Effects Of Methylation Of Pig UCP3 Gene Promoter On Gene Expression And Lean Meat Percentage

The lean meat percentage is a critical economic trait in pigs,significantly impacting their overall value.We previously discovered a significant difference in UCP3 gene expression between high-lean(DLG)and low-lean(DLD)Dulu pigs,indicating a potential influence of this gene on pig muscle growth and development.Despite its importance,there is limited research on the role of the UCP3 gene in porcine skeletal muscle development.Thus,our study compared and analyzed the promoter activity of the UCP3 gene between the DLG and DLD groups,identifying the core promoter region of the UCP3 gene in Dulu pigs.Additionally,we investigated the relationship between methylation of the UCP3 gene core promoter region and gene expression and lean meat percentage.We also studied the effect of UCP3 gene expression on the proliferation and differentiation of porcine skeletal muscle cells by interfering with and overexpressing the UCP3 gene.Below are key research findings:1.The dual luciferase reporter gene experiment results showed that both the DLG and DLD groups had active UCP3 gene promoters.However,the DLG group displayed significantly higher promoter activity than the DLD group(P<0.05).Notably,the core promoter of the UCP3 gene was located within the-250 ～ +90 bp region.2.Real-time fluorescent quantitative PCR analysis revealed that the expression level of the UCP3 gene was significantly higher in the DLG group than in the DLD group(P<0.01).The UCP3 gene in Dulu pigs was found to have a 153 bp core promoter region that contained a CpG island with 12 CpG sites.BSP sequencing indicated that the overall methylation level of the DLG group was lower than that of the DLD group,but the methylation level was not significantly correlated with gene expression and lean meat percentage(P>0.05).However,the methylation level of CpG-3,located in the transcription factor Sp1 binding site,showed a highly significant negative correlation with gene expression(P<0.01).Furthermore,the core promoter activity of the DLG group was significantly higher than that of the DLD group(P<0.01).This suggests that the methylation of the UCP3 gene promoter could potentially interfere with the binding of Sp1 to the promoter,leading to a decrease in the activity of the UCP3 core promoter and inhibition of the expression of the UCP3 gene.3.After overexpressing the UCP3 gene in porcine skeletal muscle cells,cell viability decreased while the expression levels of cell proliferation marker genes such as Pax3,PCNA,and CCND1 were significantly increased(P<0.01).At the same time,the expression levels of cell apoptosis marker genes Bax and Caspase3 decreased considerably(P<0.01).Conversely,interfering with the UCP3 gene resulted in the opposite effect.Moreover,myogenic induction was performed after overexpressing and interfering with the UCP3 gene in porcine skeletal muscle cells,which showed that overexpression of the UCP3 gene significantly increased the expression levels of muscle cell differentiationrelated genes such as MyoD,MyoG,and Myf5(P<0.01).Conversely,UCP3 gene interference significantly decreased the expression levels of MyoD,MyoG,and Myf3(P<0.01).These results indicate that the UCP3 gene plays a crucial role in promoting the proliferation and differentiation of porcine skeletal muscle cells.In conclusion,the porcine UCP3 gene regulates the proliferation and differentiation of porcine skeletal muscle cells by regulating its gene expression through promoter methylation,which affects the leanness of pig carcasses.In summary,the porcine UCP3 gene regulates its gene expression through promoter methylation,regulates the proliferation and differentiation of porcine skeletal muscle cells,and thus affects the lean meat percentage of the porcine carcass.