猪微小RNA⁃15b前体突变对其生物学加工过程及前体脂肪细胞分化的影响

Effects of Porcine MicroRNA-15b Precursor Mutation on Its Biological Processing and Precursor Adipocytes Differentiation

基于在猪微小RNA⁃15b(miR⁃15b)前体(pre⁃miR⁃15b)基因位点第58位碱基上发现了1个C>T突变(+58C>T),本试验旨在探究此突变对微小RNA(miRNA)生物学加工过程以及对猪前体脂肪细胞分化的影响。试验通过构建野生型(pmR⁃miR⁃15bW)和突变型(pmR⁃miR⁃15bM)miR⁃15b过表达载体,分别转染猪原代前体脂肪细胞进行miR⁃15b的过表达试验,然后利用实时荧光定量PCR对比2组细胞pre⁃miR⁃15b和miR⁃15b成熟体的表达量,在体外细胞水平明确+58C>T对miR⁃15b生物学加工过程的影响;随后,诱导转染了不同基因型miR⁃15b过表达载体(pmR⁃miR⁃15bW和pmR⁃miR⁃15bM)的猪前体脂肪细胞成脂分化,利用实时荧光定量PCR对比2组细胞miR⁃15b靶基因叉头框蛋白O1(FOXO1)(可抑制脂肪细胞分化)以及成脂分化相关基因的相对表达量,同时结合油红O染色,明确此突变对猪前体脂肪细胞分化的影响。结果表明:2组细胞miR⁃15b初级转录本(pri⁃miR⁃15b)的表达量没有显著差异(P>0.05),而突变组pre⁃miR⁃15b、miR⁃15b和微小RNA⁃16(miR⁃16)表达量极显著低于野生组(P<0.01);突变组分化前FOXO1相对表达量极显著高于野生组(P<0.01),分化后成脂分化相关基因固醇调节元件结合蛋白-1c(SREBP⁃1c)和过氧化物酶体增殖物激活受体γ(PPARγ)相对表达量极显著低于野生组(P<0.01),脂肪细胞蛋白2(aP2)相对表达量低于野生组(P>0.05);油红O染色结果表明突变组细胞脂滴含量明显低于野生组。综上可知,+58C>T阻碍了pri⁃miR⁃15b到pre⁃miR⁃15b的生物学加工过程,导致miR⁃15b成熟体表达量下降,其靶基因FOXO1相对表达量增高,从而抑制猪前体脂肪细胞分化。

Based on the discovery of a C>T mutation at the 58th base(+58C>T)of the porcine microRNA-15b(miR15b)precursor(premiR-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(pmRmiR-15bW)and mutant type(pmRmiR-15bM)miR15b overexpression vectors were constructed and transfected into porcine primary precursor adipocytes respectively for miR-15b overexpression test.Then realtime fluorescent quantitative PCR was used to compare the expression levels of premiR-15b and miR-15b mature in the cells in 2 groups,and the influence of+58C>T on the biological processing of miR15b was determined at the cell level in vitro.Subsequently,porcine precursor adipocytes transfected with different genotypic miR-15b overexpression vectors(pmRmiR-15bW and pmRmiR-15bM)were induced to lipogenic differentiation.Realtime 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 differentiation 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 miR15b(primiR15b)between 2 groups(P>0.05),but the expression levels of premiR-15b,miR-15b and microRNA-16(miR-16)in mutant 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 elementbinding protein1c(SREBP1c)and peroxisome proliferatoractivated receptorγ(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 conclusion,+58C>T can hinder the biological processing from primiR-15b to premiR-15b,resulting in a decrease 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.