Effects of Porcine MicroRNA-15b Precursor Mutation on Its Biological Processing and Precursor Adipocytes Differentiation
Based on the discovery of a C＞T mutation at the 58th base(+58C＞T)of the porcine microRNA-15b(miR-15b)precursor(pre-miR-15b)gene locus,this study aimed to explore the effects of this mutation on the biological processing of microRNA(miRNA)and the differentiation of porcine precursor adipocytes.Wild type(pmR-miR-15bW)and mutant type(pmR-miR-15bM)miR-15b overexpression vectors were con-structed and transfected into porcine primary precursor adipocytes respectively for miR-15b overexpression test.Then real-time fluorescent quantitative PCR was used to compare the expression levels of pre-miR-15b and miR-15b mature in the cells in 2 groups,and the influence of+58C＞T on the biological processing of miR-15b was determined at the cell level in vitro.Subsequently,porcine precursor adipocytes transfected with different genotypic miR-15b overexpression vectors(pmR-miR-15bW and pmR-miR-15bM)were induced to lipogenic differentiation.Real-time fluorescent quantitative PCR was used to compare the relative expression levels of the miR-15b target gene Forkhead box protein O1(FOXO1,which could inhibit adipocyte differentiation)and the genes related to adipogenic differentiation in the cells in 2 groups,and the effects of this mutation on the differ-entiation of porcine precursor adipocytes was determined by oil red O staining.The results showed that there was no significant difference in the expression level of primary transcript of miR-15b(pri-miR-15b)between 2 groups(P＞0.05),but the expression levels of pre-miR-15b,miR-15b and microRNA-16(miR-16)in mu-tant group was extremely significantly lower than that in wild group(P＜0.01).The relative expression level of FOXO1 in mutant group was extremely significantly higher than that in wild group before differentiation(P＜0.01),the relative expression levels of sterol regulatory element-binding protein-1c(SREBP-1c)and peroxi-some proliferator-activated receptor γ(PPARγ)in the adipogenic differentiation related genes were extremely significantly lower than those in wild group after differentiation(P＜0.01),and the relative expression level of adipocyte protein 2(aP2)was lower than that in wild group(P＞0.05).The results of oil red O staining showed that the content of lipid droplets in mutant group was significantly lower than that in wild group.In con-clusion,+58C＞T can hinder the biological processing from pri-miR-15b to pre-miR-15b,resulting in a de-crease in the expression of miR-15b mature and an increase in the relative expression of its target gene FOXO1,thus inhibiting the differentiation of porcine precursor adipocytes.[Chinese Journal of Animal Nutrition,2024,36(3):1892-1903]
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