Function Study Of Bovine MiR-204 On Proliferation,Differentiation And Apoptosis In Adipocyte

Adipose is a complex,extremely active tissue that is involved in the regulation of various biological processes in the body under the control of numerous transcription factors,such as energy balance,inflammation and immunity.In addition,once the function of adipose tissue is disordered,various related diseases will also appear in the body,such as obesity,cardiovascular disease,type II diabetes,etc.micro RNAs(miRNAs)are small single-stranded non-coding RNAs(about 22 nucleotides),they play biological functions by degrading the m RNA of a target gene or blocking its translation.Recent studies have found that miRNAs also play a key role in the body's adipose growth and development,but most of them focus on the study of obesity and related metabolic diseases,and there are relatively few studies on miRNAs' functions in bovine adipose.Therefore,this study firstly examined the RNA expression of miR-204 in heart,liver,spleen,lung,kidney,muscle and adipose tissue of fetal,calf and adult cattle,respectively.Then,miR-204 mature sequences of several species were analyzed by on-line analysis software,and their conservation among species was studied.Using qPCR,CCK8,Ed U,flow cytometry,Oil Red O staining,dual luciferase reporter system,Western blot and other methods to study the role of miR-204 in adipocyte growth and development,target genes and genetic variations.The main results of the study were indicated as follows:1.Spatio-temporal expression profiling of bovine miR-204 and the sequence conservation analysis among speciesmiRNA expression in heart,liver,spleen,lung,kidney,muscle and adipose tissues of Qinchuan fetal,calf and adult bovine was examined using qPCR,respectively.The expression of miR-204 in kidneys at three different stages was the highest,but only significant differences in liver and adipose tissues were found(P < 0.05),indicating that miR-204 may play an important role in adipocyte growth and development.Through bioinformatic software comparisons,miR-204 mature sequences were highly conserved among human,chicken,cattle and mice.2.The function of miR-204 in adipocytes2.1 The role of miR-204 in adipocytes proliferationmiR-204 mimics and NC(negative control)were transfected into 3T3-L1 cells for 24 h,respectively,then,cells were harvested.qPCR was used to detect the m RNA expression levels of adipocyte proliferation marker genes Cyclin D1,Cyclin E and CDK2.CCK8,Ed U,flow cytometry and Western blot results indicated that overexpressed miR-204 had no significant effect on adipocytes proliferation.2.2 The role of miR-204 in adipocytes differentiationqPCR was used to detect m RNA expression levels of differentiation key genes PPAR?,AP2 and C/EBP?.It was found that overexpression of miR-204 significantly promoted the expression of PPAR? and AP2 genes(P < 0.05).It was found that it can promote the formation of lipid droplets by Oil Red O staining.Western blot showed that over-expression of miR-204 can significantly promote the expression of FABP4 in protein level.The above results indicated that miR-204 promoted the differentiation of adipocytes.2.3 The role of miR-204 in adipocytes apoptosisThe m RNA expression of Caspase3,Bax and Bcl-2 was detected by qPCR,respectively.It was found that overexpression of miR-204 significantly increased the expression of Caspase3 and Bax genes,and significantly decreased the expression of Bcl-2 gene(P < 0.05).Flow cytometry showed that miR-204 significantly increased the number of cells in the apoptotic phase(P < 0.05).Western blot analysis showed that overexpression of miR-204 significantly increased the expression of Bax and significantly decreased the expression of Bcl-2 in protein level.The above results indicated that miR-204 promoted apoptosis of adipocytes.3.miR-204 target gene validationUsing Target Scan software to predict all target genes of miR-204,seven fat-related target genes were initially screened out(ELOVL6,TGFBR2,KLF12,ACSL4,DMTF1,TGFBRAF1 and PPARGC1A).The wild-type binding site and mutant-type binding site psi-Check2 vectors that bind to the 3?UTR of the 7 genes and the miR-204 seed region were constructed,respectively,and the change of corresponding fluorescence value was detected by the dual luciferase reporter system on 293 T cells.qPCR confirmed that the TGFBR2 and ELOVL6 were the target genes of miR-204.4.Genetic variations of miR-204 and related regulatory network genesUsing the method of DNA mixed pool,the sequences of miR-204 and its regulatorynetwork genes(SIRT1 and ADNCR)were scanned and genetic variations were found.Genotyping was performed on a total of 436 individuals of Qinchuan and Xianan cattle.Then,it was found that the genetic variations of miR-204 and its regulatory network genes : SIRT1 and ADNCR were all significantly correlated with the hucklebone width of Qinchuan cattle,besides,miR-204 was also significantly correlated with the body length of Qinchuan cattle(P < 0.05).In summary,this study revealed the positive role of miR-204 in the differentiation and apoptosis of adipocytes,screened and verified miR-204 target genes TGFBR2 and ELOVL6,and the genetic variations of miR-204 and its regulatory network genes: SIRT1 and ADNCR were also studied.The above results not only would lay a theoretical foundation for the further studying of the regulatory network of adipocyte growth and development and improving animal meat quality,but also would provide a theoretical reference for the treatment of obesity and other related diseases.