The Mechanism Of Bta-miR-200b Targeting MYB To Regulate The Proliferation And Apoptosis Of Bovine Endometrial Epithelial Cells

Embryo death is considered to be one of the main causes of decreased reproductive rate in cattle industry,and endometrial receptivity is the main factor leading to implantation failure and loss of embryo.Apoptosis of endometrial epithelial cells is an important process of endometrial receptivity and embryo implantation.A large number of studies have shown that miRNAs play a certain role in the regulation of embryo implantation and embryo growth and development.In mammalian endometrial epithelial cells,miRNAs are involved in many cellular processes,including cell proliferation and apoptosis.In this study,the role and regulatory mechanism of uterine fluid exosome miRNAs in embryo implantation and development were investigated to find molecular markers that can be used as markers of early pregnancy in cattle.At the early stage of the experiment,transcriptome sequencing was performed on the extracellular miRNAs of uterine fluid from 3 donor and 3 recipient cattle before embryo transfer,and a large number of different miRNAs were screened.In this study,differentially expressed bta-miR-200 b was selected as the research object.After differentially expressed verification,bta-miR-200 b was overexpressed.The effects of bta-miR-200 b on the proliferation and death of bovine endometrial epithelial cells were investigated by RT-q PCR,Western blot,flow cytometry and CCK-8.MYB was identified as the target gene of bta-miR-200 b using a dual luciferase reporter system and interfered with MYB gene to analyze its effect on endometrial epithelial cells.Through the above experiments,we analyzed the mechanism of bta-miR-200 b targeting MYB to regulate the proliferation and apoptosis of bovine endometrial epithelial cells,which can be used as a candidate molecular marker for bovine early pregnancy markers.The main research results are as follows:(1)After overexpression of bta-miR-200 b in bovine endometrial epithelial cells,m RNA and protein expressions of apoptotic gene-related X(BCL2),apoptotic regulatory factor(BAX)and endometrial receptive marker gene(OPN)were significantly up-regulated.In addition,overexpression of bta-miR-200 b can inhibit the proliferation of bovine endometrial epithelial cells,promote cell apoptosis,and arrest the cell cycle in the G0/G1 phase.(2)MYB proto-oncogene(MYB)was confirmed to be a target of bta-miR-200 b in bovine endometrial epithelial cells by dual luciferase reporting assay.By using bioinformatics software Targetscan,differentially expressed genes predicted by transcriptome sequencing and literature review,the potential target gene MYB of bta-miR-200 b that may affect the proliferation and marcelation of bovine serophytic cells was screened.Double luciferase reporter vectors of wild-type(WT)and mutated(MUT)of the potential binding site between the gene 3 ’UTR region and bta-miR-200 b were constructed.MYB was identified as the target gene of bta-miR-200 b by RT-q PCR and double luciferase reporting assay.(3)Transfection of bta-miR-200 b mimics decreased MYB m RNA and protein expression.Overexpression of MYB reduced the effect of bta-miR-200 b on apoptosis of bovine endometrial epithelial cells.Our results suggest that bta-miR-200 b can affect apoptosis of bovine endometrial epithelial cells by targeting MYB gene.This study revealed the influence of bta-miR-200 b and its target gene MYB on the proliferation and apoptosis of bovine endometrial epithelial cells,and further verified the targeted silencing effect of bta-miR-200 b on MYB.These results suggest that MYB can regulate the proliferation and apoptosis of bovine endometrial epithelial cells through related genes,and can be used as a candidate molecular marker for bovine early pregnancy.