调节猪TNF-α表达的miRNAs鉴定

Identification miRNAs That Regulate Porcine TNF-α Expression Through Targeting TNF-α UTR

肿瘤坏死因子α(TNF-α)是脂肪细胞的分泌产物之一,在脂肪中行使着复杂的调节功能,为了研究猪脂肪组织中TNF-α的表达受到哪些miRNA的调控,从猪脂肪组织基因组中获得猪TNF-α3'端非翻译区(UTR)序列,与荧光素酶质粒PGL3-control连接构建猪TNF-α的萤光素酶表达质粒PGL3-TNF-α3'UTR。生物信息学预测miR-19a,miR-124,miR-130a,miR-301,miR-506等miRNAs均靶向猪TNF-α,将这些miRNA分别与PGL3-TNF-α3'UTR质粒共转到细胞中,以乱序序列作为阴性对照(NC),检测miRNA对质粒荧光素酶活性的作用。结果发现miR-19a,miR-124和miR-130a均能够显著抑制萤光素酶的活性(P<0.01),为了验证这3个miRNA是否通过各自种子序列起调控作用,突变了PGL3-TNF-α3'UTR中这3个miRNA种子序列的结合位点,结果发现miRNAs对突变质粒中的荧光素酶均无明显抑制作用(P>0.05)。结果证明,miR-19a,miR-124和miR-130a与猪TNF-α均有直接的靶向关系并通过各自种子序列抑制TNF-α的表达。

TNF-α is one of the secretory products of adipocyte and is a multifunctional cytokine that plays an important role in regulating lipogenesis. miRNA is a kind of endogenous RNA with length of about 22 nt,which regulates 60% of mammalian gene via its seed sequence. Author＇s previous work found miR-181 a,which targeting porcine TNF-α,is highly expressed in a fat-rich pig breed and has an effects on adipocyte differentiation by regulation of TNF-α. In order to find out which miRNAs that targeting the porcine TNF-α,3 end untranslated region of porcine TNF-α were amplified and the PCR product was digested with Xba I and Hpa I,then ligated to pGL3-control at the corresponding sites to construct the luciferase expression plasmid pGL3-TNF-α-UTR. Then five miRNA miR-19 a,miR-124,miR-130 a,miR-301, miR-506 were predicted to target TNF-α using bioinformatic analysis（ softwares of TargetScan：http： / /www. targetscan. org /、miRanda：http： / /www. microrna.org /microrna /home. do and Pictar：http： / /pictar. mdc-berlin. de /）. Mimics of these five miRNAs were synthesis and cotransfected with PGL3-TNF-α-UTR into CHO cells respectively,taking scrambled sequence as negative control. The results showed that TNF-α was the target of miR-19 a,miR-124 and miR-130 a by a dual luciferase assay（P 0. 01）. To verify whether TNF-α expression was inhibited by the seed sequences of these three miRNAs,binding sites of miR-19 a,miR-124 and miR-130 a on TNF-α3＇ UTR were mutated to design the ritedirected mutagenesis primers,after PCR amplifying,the products were digested with Dpn I to remove the originally un-mutated template. Then the mutated vectors PGL3-TNF-α-UTR-mutant1（ miR-19 /130a） and 2（miR-124）were constructed. The mutated plasmids were cotransfected with miR-19 a,miR-124 and miR-130 a respectively,and the result of dual luciferase assay showed that they all have no significant effect on depressing the expression of TNF-α（P 0. 05）. The result showed that porcine TNF-α is the target of miR-19 a,miR-124 and miR-130 a and they all inhibit the expression of TNF-α by their seed sequences,which provided the evidence of interrelationship between TNF-α and the three miRNAs.