Abstract: 【Objective】 The aim of this study was to investigate the function and mechanism of miR-140-5p in the differentiation of preadipocytes 3T3-L1.【Method】 When the convergence degree of 3T3-L1 cells reached 100%,the adipogenic differentiation was induced.The cells of differentiation day ―1 (the day before differentiation induction),days 0,1,2,3,5 and 7 were collected,and the expression of miR-140-5p was detected by Real-time quantitative PCR.miR-140-5p mimics and NC were transfected into 3T3-L1 cells to induce adipogenic differentiation.Oil red O staining was used to observe the formation of lipid droplets.Real-time quantitative PCR was used to detect the relative expressions of adipogenic marker genes CAAT enhancer binding protein β (C/EBPβ),CAAT enhancer binding protein δ (C/EBPδ) and peroxisome proliferator-activated receptor γ (PPARγ).The target gene of miR-140-5p was predicted by miRandn and TargetScan online websites,the sequence conservation of the binding site sequence of P300/CBP-related factor (PCAF) 3'-UTR sequence and miR-140-5p in different species such as mice and humans was analyzed by comparing the sequence differences.miR-140-5p mimics,inhibitor and NC were transfected into 3T3-L1 cells and the cells were induced to differentiate into adipocytes.The relative expression of miR-140-5p and PCAF was detected by Real-time quantitative PCR,and the protein level of PCAF was detected by Western blotting.PEGFP-N1-PCAF and PEGFP-N1 were transfected into 3T3-L1 cells.Oil red O staining was used to observe the formation of lipid droplets.Real-time quantitative PCR was used to detect the relative expression of PCAF,C/EBPδ and PPARγ genes.Western blotting was used to detect the expression levels of C/EBPβ,C/EBPδ and PPARγ proteins.Three PCAF siRNAs (siRNA1,siRNA2 and siRNA3) and siRNA NC were transfected into 3T3-L1 cells.The protein level of PCAF was detected by Western blotting to screen the best PCAF siRNA.Optimal PCAF siRNA and siRNA NC were transfected into 3T3-L1 cells,and the formation of lipid droplets was observed by oil red O staining.The relative expression of PCAF,C/EBPβ,C/EBPδ and PPARγ genes were detected by Real-time quantitative PCR,and the expression of C/EBPβ and PPARγ protein were detected by Western blotting.miR-140-5p mimics,NC,PGL0-PCAF 3'-UTR vector and PGLO empty vector were co-transfected into 293T cells,respectively.The targeting relationship between miR-140-5p and PCAF gene was detected by double luciferase report test.【Result】 In the process of inducing adipogenic differentiation of 3T3-L1 cells,compared with the day before inducing differentiation,the relative expression of miR-140-5p was extremely significantly increased on the day 1 and day 2 of adipogenic differentiation (P＜0.01) and was significantly increased on the day 3 day of differentiation (P＜0.05).Compared with NC group,the number of lipid droplets in miR-140-5p mimics group was increased significantly,and the relative expression of lipid marker genes C/EBPδ and PPARγ in miR-140-5p mimics group were extremely increased significantly (P＜0.01).The result of target gene prediction showed that miR-140-5p had the expected binding site with PCAF.The results of conservation analysis showed that the binding site sequence of target gene PCAF was highly conserved among different species.Compared with NC group,the relative expressions of miR-140-5p and PCAF gene in mimics group were extremely significantly increased (P＜0.01),while those in inhibitor group were extremely significantly decreased (P＜0.01),PCAF was significantly decreased (P＜0.05),and the expression of PCAF protein was significantly increased (P＜0.05).Compared with PEGFP-N1 group,the number of lipid droplets in PEGFP-N1-PCAF group was increased,and the relative expression levels of PCAF and PPARγ genes and the levels of C/EBPβ,C/EBPδ protein were extremely significantly increased (P＜0.01),PPARγ protein was significantly increased (P＜0.05).PCAF siRNA1,siRNA2 and siRNA3 all extremely significantly inhibited the expression of PCAF protein (P＜0.01),and siRNA3 had the most significant effect,so siRNA3 was selected for the follow-up test.Compared with NC group,the number of lipid droplets in 3T3-L1 cells in PCAF siRNA3 group was less,the expression of PCAF,C/EBPβ,C/EBPδ and PPARγ genes and the level of C/EBPβ protein were extremely decreased significantly (P＜0.01).The results of double luciferase report test showed that there was no target relationship between miR-140-5p and PCAF gene.【Conclusion】 The experiment proved that the expression of endogenous miR-140-5p was increased during the differentiation of 3T3-L1 cells.miR-140-5p might promote the adipogenic differentiation of 3T3-L1 cells by indirectly up-regulating the expression of PCAF gene.

Key words: miR140-5p; P300/CBP-related factors; 3T3-L1 cells; adipogenic differentiation