Regulation Mechanism Of MiR-29 Family To Lactation Of Dairy Cow Mammary Epithelial Cells

With the rapid development of dairy industry and improvement of human living standards, the consumption market of dairy products constantly expanding, how to increase the milk yield and optimize milk quality has become the focus research of dairy industry. Micro RNA is a class of important post transcriptional regulation and non-coding small RNA molecules. In animals, mi RNA can through combining target gene 3 ’UTR region to inhibit target gene translation or specificly cleavage its target mRNA and degrade target m RNA and accordingly regulate gene expression. mi RNA play an important role in individual development, cell proliferation, cell apoptosis and cell differentiation. Many studies showed that mi RNA has an important role in the regulation of mammalian mammary development and lactation.At present, as the focus in life science research field, epigenetic has been shown to play an important role in the expression of milk protein genes and animal mammary gland development or remodeling. DNA methylation is one of the major epigenetic genetic modifications. Under the action of DNA methyl transferase(DNMT), Cp G dinucleotide 5’ position hydrogen is substituted by active methyl of S-adenosylmethionine and changed into 5’-methyl cytosine, DNA methylation through this mechanism regulate genes expression of tissues and cells with no change of DNA sequence. In mammals, DNA methyltransferases mainly contain DNMT1, DNMT3 A and DNMT3 B. As the de novo DNA methyltransferases, DNMT3A/3B can methylate the unmethylated DNA double chain without the guide of the parent chain and have an important guiding roles in the abnormal occurrence of genomic DNA methylation. DNA methylation is involved in regulation of many important physiological and pathological phenomenons, some studies have shown that DNA methylation is closely related to the regulation of milk protein genes expression in dairy cow mammary gland, which provides a new direction for exploring the mechanisms of mammary gland development and the regulation of lactation function.The majority mi RNA regulations on DNA methylation patterns are focused in the studies of tumor diseases, but the related researches has been rarely reported in dairy cow mammary gland. Our previous studies found that the expression of mi RNA were significant different between high-milk-quality and low-milk-quality dairy cow mammary tissues. Among the up-regulated mi RNAs in high-milk-quality dairy cow mammary tissue, mi R-29 family(mi R-29 a, mi R-29 b, mi R-29c) have complementary sequences with 3’UTR of Dnmt3 a and Dnmt3 b which play a key role in DNA methylation. Whether mi R-29 s can influence dairy cow mammary development and lactation through DNA methylation regulation become the focus of our study.In this study, we used primary culture bovine mammary epithelial cells(DCMECs) as the cell model in vitro, and transfected mi R-29 s mimics or inhibitor using liposomal transfection method. First, we validated the target genes of mi R-29 s through the detection of dual luciferase activity and green fluorescent protein(EGFP) expression. We detected the expressions of DNMT3A/3B and lactation related genes with quantitative real time polymerase chain reaction(q RT-PCR) and western blotting technology. We analyzed DNA methylation changes of lactation related genes promoter using bisulfite sequencing PCR(BSP). We also observed the effects of methyltransferase inhibitor 5-Aza-dc on expression of mi R-29 s and lactation related genes. We detected changes of viable cells number and cell viability, and detected effects of mi R-29 s on cell proliferation with Ed U marker technique. Meanwhile, we detected the content of ?-casein, lactose,and triglyceride secretion respectively using ?-casein assay kit, lactose/galactose(rapid) kit and TG detection kit. Experiment results showed that:(1)The results of q RT-PCR were consistented with the results of small RNA sequencing, both of them showed that mi R-29 s expression in high-milk-quality dairy cow mammary tissue were significantly increased compared with low-milk-quality and non-lactating groups(P<0.05).(2)The experiments of the identification of target genes showed that DNMT3A/3B are the target genes of mi R-29 s in DCMECs, and mi R-29 s could reduced genomic global DNA methylation level and decreased activity of methyltransferases through directly negative regulation of DNMT3A/3B expression.(3)The results of q RT-PCR and western blotting showed that mi R-29 s influenced mRNA and protein expressions of lactation related genes in DCMECs. The expression of AKT1, m TOR, ELF5, CSN1S1, SREBP1, PPARγand GLUT1 were significantly decreased after overexpression of mi R-29 s in DCMECs. When we inhibited mi R-29 s expression, the expression of AKT1, mTOR, ELF5, CSN1S1, SREBP1, PPARγand GLUT1 were significantly increased. We guessed mi R-29 s have important roles in the regulation of milk compositions synthesis and secretion.(4)Bisulfite sequencing results showed that DNA methylation levels of Elf5, Csn1s1, Srebp1, Ppar G, Glut1 gene promoter regions were markedly increased after silencing of mi R-29 b in DCMECs, while DNA methylation levels of Akt1 and m TOR promoter regions had no obvious changes. Then we used q RT-PCR method to detect Elf5, Csn1s1, Srebp1, Ppar G, Glut1, Akt1, m TOR m RNA expressions after treating DCMECs with 5-Aza-dc, found that Elf5, Csn1s1, Srebp1, Ppar G, Glut1 mRNA expressions were significantly increased in 5-Aza-dc treatment group when compared with the blank control group, yet Akt1, m TOR mRNA expression levels had no significant changes. The experimental results indicated that mi R-29 s mainly through regulating DNA methylation pattern influenced the expressions of Elf5, Csn1s1, Srebp1, Ppar G, Glut1 genes which were important for the regulation of lactation, but the regulation mechanism of Akt1, m TOR still need further researches.(5)Compared with the blank control group, mi R-29 b expression was significantly increased in 5-Aza-dc treatment group, while the change of mi R-29 a and mi R-29 c expressions were not obvious. Bisulfite sequencing results showed that DNA methylation level of mi R-29 b promoter region was increased in 5-Aza-dc treatment group when compared with the blank control group. The results suggested that mi R-29 b expression was regulated by DNA methylation in DCMECs.(6)CASY and Ed U marker test results showed that mi R-29 s promoted cell viability and proliferation of DCMECs. The detection results of β-casein, lactose and TG secretion in DCMECs showed that mi R-29 a,-29 b and-29 c overexpression could promote the synthesis of milk protein, milk fat and lactose, silence of mi R-29 a,-29 b and-29 c inhibited the secretion of β-casein, lactose and TG, and 5-Aza-dc could part reversal or recover the inhibitory effects of mi R-29 s inhibitor on the synthesis of milk composition. These results suggested that the regulation of mi R-29 s in lactation of cow mammary gland was closely linked to DNA methylation.Taken together, mi R-29 a, mi R-29 b and mi R-29 c as members of the mammary lactating regulatory network, affected DNA methylation level of DCMECs through targeting the expressions of Dnmt3 a and-3b. The changes of DNA methylation level of lactation associated genes promoters caused aberrant genes expressions, and then influenced cell proliferation and milk composition secretion of DCMECs. The clarification of mi R-29 s involved in regulation of bovine mammary lactation by DNA methyl regulation of DCMECs provided new theory references for the future of milk quality optimization in dairy industry and mammary gland lactation biology researches.