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| Identification miRNAs That Regulate Porcine TNF-α Expression Through Targeting TNF-α UTR |
| LI Hong-yi1, XI Qian-yun2, ZHANG Yong-liang2 |
1. Fujian Provincial Key Laboratory of Preventive Veterinary Medicine and Biotechnology, College of Life Science, Longyan University, Fujian 364012, China;
2. College of Animal Science, South China Agricultural University, Guangzhou 510642, China |
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Abstract 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 22nt, which regulates 60% of mammalian gene via its seed sequence. Author's previous work found miR-181a, 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-19a,miR-124,miR-130a,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-19a, miR-124 and miR-130a 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-19a, miR-124 and miR-130a on TNF-α3' UTR were mutated to design the rite-directed 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-19a, miR-124 and miR-130a 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-19a, miR-124 and miR-130a 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.
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Received: 22 July 2014
Published: 25 October 2014
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