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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2014, Vol. 34 Issue (10): 35-40    DOI: 10.13523/j.cb.20141006
研究报告     
调节猪TNF-α表达的miRNAs鉴定
李虹仪1, 习欠云2, 张永亮2
1. 龙岩学院生命科学学院 预防兽医学与生物技术福建省高等学校重点实验室 龙岩 364012;
2. 华南农业大学动物科学学院 广州 510642
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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摘要：

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-α 3'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-α 3'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-α 3'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-α 3'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.
关键词： 肿瘤坏死因子α;  miRNA荧光素酶    
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.
Key words: TNF-α    miRNA    Luciferase assay
收稿日期: 2014-07-22 出版日期: 2014-10-25
ZTFLH:  Q786  
基金资助:

转基因生物新品种培育科技重大专项重点项目(2014ZX08009048B)、国家自然科学基金(31272529)资助项目
通讯作者: 张永亮     E-mail: zhangyl@scau.edu.cn
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引用本文:

李虹仪, 习欠云, 张永亮. 调节猪TNF-α表达的miRNAs鉴定[J]. 中国生物工程杂志, 2014, 34(10): 35-40.

LI Hong-yi, XI Qian-yun, ZHANG Yong-liang. Identification miRNAs That Regulate Porcine TNF-α Expression Through Targeting TNF-α UTR. China Biotechnology, 2014, 34(10): 35-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141006    或    https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I10/35


[1] Borzecka K, Plociennikowska A, Bjorkelund H, et al. CD14 mediates binding of high doses of LPS but is dispensable for TNF-alpha production. Mediators Inflamm, 2013,2013:824919.

[2] Morioka S, Broglie P, Omori E, et al. TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation. J Cell Biol, 2014,204(4):607-623.

[3] Takada I, Kouzmenko A P, Kato S. PPAR-gamma signaling crosstalk in mesenchymal stem cells. PPAR Res, 2010,2010.

[4] Bluher M, Bashan N, Shai I, et al. Activated Ask1-MKK4-p38MAPK/JNK stress signaling pathway in human omental fat tissue may link macrophage infiltration to whole-body insulin sensitivity. J Clin Endocrinol Metab, 2009,94(7):2507-2515.

[5] Tili E, Michaille J J, Cimino A, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol, 2007,179(8):5082-5089.

[6] El Gazzar M, Church A, Liu T, et al. MicroRNA-146a regulates both transcription silencing and translation disruption of TNF-alpha during TLR4-induced gene reprogramming. J Leukoc Biol, 2011,90(3):509-519.

[7] Liu M, Wang Z, Yang S, et al. TNF-alpha is a novel target of miR-19a. Int J Oncol, 2011,38(4):1013-1022.

[8] Lorente-Cebrian S, Mejhert N, Kulyte A, et al. MicroRNAs regulate human adipocyte lipolysis: effects of miR-145 are linked to TNF-alpha. PLoS One, 2014,9(1):e86800.

[9] Li H Y, Chen X, Guan L Z, et al. MiRNA-181a regulates adipogenesis by targeting tumor necrosis factor-alpha (TNF-alpha) in the porcine model. PLoS One, 2013,8(10):e71568.

[10] Zhao J, Gong A Y, Zhou R, et al. Downregulation of PCAF by miR-181a/b provides feedback regulation to TNF-alpha-induced transcription of proinflammatory genes in liver epithelial cells. J Immunol, 2012,188(3):1266-1274.

[11] Chen Y, Wang C, Liu Y, et al. miR-122 targets NOD2 to decrease intestinal epithelial cell injury in Crohn's disease. Biochem Biophys Res Commun, 2013,438(1):133-139.

[12] Menon B, Sinden J, Franzo-Romain M, et al. Regulation of LH receptor mRNA binding protein by miR-122 in rat ovaries. Endocrinology, 2013,154(12):4826-4834.

[13] Karbiener M, Fischer C, Nowitsch S, et al. microRNA miR-27b impairs human adipocyte differentiation and targets PPARgamma. Biochem Biophys Res Commun, 2009,390(2):247-251.

[14] Lee E K, Lee M J, Abdelmohsen K, et al. miR-130 suppresses adipogenesis by inhibiting peroxisome proliferator-activated receptor gamma expression. Mol Cell Biol, 2010,31(4):626-638.
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