandstructureofhightiter lentivirus-vectors of mi R-let-7c overexpression and silencing expression.2.Successfully establish optimal infection conditions in Mouse spermatogonial cell line GC-1spg.3.miR-let-7c inhibits the proliferation of GC-1spg.4.miR-let-7c negatively regulates the expression of CDKN1A mRNA and protein in GC-1spg.