The Role And Mechanism Of MiR-99b-5p In The Proliferation And Differentiation Of 3T3-L1 Preadipocytes

The number and distribution of adipocytes directly affect the quality of livestock meat products.The analysis of adipogenesis mechanism is the basis for improving meat quality.The production of adipocytes mainly includes two processes: proliferation and differentiation,which are regulated by a variety of transcription factors,growth factors,cytoinflammatory factors,signal pathways,and non-coding RNAs including miRNAs.miRNA is a short ncRNA(non-coding RNA,20-22 nt)that regulates gene expression at the post-transcriptional level,and can participate in various basic biological processes.Studies have shown that miR-99b-5p is differentially expressed in the subcutaneous adipose tissue of piglets and adult pigs(P<0.05).Therefore,this study predicts that miR-99b-5p may play an important role in the lipid deposition process of adipose tissue effect.This thesis takes 3T3-L1 preadipocytes as the research object,using real-time fluorescent quantitative PCR,western blotting,EdU staining,flow cytometry,CCK8 test analysis,oil red O and Bodipy staining,proteomics sequencing,and dual fluorescence The effects of miR-99b-5p on the proliferation and differentiation of preadipocytes and their mechanisms have been studied using techniques such as prime enzyme report analysis.The main results are as follows:1.Tissue expression profile analysis showed that miR-99b-5p expressed the highest in mouse lungs and moderately expressed in adipose tissue;the expression of miR-99b-5p continued rise during the proliferation of 3T3-L1 preadipocytes,but in the process of differentiation,it first rises and then falls.These results indicate that miR-99b-5p may play a role in the proliferation and differentiation of preadipocytes.2.Overexpression of miR-99b-5p significantly increases the number of positive cells and cell viability of EdU staining and CCK8,as well as the number of cells in S phase,and increases the m RNA expression of Cyclin D,Cyclin E(P<0.01)and CDK4(P<0.05),inhibit the expression of p21(P<0.001);at the protein level,the expression of Cyclin D,Cyclin E(P<0.05),and CDK4(P<0.05)increased significantly,and the expression of p21 decreased significantly(P<0.05).On the contrary,interference miR-99b-5p shows the opposite result to overexpression.The above results shows that miR-99b-5p promoted cell proliferation.3.Overexpression of miR-99b-5p significantly reduces the number of lipid droplets in Oil Red O staining and Bodipy staining,and reduces PPAR?(P<0.01),C/EBP?(P<0.001),Ap2,ATGL(P<0.01),HSL(P<0.001)m RNA expression,at the protein level,the expression of SREBP1,PPAR?,Ap2,HSL(P<0.05)also decreased significantly.In contrast,interference with miR-99b-5p shows the opposite result to overexpression.The above results indicate that miR-99b-5p can inhibit cell differentiation.4.miR-99b-5p inhibits the differentiation of 3T3-L1 preadipocytes through SCD1 and Lpin1.In summary,miR-99b-5p can promote the proliferation of preadipocytes and inhibit the differentiation process of preadipocytes by targeting SCD1 and Lpin1.This study provides a theoretical basis for improving the quality of livestock meat.