Effect of miR-455-3p on Milk Fat Metabolism in Chemically Induced Mammary Epithelial Cells in Dairy Goats
[Objective]This study was aimed to explore the effect of miR-455-3p on milk fat metabolism in chemically induced mammary epithelial cells(CiMECs)of dairy goats,so as to provide a theoretical basis for the study of microRNA(miRNA)regulation of mammary gland function in goats.[Method]Goat fibroblasts(GEFs)were induced by a single small molecule compound RepSox(TGFβR-1/ALK5 inhibitor),and the morphological changes of the cells were observed after 8 days of induction,and immunofluorescence staining and oil red O staining,which were specific markers,were used to identify whether the mammary epithelial cells were successfully induced.The overexpression vectors(miR-455-3p mimics and miR-455-3p mimics-NC)and interference vectors(miR-455-3p inhibitors and miR-455-3p inhibitors-NC)for miR-455-3p were transfected into CiMECs using liposomes,which were analyzed by the cell proliferation and toxicity assay(CCK-8)kit,triglyceride assay kit and Real-time quantitative PCR to detect the proliferation rate,triglyceride secretion and the expression of milk fat metabolism genes of miR-455-3p for CiMECs.[Result]GEFs induced the formation of insular,multinucleated and cobblestone cell shapes similar to goat mammary epithelial cells after 8 days of induction.The immunofluorescence results showed that CiMECs could express epithelial-specific marker antigens,including cytokeratin 14(CK14)and CK18.The results of oil-red O staining showed that CiMECs possessed the function of secretion of lipid droplets.The CiMECs transfection results showed that compared with control group,miR-455-3p mimics extremely significantly promoted the expression of miR-455-3p(P＜0.01),and miR-455-3p inhibitors significantly inhibited the expression of miR-455-3p expression(P＜0.05).The results of CCK-8 and triglyceride assay showed that compared with control group,the cell proliferation rate and triglyceride secretion were extremely significantly decreased in miR-455-3p mimics group(P＜0.01),and the cell proliferation rate and triglyceride secretion were significantly increased in miR-455-3p inhibitors group(P＜0.05).Real-time quantitative PCR results showed that compared with control group,the expression of HADHB gene was extremely significantly decreased in CiMECs after induction(P＜0.01),was extremely significantly increased in miR-455-3p mimics group(P＜0.01),and was significantly decreased in miR-455-3p inhibitors group(P＜0.05).[Conclusion]The inhibition of miR-455-3p promoted CiMECs proliferation and reduced the expression of the fat metabolism gene HADHB in dairy goats,thereby increasing the fatty acid content of goat milk.The results could provide a basis for the subsequent use of miRNA to improve the milk quality in goats.
国家科技期刊平台
NSTL
万方数据
维普
