Identification Of MiRNA In Muscle And Adipose Tissue In Domestic Pigs And Functional Studies Of Mir181a And Mir-181d-5p

Muscle and fat,as one of the most important economic traits of pigs,are the focus of pig genetics research and breeding work,and their molecular regulation mechanism has always been regarded as an important research content in pig molecular breeding.There are also obvious differences in the production traits of different breeds of pigs.Micro RNA(miRNA)is an endogenous single-stranded non-coding RNA with the function of regulating gene expression.The length can reach 20-22 nt.Elucidating porcine micro RNAs is critical for explaining phenotypic differences across species and for understanding the genetic architecture of complex traits.In this study,miRNA were extracted from skeletal muscle and adipose tissue,and differences in miRNA expression levels were compared between one western breed pig(Yorkshire)and seven Chinese native breed pigs.Then,according to the sequencing results,it was found that miR-181 a and miR-181d-5p were specifically highly expressed in adipose tissue.Taking the subcutaneous adipose tissue and cells of the back of Chenghua pig as the research object,the bioinformatics analysis method was used to predict miR-181 a and miR-181d-The target genes of 5p,GPD2 and CREB1,were used to investigate the regulatory functions of miR-181 a and miR-181d-5p on porcine adipocyte differentiation by real-time quantitative PCR,dual-luciferase reporter assay and cell transfection assay.The main research results are as follows:(1)The expression of 172 known miRNA and 181 novel miRNA was detected.Differential expression analysis found that there were 92 and 12 differentially expressed miRNA in adipose and muscle tissues,respectively.The differential miRNA and target m RNAs of adipose and muscle tissue were obtained through the bioinformatics database,and the study found that 302 pairs of miRNA and DE genes were negatively correlated in adipose tissue,and 143 pairs of miRNA and DE genes were negatively correlated in muscle tissue.(2)By studying the miRNA that regulate the phenotypic traits of Chinese breed pigs and foreign breed Yorkshire pigs,it was found that 520 pairs of miRNA in adipose tissue were significantly related to phenotypic traits,and 39 pairs in muscle tissue were associated with phenotypic traits.Significant correlation.(3)The study found that miR-181 a and miR-181d-5p are highly related to the function of fat development,and by comparing the seed sequences of miRNA of various species,it was found that there is a high degree of sequence conservation among species,which can be used as key research objects.Experiments show that the expression levels of miR-181 a and miR-181d-5p in pig back fat are significantly higher than those in muscle tissue(4)The target genes related to adipogenic differentiation were predicted through the database,combined with the results of the binding free energy analysis of miRNA and target genes and the double fluorescent reporter gene experiment,and finally determined that miR-181 a and miR-181d-5p can be targeted and bound respectively.GDP2 and CREB1 3’UTR region sequences,inhibit the expression of GDP2 and CREB1 genes(P<0.01).(5)The expression results of miR-181 a,miR-181d-5p and their target genes during the differentiation of porcine adipocytes showed that compared with undifferentiated adipocytes,the expression of miR-181 a increased significantly after induction of differentiation(P<0.05),to A very significant difference was reached on the 6th day(P<0.01);the expression of miR-181d-5p was extremely significantly increased after the2 nd day compared with the undifferentiated adipocytes(P<0.01);the target genes GPD2 and CREB1 were differentiated on the 4th day The subsequent expression levels were significantly decreased compared with undifferentiated adipocytes(P<0.01).(6)The expression patterns and cell phenotypes of miR-181 a,miR-181d-5p,GPD2,CREB1,PPARγ and C/EBPα during the induced differentiation of subcutaneous preadipocytes in Chenghua pigs showed that miR-181 a and miR-181d-5p could inhibit the expression of GPD2 and CREB1 genes,enhance the differentiation of adipocytes,and promote the differentiation of adipocytes cell and promote lipogenesis.The results of oil red O staining showed that cells transfected with miRNA mimics and si RNAs corresponding to the target genes had more and denser lipid droplets compared with the negative control,while cells transfected with miRNA inhibitors had fewer lipid droplets compared with the negative control.In summary,this study provides a comprehensive analysis of miRNA-m RNA expression profiles in muscle and adipose tissue of seven native Chinese breeds and one western commercial breed.These two groups of pigs with different phenotypic differences in meat quality and yield were found to have different miRNA expression profiles between them.And two interaction networks were identified,miRNA-m RNA regulatory network and miRNA-phenotypic trait network,and two miRNA were selected for functional validation based on sequencing data.miR-181 a and miR-181d-5p bound target genes GPD2 and CREB1 3′UTR region sequences,respectively,to reduce target gene expression and promote adipocyte lipogenesis differentiation.