Regulation Of MiR-152to Gene Dnmtl Expression In Dairy Cow Mammary Gland Development And Lactation

MicroRNAs (miRNAs) are endogenous, non-coding, functional RNAs. Mature miRNA is a double-stranded RNA that contains approximately22nucleotides. miRNAs are post-transcriptional regulators that bind by complementary base pairing to sequences in the3’untranslated regions (3’-UTRs) of target mRNAs by translational repression or degradation of the transcript. Much evidence, such as cell differentiation, cell growth, and cell death has shown that miRNAs have an important influence on different physiological processes. But the mechanisms of miRNAs regulate mammary gland development and lactation was not known. Using qRT-PCR, we found that the miR-152expression is different during different lactation periods in cow mammary gland tissue. Therefore, it was proposed that miR-152could regulate mammary gland development and lactation at the post-transcriptional level in Holstein dairy cows.DNA methylation is an important epigenetic modification that participates in the regulation of gene expression. DNA methylation is catalyzed by enzymes in the DNA methyltransferase (DNMT) family, which have three members:DNMT1, DNMT3a and DNMT3b. During DNA replication, though the DNMT1enzymatic activity affects the maintenance of DNA methylation, DNMT3a and DNMT3b are responsible for unmodified DNA de novo methylation. Each enzyme catalyzes the methyl group transformation at the carbon5position (5meC) in DNA from S-adenosyl-L-methionine to a cytosine base. Methylation is often found in CpG and CpHpG (H=A, T, C) sequences. When it is located at gene promoters, methylation usually represses the expression of genes.In this study, use dairy cow mammary epithelial cells (DCMECs) as cell model in vitro. Transfected miR-152mimics/inhibitor with liposomal transfection method. Dnmtl and its related lactation pathways genes were detected by using qRT-PCR in DCMECs. Western blotting was used to detect Dnmtl and its related lactation pathways genes protein expression changes in DCMECs. By using an electronic Coulter counter (CASY-TT) and flow cytometer, we detect the viability and multiplication capacity of DCMECs. At the same time the application the ELISA kit to detect cells beta-casein lactose and triglyceride secretion. The experiment results showed that:①Real-time quantitative PCR results consistently with the chip showed results. miR-152expressed in mammary gland tissues of cows with different milk qualities. In the mammary gland tissues of cows producing high quality milk (H), the expression of miR-152was highest compared to pregnancy, and cows producing low quality milk (L). The results indicated that miR-152could regulate the cyclical changes of the dairy cow mammary gland development and lactation.②Constructing a pMIR-Report-Dnmtl vector, which contains Dnmtl3’UTR. The pMIR-Report-Dnmtl was transfected into DCMECs with miR-152mimics, and the cells were collected and analyzed using the Dual-Luciferase Reporter Assay System. The result showed that Dnmtl is a direct target of miR-152. miR-152down-regulates Dnmtl in DCMECs.③Using quantitative PCR and Western Blotting test the change expression of Dnmtl and related lactation genes after transfection with miR-152mimics/inhibitor. The expression of STAT5、ELF5、AKT1、mTOR、S6K1、SREBP1、PPARγ、GLUT1、CyclinD1were increased after overexpress miR-152. However, the expression of4EBPlwas decreased. When inhibition of miR-152, it is showed opposite results. According to the results, we speculate that miR-152can regulate DNMT1expression, which is associated with milk protein、lactose and the expression of butterfat signaling pathway genes.④CASY and flow cytometer detected the viability and multiplication capacity of DCMECs. the ELISA kit detect the secretion changes of cells beta-casein、lactose and triglyceride. The results showed that miR-142-3p promoted cell viability and proliferation, and enhanced the secretory ability of the epithelial cells secreted the beta-casein、lactose and triglyceride.