The Study Of Chi-miR-182 Regulation On NTS,TES And Endometrial Epithelium Cells

In this study,the relationship between miR-182 and NTS/TES was studied in dairy goat endometrial epithelial cells(EEC).Real-time PCR and Western Blot were used to study the effects of miR-182 on the expression of NTS,TES,Caspase-8 and p38.The pcDNA3.1(+)vectors were constructed to study the interaction between NTS and TES,the regulations of NTS and TES on the protein expression of Caspase-8 and p38,and the effects of NTS and TES on the apoptosis of EEC.The main findings were showed as following:1.Verify the regulatory relationship between miR-182 and NTS/TESBioinformatics predicted that the miR-182 target site existed in the 3’UTR of the dairy goat NTS gene,and the CDS region of the TES gene.Construction of double luciferase reporter vectors,co-transfected with mi R-182 mimics,or Negative control(NC)into 293 T cells.The results showed that miR-182 mimics significantly inhibited luciferase activity compared with NC,indicating that mi R-182 decreased the activity of luciferase(P < 0.05)by interaction with NTS-3’UTR and TES-CDS(P < 0.05).Based on this study,NTS and TES were identified as the target genes of miR-182 in EEC.2.To study the effect of mi R-182 on the expression of NTS,TES,Caspase-8 and p38Real-time PCR and Western Blot were used to detect the expression of NTS and TES genes.The results showed that miR-182 significantly inhibited the expression of mRNA and protein of NTS and TES compared with NC(P < 0.05).Western blot analysis showed that mi R-182 significantly increased the expression of Caspase-8 protein(P < 0.05)and decreased p38 protein(P < 0.05).3.The effects of NTS and TES on the expression of Caspase-8 and p38.The pcDNA3.1(+)-NTS and pcDNA3.1(+)-TES overexpression vectors were constructed and transfected into EEC.Western blot was used to detect the expression of Caspase-8 and p38.The results showed that overexpression of NTS and TES significantly reduced the expression of Caspase-8 protein(P < 0.05)and increased p38 protein(P < 0.05).These results were in contrast to the effects of miR-182 on the expression of Caspase-8 and p38.4.To verify the interaction between NTS and TES and the effects of NTS and TES on EEC apoptosisThe pcDNA3.1(+)-NTS and pcDNA3.1(+)-TES overexpression vectors were transfected into EEC.The expression of NTS and TES were detected by real-time PCR and Western Blot.The results showed that overexpression of NTS significantly inhibited the expression of TES mRNA and protein(P < 0.05).The overexpression of TES gene only reduced the expression of NTS protein(P < 0.05),but had no significant effect on NTS mRNA(P > 0.05).The effects of NTS and TES on EEC apoptosis were detected by flow cytometry,and the results showed that both NTS and TES significantly inhibited EEC apoptosis(P < 0.05).The above results indicated that NTS and TES were the target genes of mi R-182,and mi R-182 may affect the apoptosis of endometrial epithelial cells of dairy goat by down-regulating NTS and TES,and up-regulating Caspase-8.