Bovine Sperm-specific High Expressed Bta-miR-182 Promotes Early Embryonic Development By Regulating CFL1

MicroRNA(miRNA)is a type of non-coding single-stranded RNA molecule with a length of about 18 to 24 nt.miRNA affects the expression of genes by regulating the transcription level of genes,the stability of mRNA and post-transcriptional translation,and has an important impact on apoptosis,cell differentiation,senescence,and canceration.During the development of cloned bovine embryos,abnormal embryonic development may occur,such as developmental blockage and abnormal embryo morphology and quality,especially in the early development of cloned cattle embryos.It may be no sperm involved in the cloned embryo,and the regulation of the sperm-derived substance is missing,thereby causing abnormal development of the cloned embryo.Therefore,the role of miRNAs with high sperm-specific expression should be studied in the early development of bovine embryos.The main results obtained in this experiment are as follows:1.Identify Bta-miRNA-182 as sperm-specific highly expressed miRNA through miRNA quantitative,and screen out target genes ACVR1,CFL1 and ZFP36L1 by bioinformatics,these genes may play regulatory roles in early embryo development.2.The dual-fluorescence reporter vectors of target genes ACVR1,CFL1 and ZFP36L1 were constructed respectively,and the expression of the target genes was detected using the dual-luciferase reporter gene method.Lip 2000 was used to co-transfect the target gene plasmid and miRNA mimic into 293T cells.Compared with the control group,the fluorescence intensity of CFL1 and ACVR1 in the co-transfected miRNA-182 mimic group was significantly reduced(p<0.05),while the fluorescence intensity of ZFP36L1 was not significantly changed.After that,real-time quantitative PCR(qPCR)was used to detect the changes of ACVR1,CFL1 and ZFP36L1.The results showed that the expression levels of CFL1,ZFP36L1 and co-transfection with miRNA-182 were significantly reduced(p<0.05).Combined with the related functional analysis of genes,CFL1 was screened as the target gene of miRNA-182.Western Blot test transfected mimic into fibroblasts.The results showed that,compared with Negative control mimic(NC mimic)and inhibitor group,the protein expression level of CFL1 in miR-182 mimic electrotransfected group was significantly reduced(p<0.05),proving that CFL1 is the target gene of miRNA-182.3.Inject miR-182 mimic into the embryo according to the gradient concentration,and select the most suitable miR-182 mimic concentration by qPCR.When the optimal concentration of miR-182 mimic was injected into early embryos,the results of immunofluorescence staining showed that the expression level of CFL1 in the embryo 2-cell and 4-cell stage was significantly reduced(p<0.05).The results of phalloidin staining showed that the miR-182 mimic injection group had significantly increased actin expression levels at the 2-cell and 4-cell stages(p<0.05).It shows that miR-182 can regulate the expression of F-actin by targeting CFL1.In addition,compared with the cleavage time of SCNT and parthenogenetic embryos,the cleavage time of miR-182 mimic injection group was closer to IVF embryos.After injection,the embryo blastocyst rate was increased(p<0.05),and the embryo blastocyst apoptosis cells were reduced(p<0.05).Then screened the small molecule interfering RNA fragment si-429 with obvious inhibitory effect on CFL1.After injection of si-429,it was found that the expression level of CFL1 in the 2-cell period was significantly reduced(p<0.05),and the blastocyst rate was significantly increased(p<0.05).The experiment showed that miR-182 with high sperm-specific expression affects the expression of actin by regulating the target gene CFL1.The cleavage time is close to that of IVF embryos,which increases the blastocyst rate of early embryos.