The Effects Of MiR-101 On ING3 And Bovine Nuclear Transfer Embryo Development

The SCNT(somatic cell nuclear transfer) technique is a key technology of modern biotechnology, has been widely used in livestock multiplication of excellent individuals, transgenic technology and bio-pharmaceuticals. However, due to incomplete reprogramming factors, a low birth rate of cloned animals, a higher proportion of abnormal individuals, affect the further development of SCNT technology. It is a hot topic to improve the efficiency of nuclear transfer reprogramming and promote normal development of nuclear transfer embryos in the field of biological reproduction. Accumulating evidence suggests that some microRNAs(miRNAs) are involved in the development potential of SCNT embryos. As the abundant miRNA-101 in SCNT donor-cell and bovine oocytes, numerous studies have demonstrated that miR-101 can regulates cell proliferation and apoptosis. However, what its role in bovine SCNT donor cells and embryo are not know. In order to elucidate the role of mi R-101 in in bovine SCNT donor cells and embryo, Luciferase Reporter assay and Western blot identified ING3 as a novel target of miR-101 in bovine fibroblasts. Real time PCR and Flow cytometry were introduced to understand the roles of miR-101 and ING3 in bovine skin fibroblasts. Finally, the effects of miR-101 on development of SCNT embryo and blastocyst quality were evaluated. The following results were achived:1. In this study, we amplified deoxynucleotide sequence of mi R-101 and fragment that containing seed sequence of predicted target gene, inserted those into PCDH-513-B1 overexpression vector and PsiCHECKTM-2 reporter vector respectively. Over extension PCR was performed to produce fragment contain seed sequence mutation, and connected to PsiCHECKTM-2 reporter vector. Positive recombinants mi R-101-513-B1, WT-ING3-PsiCHECKTM-2 and MUT-ING3-PsiCHECKTM-2 were verified by sequencing. To determine whether ING3(inhibitor of growth proteins) is the miR-101 predicted targets, luciferase reporter assays were done in 293 T. The results demonstrated the 3’-UTR in ING3 mRNA contained a complementary site for the seed region of miR-101. ING3 expression was detected in level of translation by western blot in bovine fibroblasts, the result showed mi R-101 could down-regulate ING3 protein expression.2. Stably transfected PCDH-miR-101 or PCDH-513-B1 and screened clone cells. The fuctions of miR-101 and ING3 in bovine fibroblasts were detected by real-time PCR, flow cytometry and cell proliferation assay curve. The results showed miR-101 reduced ING3 mRNA level, also inhibited expression of P53、P21、UHRF1、BAX �'� DNMT3 A. mi R-101 promotes bovine fetal(BFF) fibroblasts growth and decreased its apoptosis by down-regulating ING3 expression, it consistent with proliferation detected.3. In our study, by utilizing donor-cell with overexpressed mi R-101, the blastocyst rate of bovine SCNT embryos in mi R-101 group(35.75%) was significantly enhanced than the control group(26.06%)(P<0.05), while the cleavage rate was no significant change(P>0.05). And the apoptotic rate in SCNT blastocysts was reduced in miR-101 group compared to the control group(P<0.05). The genes with early embryo development were detected, both higher expression of OCT4 and SOX2 were found in miR-101 group SCNT blastocysts than the control group(P<0.05).