| Obesity is characterized by metabolic imbalance leading to adipocyte hypertrophy and hyperplasia,and the excess accumulation of adipose tissue.Adipocytes are the primary component of adipose tissue,and are formed through adipogenesis from precursor mesenchymal stem cells.While the major molecular pathways of adipogenesis are understood,little is known about the non-coding RNA networks involved in adipogenesis.Long non-coding RNAs are one of the most abundant types of non-coding RNA and have vital roles in differentiation.This study is aimed at investigating the role of LncRNA in differentiation process of adipocytes.By taking the specific LncRNA as the object,we studied its regulation of differentiation of adipocytes and specific molecular mechanisms.Methods:1.PGC1β-OT1 with differentiation expression in differentiation process of adipocytes was screened by using Microarray technology,the abundance changes of PGC1β-OT1 in the differentiation process of adipocytes were detected by RT-qPCR,5’RACE and 3’RACE techniques and Bioinformatics method were used to analyze the basic characteristics of PGC1β-OT1.2.Construct and design overexpression vector and siRNA corresponding to PGC1β-OT1.The empty vector and over expression vectors or negative control(NC)and siRNA was transfected into ST2 mouse bone marrow stromal cells respectively,and then followed by adipogenic differentiation.The process of differentiation of adipocytes and extent of differentiation of adipocytes of the transfected cells were evaluated by the methods such as oil red O staining,RT-qPCR,and Western Blotting,thus to determine impact of PGC1β-OT1 on differentiation of adipocytes in function access and function loss state.3.Bioinformatics predictions were used to search for miRNAs(miR-148a-3p)that could bind to PGC1β-OT1,construction of reporter gene vectors for PGC1β-OT1 and PGC1β-OT1 with miR-148a-3p binding site mutation,luciferase was used to verify whether there’s direct binding between PGC1β-OT1 and miR-148a-3p.4.RT-qPCR was used to detect the synchronization changes of the expression of PGC1β-OT1 and miR-148a-3p.RT-qPCR and Western Blotting methods were used to detect mRNA and protein expression of the miRNA target gene,which was to verify whether PGC1β-OT1 can affect the protein expression level of the target gene by binding to miR-148a-3p.5.Transfect PGC1β-OT1 or/and miR-148a-3p in ST2 cells to carry out the rescue experiment.Detected the adipocyte differentiation by oil red O staining,RT-qPCR and Western Blotting method,and verified whether PGC1β-OT1 could affect the differentiation of adipocytes through interaction with miR-148a-3p.Results:1.According to Microarray results,PGC1β-OT1’s expression in the process of MSC and ST2 cells adipogenesis were increased.5’RACE and 3’RACE determine that the full length of PGC1β-OT1 is 1759 bp,and the Coding potential calculator online software analysis shows protein cannot be encoded by PGC1β-OT1.2.After PGC1β-OT1 overexpression vector was transfected into ST2 cells,there’s declined extent for cells differentiated into mature adipocytes,and the mRNA and protein expression of adipocytes-specificity gene in cells decreased.After PGC1β-OT1’s endogenous knockdown siRNA transfected into ST2,the neutral lipids formed by cells increased,mRNA and protein expression of adipocytes-specificity genes increased accordingly.3.Bioinformatics analysis showed that there’s potential miR-148a-3p binding site on PGC1β-OT1.The dual luciferase experiment showed that miR-148a-3p can reduced the luciferase activity of wild type PGC1β-OT1.When the miR-148a-3p binding site on PGC1β-OT1 was mutated,experiments showed that there’s no significant decrease in luciferase activity.This indicates that there’s binding effect between PGC1β-OT1 and miR-148a-3p.4.RT-qPCR results showed that PGC1β-OT1 may affect miR-148a-3p expression to some extent.In addition,PGC1β-OT1 can increase the protein expression of the target gene KDM6 B of miR-148a-3p.However,when the miR-148a-3p binding site on PGC1β-OT1 was mutated,the protein expression of KDM6 B almost unchanged.This indicates that PGC1β-OT1 can regulate the protein level of KDM6 B through miR-148a-3p.5.miR-148a-3p can promote the differentiation of adipocytes,PGC1β-OT1 can inhibit the differentiation of adipocytes,enhance the endogenous expression level of miR-148a-3p can reverse the inhibition of adipogenic differentiation by PGC1β-OT1,while the overexpression of PGC1β-OT1 attenuated the effect of miR-148a-3p on promoting differentiation of adipocytes.This indicates that PGC1β-OT1 has a negative regulation of the differentiation of adipocytes through through binding and interacting with miR-148a-3p.Conclusion:1.PGC1β-OT1 is lnRNA molecules with a negative regulation of differentiating of adipocytes;2.PGC1β-OT1 can be used as endogenous competitive molecule of miR-148a-3p to regulate differentiation of adipocytes. |
