The Function Of MiRNA Let-7a-5p On The Development Andcell Pluripotency Of Preimplantation Embryo In Mice

MicroRNAs(miRNAs)are small non-coding RNAs that regulate gene expression at the post-transcriptional level.Studies have shown that the miRNA let-7family can regulate the development time of nematode,suppress tumors by targeting oncogenes,and inhibit the self-renewal of stem cells.The expression of miRNA let-7family members can be detected in early embryos in mouse,but its role and regulatory mechanism in maternal zygote transition(MZT)have not been clarified.In this study,the expression of the miRNA let-7 family in mouse tissues were firstly detected,and results showed that there were highly expressed in highly differentiated tissues such as heart,lung,and muscle,indicated that miRNA let-7family is related to cell differentiation.The expression of let-7 family were also detected in mouse oocytes and embryos at 2-cell,4-cell,8-cell,morula and blastocysts stages,the expression of let-7 family showed a dynamic change with gradually decreased from oocytes to 8-cell stage,and increased from morula to blastocyst stage.To investigate the role of let-7 family in early embryonic development,a family members,let-7a-5p miRNA,were selected and studied.When miRNA let-7a-5p mimic were injected into mouse pronuclear embryo,the results showed that the development rate of 2-cell embryo was significantly lower than that of the control group.The qPCR results showed that the ZGA-related genes Zscan4 d,Elif1a,MuERV-L and Hsp1 b were significantly down-regulated in 2-cell stage embryos afterinjection.For those embryos developed to blastocyst stage after injection,the core pluripotent gene Sox2 and the auxiliary pluripotent gene Esrrb were significantly down-regulated,and the trophectoderm-related gene Cdx2 was significantly down-regulated,the mesoderm gene T was significantly down-regulated,and the primitive endoderm genes sox17,Gata6 were significantly up-regulated,differentiation-related genes Map2 and Hand1 were significantly up-regulated,cardiac development-related transcription factor Gata4 was significantly up-regulated,and DNA methylation transferase gene Dnmt3 a was up-regulated,A total of 6let-7a-5p mimic ESCs and 4 NC ESCs cell lines were obtained by establishing let-7a-5p mimic ESCs cell lines.It was found that let-7a-5p mimic ESCs(50.00%)had a lower establishment rate than NC ESCs(66.67%)cell lines,the expression of pluripotency-related genes Oct4,Nanog,Sox2,and Esrrb were also lower than those of NC ESCs.To further study the regulatory mechanism of let-7a-5p in pre-implantation embryo development,In this study,the targetscan,miRDB and Diana software were used to predict the downstream target genes of let-7a-5p.A total of 503 target genes were obtained,candidate molecules were screened out by KEGG signal pathway enrichment analysis and GO enrichment analysis,including Lin28 a,Lin28b,NRAS,IGF1 R,HAND1 and IGF1.Subsequently,the luciferase assay(Luciferase assay)was used to investigate the interaction between let-7a-5p and the RNA binding protein Lin28 a,also its homolog Lin28 b.The results showed that Lin28 a and its homologous Lin28 b were the target genes of let-7a-5p.In summary,let-7a-5p is gradually down-regulated during the period from oocyte to 8-cell,and up-regulated from morula to blastocyst stage,let-7a-5p mimic upregulated let-7a-5p during pronuclear injection.After expression,some embryos are blocked in the 2-cell stage,indicating that miRNA let-7a-5p is activated in the embryonic genome.After injection,the pluripotency genes in the embryos that developed to the blastocyst stage were down-regulated,and the differentiation-relatedgenes were up-regulated.The use of blastocysts after injection to establish lines found that compared with the NC group,the establishment rate was reduced,and the pluripotent genes were down-regulated,indicating that miRNA let 7a-5p plays a role in embryonic cell pluripotency.KEGG and GO analysis showed that the predicted target genes of let-7a-5p were involved in a variety of biological processes,and those genes were enriched in PI3K-Akt,MAPK,FoxO,Ras and other signaling pathways.Results from luciferase report assay suggested that let-7a-5p and Lin28a/b may have a complex interactions.