Polymorphism Of LncRNA MEG3 And Its Effect On Differentiation Of Precursors In Yanbian Yellow Cattle

This experiment was conducted to explore the correlation between MEG3 single nucleotide polymorphism of long non-coding RNA and growth traits of Yanbian Yellow cattle,the effects of MEG3 and miR-24-3p on adipogenic differentiation,proliferation and apoptosis of Yanbian Yellow cattle precursor adipocytes,and their interaction.MEG3 gene polymorphisms of 90 24-month-old Yanbian Yellow cattle were analyzed by direct DNA sequencing and high-resolution melting curve technology,and 12 growth traits including chest width,hip width and abdominal circumference were analyzed for association analysis.After interference and overexpression of Yanbian Yellow cattle precursor fat cells MEG3 and miR-24-3p,the effects of the two on adipogenic differentiation,proliferation and apoptosis of Yanbian Yellow cattle precursor fat cells were detected at molecular and cellular levels by real-time fluorescence quantitative PCR,oil red O staining,triglyceride content determination,CCK-8 method and flow cytometry.Finally,the targeting relationship between MEG3 and miR-24-3p was determined by double luciferase reporter gene test,all were repeated three times.The results show:1.The fifth exon of MEG3 contains two mutations,g.65745819 G>A and g.65745850 C>A,both of which have two alleles,with frequencies of G,A and C and A being 0.661 and 0.339 and 0.778 and 0.222 respectively.The frequencies of GG,GA,AA and CC,CA,AA were 0.422,0.478,0.100 and 0.611,0.333,0.056,respectively.Chi-square adaptation test showed that the two loci did not deviate from hardy-Weinberg equilibrium(P>0.05).The genetic homozygosity,heterozygosity,number of effective alleles and polymorphic information content of the two loci were0.552,0.448,1.812,0.348 and 0.286,0.346,1.528,0.286,respectively,which were moderately polymorphic(0.25<PIC<0.5),g.65745819 G>A locus.The breast width of GA genotype was significantly higher than that of AA genotype(P<0.05),and the hip width of AA genotype was significantly higher than that of GG and GA genotype(P<0.05).g.65745850 C>A locus,the abdominal circumference of AA genotype was significantly higher than that of CA genotype(P<0.05),but there were no significant differences among other genotypes(P>0.05).2.Compared with the meaningless siRNA transfection group(si-NC group),the expression levels of siRNA group with interference efficiency was transfected(si-48)marker genes FASN and LPL,C/EBPα,proliferation-related gene Cyclin D1 and PCNA were significantly increased(P<0.01),respectively(P<0.05).Apoptosis promoting gene BAX was significantly decreased(P<0.05),but other genes were not significantly affected,cell viability was significantly increased(P<0.01),lipid content,triglyceride content and apoptosis rate were not significantly affected.Compared with the over-NC group,PPARγ,C/EBPα,LPL,PCNA and BCL2 in the over-MEG3 group were significantly decreased(P<0.05),FASN was significantly decreased(P<0.01),BAX was significantly increased(P<0.05),and other genes were not significantly affected.Lipid content and triglyceride content were significantly decreased(P<0.05),cell viability was significantly decreased(P<0.01),apoptosis rate was significantly increased(P<0.05),and other indexes were not significantly affected.3.Compared with the mimics NC group,the miR-24-3p mimics group had significantly increased Cyclin D1 and BCL2(P<0.01),extremely significantly increased PCNA(P < 0.01),and significantly decreased MEG3(P<0.05),with no significant effect on other genes.Cell viability was significantly increased(P<0.01),but other indexes were not significantly affected.Compared with inhibitor NC group,Cyclin D1 and BCL2 in miR-24-3p inhibitor group were significantly decreased(P<0.05),PCNA was extremely significantly decreased(P<0.01),MEG3 was significantly increased(P<0.05),and other genes had no significant effects.Cell viability was significantly decreased(P<0.01),apoptosis rate was significantly increased(P<0.05),and other indicators were not significantly affected.4.Rluc/ fluc in the mimics NC+MEG3-wt group was significantly higher than that in the bta-miR-24-3p+MEG3-wt group(P<0.001).There was no significant difference between the mimics NC+MEG3-mut group and the bta-miR-24-3p+MEG3-mut group(P>0.05).In conclusion,lncRNA MEG3 g.65745819 G>A and g.65745850 C>A polymorphism are related to breast width,hip width and abdominal circumference of Yanbian Yellow cattle,respectively,and can be used as candidate genes for marker-assisted breeding of Yanbian Yellow cattle.lncRNA MEG3 can affect adipogenesis,proliferation and apoptosis of Yanbian Yellow cattle precursor adipocytes.miR-24-3p can affect the proliferation and apoptosis of Yanbian Yellow cattle preadipocytes.miR-24-3p and lncRNA MEG3 have targeted binding effect,which provides reference for the analysis of adipogenic differentiation,proliferation and apoptosis of bovine adipocytes.