Abstract: 【Objective】 The aim of this study was to investigate the role and mechanism of miR-181a and miR-181d-5p in the differentiation of primary precursor adipocytes in Rongchang pigs.【Method】 The sequence conservation of miR-181a and miR-181d-5p among different species of pig, human, rat and mouse was analyzed by comparing seed sequence differences.miR-181a and miR-181d-5p expression in pig muscle and subcutaneous adipose tissue was detected by Real-time quantitative PCR.miRandn and TargetScan online softwares were used to predict miR-181a and miR-181d-5p target genes with GO and KEGG enrichment analysis.The free energy of miR-181a and miR-181d-5p binding to their target genes was predicted using RNAhybrid online software.Primary precursor adipocytes were isolated from subcutaneous adipose tissue on the back of 7-day-old male Rongchang pigs.Wild-type and mutant vectors of miR-181a and miR-181d-5p target genes were designed and cotransfected with the corresponding miRNAs in the precursor adipocytes.A dual luciferase reporter gene assay was performed to verify the targeting relationship between miRNA and target genes, and the binding of miR-181a and miR-181d-5p to their target genes was measured.miR-181a and miR-181d-5p mimics (miR-181a mimics, miR-181d-5p mimics), inhibitor (miR-181a inhibitor, miR-181d-5p inhibitor), negative control (NC) and interfering siRNAs of their target genes (Gene-siRNA) were constructed, transfected the cells.The transfected cells were induced to differentiate 6 h later.After 6 d, gene expression and triglyceride production were determined by Real-time quantitative PCR, oil red O staining was used to detect triglyceride in the treated cells, respectively.【Result】 The results of conservativeness analysis showed that miR-181a and miR-181d-5p were highly sequence conserved among pig, human, rat and mouse.miR-181a and miR-181d-5p were quantified in different tissues of pigs, which showed that miR-181a and miR-181d-5p were specifically highly expressed in porcine adipose tissues.The results of target gene prediction showed that the target genes of miR-181a and miR-181d-5p were mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) and cyclic adenylate response element (CREB1), respectively.GO and KEGG functional enrichment analysis revealed that the target genes of miR-181a and miR-181d-5p could inhibit triglyceride production and regulate adipocyte differentiation, and miR-181a and miR-181d-5p.The free binding energy of both miR-181a and miR-181d-5p with their target genes was less than -87.8 kJ/mol.The results of dual luciferase reporter gene assay showed that mimics transfected with miR-181a and miR-181d-5p could extremely significantly inhibit luciferase activity in the target gene 3'-UTR wild-type dual luciferase reporter vector (P<0.01), but did not affect the target gene mutant type.However, it did not affect the luciferase activity in the target gene mutant dual luciferase reporter vector.The induction of differentiation showed that miR-181a was significantly higher at day 4 (P<0.05) and reached a extremely significant difference at day 6 (P<0.01) compared with undifferentiated adipocytes (0 d).miR-181a and miR-181d-5p, as well as GPD2 and CREB1 genes, were extremely significantly higher at days 4 and 6 of differentiation (P<0.01).miR-181d-5p cell transfection assays both yielded similar results.miR-181a and miR-181d-5p expressions were both extremely significantly increased in mimics group (P<0.01) and miR-181a and miR-181d-5p expressions were both extremely significantly decreased in inhibitor group (P<0.01) compared with the NC group.The expressions of GPD2 and CREB1 genes were extremely significantly decreased in both mimics and siRNA groups (P<0.01), and the expressions of GPD2 and CREB1 genes were extremely significantly increased in inhibitor group (P<0.01).The relative expressions of PPARγ and C/EBPα genes were significantly or extremely significantly increased in both mimics and siRNA groups (P<0.05 or P<0.01), and the relative amounts of PPARγ and C/EBPα genes were significantly decreased in inhibitor group (P<0.05).The results of oil red O staining showed that the number of cellular lipid droplets increased in mimics and siRNA groups and decreased in inhibitor group compared with NC group.【Conclusion】 miR-181a and miR-181d-5p could target and bind GPD2 and CREB1 genes 3'-UTR region sequences, respectively, reduce the expression of GPD2 and CREB1 genes, and promote lipogenic differentiation of porcine precursor adipocytes.

Key words: Rongchang pigs; miR-181a; miR-181d-5p; adipose cell