Regulation Of MiR-20a-5p In Adipocyte Differentiation And Its Effect On Obesity Mice

Objective microRNAs(miRNAs)are a class of short(about 22nt)non-coding RNA molecules that can regulate gene expression primarily through post-transcriptional repression.Reciprocal relationship usually exists between osteoblastogenesis and adipogenesis,with factors stimulating one of these processes at the same time inhibiting the other.Several types of microRNAs(miRNAs)have recently been defined as important regulators in adipocyte differentiation,the role of other miRNAs in the processes and the mechanisms involved remain to be explored.The effects of miR-20a-5p on adipocyte differentiation and obesity were studied and the mechanisms were exposed in the research.Methods 1.miR-20a-5p expression was quantified in primary cultured marrow stromal cells,ST2 stromal cells after adipogenic treatment and inguinal fat isolated in ob/ob mice.We studied the effects of miR-20a-5p on adipogenic differentiation by counting the numbers of adipocytes,analysing the m RNA and protein expression levels of adipocyte-specific transcription factors and marker genes.2.Furthermore,we performed bioinformatics prediction of miRNA targets and in order to test whether the potential target genes are really targeted by miR-20a-5p,the 3'UTR fragments of the m RNAs containing the target sequences were cloned into the luciferase reporter vector.Then the luciferase activity of the 3'UTR constructs were assayed.The role of the validated target gene in controlling differentiation was investigated by carrying out gain and loss function studies in stromal cells.3.4-week-old male C57BL/6 mice were fed with high fat diet to construct obesity model.The body weight of mice in each group was recorded and mice were subcutaneously injected miR-20a-5p sponge lentivirus.The glucose,triglyceride,free fatty acid and total cholesterol levels in serum was detected.Moreover,Total RNA was extracted from adipose tissue in each group and expression levels of adipogenic factors were quantified by q PCR.Results 1.miR-20a-5p was induced in primary cultured mouse marrow stromal cells and stromal cell line ST2 after adipogenic treatment.Moreover,the expression of miR-20a-5p was increased in the genetically obese ob/ob mice.2.By transfecting the miR-20a-5p mimics into primary cultured marrow stromal cells,ST2 and C3H10T1/2,we found that over-expression of miR-20a-5p could promote lipid droplets accumulation and enhance the expression of the key adipogenic transcription factors and adipocyte marker gene.Conversely,miR-20a-5p inhibitor reduced lipid droplets accumulation and decreased the expression of the key adipogenic factors.Moreover,we construct the over-expressed miR-20a-5p lentivirus and primary cultured mouse marrow stromal cells were infected by the virus.The lentivirus duplicate the adipocyte promoting program of the the mimics.3.Tgfbr2,Kdm6 b and Nfic were predicted to be the potential targets of miR-20a-5p.Then we carried out Kdm6 b and Nfic gain and loss function studies using ST2 and C3H10T1/2 cells.We found that Kdm6 b suppress adipocyte differentiation and Nfic balances adipogenic and osteogenic differentiation from progenitor cells throngh controlling canonical Wnt signaling.Promoter studies and Ch IP assay revealed that NFIC directly binds to the promoter of miR-20a-5p.We revealed that miR-20a-5p regulates adipocyte differentiation of mesenchymal stem cells through a NFIC/miR-20a-5p regulatory feedback loop.4.In vivo studies showed that miR-20a-5p sponge lentivirus treatment reduced the gain of body weight and fat,decreased the serum level of triglycerides and free fatty acid,and improved glucose tolerance and insulin resistance in HFD-fed mice.miR-20a-5p sponge lentivirus blocked induction of adipogenic factors in adipose tissue in HFD-fed mice.Conclusion 1.miR-20a-5p was highly expressed in differentiated primary cultured mouse marrow stromal cells and stromal cell line ST2 and also was expressed higher in obesity subjects than in normal.2.miR-20a-5p favors adipocyte differentiation.3.Tgfbr2,Kdm6 b and Nfic are direct targets of miR-20a-5p and restrain adipogenesis.miR-20a-5p regulates adipocyte differentiation by directly targeting Tgfbr2,Kdm6 b and Nfic.Moreover,NFIC directly binds to the promoter of miR-20a-5p.miR-20a-5p regulates adipocyte differentiation of mesenchymal stem cells through a NFIC/miR-20a-5p regulatory feedback loop.4.In vivo study showed that miR-20a-5p sponge lentivirus inhibits high fat diet induced obesity in mice by repressing adipogenesis in adipose tissue.