Effect And Effect Mechanism Of MiR-9-5p On Mammary Epithelial Cells Of Dairy Cows

MicroRNAs(miRNAs)are the RNA molecules which do not encode proteins and are composed of 21-25 nucleotides.They are the key regulators of different biological development processes.A large number of studies have shown that micro RNAs are in volved in the regulation of cell proliferation,differentiation and apoptosis,and are closely related to glucose metabolism,lipid metabolism,protein metabolism,insulin resistance and organ development and other life activities.MicroRNAs are mainly involved in various cellular activities in animal cells by regulating gene expression at the post-transcriptional level.MicroRNAs bind to the non-coding3’UTR region of target gene m RNA through some complementary base sequences,blocking the translation process of target gene m RNA or directly cracking the m RNA to regulate the expression of target gene protein.Studies have shown that miR-9-5p performs different functions in various tissues and cells.Previous studies in our laboratory have found that miR-9-5p also exists in dairy cow mammary epithelial cells(DCMECs).At present,the function and mechanism of miR-9-5p in DCMECs are not clear and have not been reported.Therefore,the role and mechanism of miR-9-5p in DCMECs were studied in this study.Firstly,we used bioinformatics methods to predict and screen the target genes of miR-9-5p in DCMECs.Three genes related to cell metabolism were screened,Adcy5,Colec12 and Tnfrsf6 b.The 3’UTR primers for Adcy5,Colec12 and Tnfrsf6 b were designed using Primer5.0 software,and the wild-type 3’UTR fragments containing miR-9-5p targetting binding sites were obtained by PCR.The target mutant 3’UTR fragment was synthesized.The wild-type and mutant-type 3’UTR fragments of Adcy5,Colec12 and Tnfrsf6 b genes were constructed into recombinant plasmids with psi CHECK2 vector respectively.After plasmid extraction,cell transfection and double luciferase activity detection,the targeting relationship of miR-9-5p with Adcy5,Colec12 and Tnfrsf6 b was verified.The experimental results showed that miR-9-5p had a significant targeting relationship with the 3’UTR of Tnfrsf66 b and Adcy5.Then,after overexpressing or inhibiting miR-9-5p in DCMECs by liposome transfection,m RNA expression levels of A dcy5,Colec12 and Tnfrsf6 b genes were detected.The experimental results showed that the m RNA expression levels of A dcy5 and Tnfrsf6b were significantly decreased after the overexpression of miR-9-5p.It was further demonstrated that miR-9-5p had a targeting relationship with Tnfrsf6 b and Adcy5,and the targeting relationship with Tnfrsf6 b was more significant,while miR-9-5p had no insignificant targeting relationship with Colec12.To explore the effects of miR-9-5p on the lactation function and proliferation of DCMECs,the mammary epithelial cells of healthy Chinese Holstein cows were selected as the experimental cell model.miR-9-5p mimics and miR-9-5p inhibitor were transfected into DCMECs by liposome transfection.Fluorescence quantitative PCR and Western blotting were used to detect and analyze the expression changes of target genes and genes related to lactation pathway on the m RNA and protein levels of target genes.The results showed that the overexpression of miR-9-5p significantly inhibited the expression of TNFRSF6 b and the expre ssion of lactation related proteins including AKT,p-AKT,m TOR,p-m TOR,STAT5,p-STAT5,S6 K and p-S6 K,as well as SREBP1 and β-casein in the DCMECs.At the same time,the changes of triglyceride synthesis was detected by triglyceride detected kit in DCMECs after overexpression or inhibition of miR-9-5p,and the experimental results showed that the overexpression of miR-9-5p significantly inhibited the synthesis of triglycerides.In order to explore whether miR-9-5p has an effect on cell proliferation,MTT assay was used to detect the effect of miR-9-5p on cell proliferation,and flow cytometry was used to detect the effect of miR-9-5p on cell apoptosis.The results showed that the overexpression of miR-9-5p significantly inhibited cell proliferation and promoted cell apoptosis.The mechanism of miR-9-5p in DCMECs was further explored.Firstly,si RNA interference technology was used to reduce the expression of Tnfrsf6 b in DCMECs.Western blotting was used to detect the expression of TNFRSF6 b protein and lactation pathway protein.The results showed that the expression levels of AKT,p-AKT and β-casein were significantly decreased after TNFRSF6 b expression was down-regulated.The expressions of TNFRSF6 b,AKT,p-AKT andβ-casein were significantly increased after co-transfection with si TNFRSF6 b and miR-9-5p inhibitor,compared with si TNFRSF6 b group.These results indicated that miR-9-5p indeed acted on AKT and β-casein through the target gene TNFRSF6 b,and finally affected the secretion function and cell proliferation of DCMECs.According to the results of bioinformatics prediction and transcriptome sequencing in our previous experiments,we also found that there was a targeting relationship between miR-9-5p and LOC783657 in DCMECs.This study also verified whether there is a targeting relationship between LOC783657 and miR-9-5p through the experimental process of vector construction,cell transfection and dual luciferase activity detection.The results showed that LOC783657 had a targeting relationship with miR-9-5p.