miR-33靶向调控鸡CHPT1基因表达
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摘要
miR-33在脂类代谢过程中具有重要作用,同时还参与葡萄糖代谢、炎症反应以及细胞周期等多种生理过程。目前对于鸡(Gallus gallus) miR-33的功能及其与预测靶基因—胆碱磷酸转移酶1基因(choline phosphotransferase 1, CHPT1)的作用关系尚不明确。本研究旨在对鸡miR-33预测靶基因CHPT1进行实验验证,探讨鸡miR-33对CHPT1的靶向调控作用。首先利用生物信息学方法,在鸡3'-UTR数据库中共预测到378个miR-33的靶基因,包括与脂类代谢相关的CHPT1基因;然后构建miR-33过表达载体和CHPT1荧光素酶报告载体,共转染小鼠(Mus musculus)成肌细胞系C2C12,结果显示,miR-33的种子区与CHPT1预测靶位点结合,CHPT1基因表达因此被抑制;点突变实验证实了miR-33与CHPT1结合的靶位点。设计并合成miR-33拮抗物LNA (locked nucleic acid)-antimiR-33,转染鸡原代肝细胞后使miR-33表达水平下降44%,而CHPT1基因表达量有一定程度上升。利用qRT-PCR技术对miR-33的表达进行分析,结果显示,miR-33在鸡不同组织中均有表达,在肌胃和心脏中的表达量较高,在其他组织中等表达;miR-33在肌胃中的表达量与脾脏、肾脏、脑和腺胃中的表达量差异显著(P<0.05),与肝脏和腿肌中的表达量差异极显著(P<0.01);miR-33在4周龄高脂肉鸡肝脏和腹脂组织中的表达量显著高于低脂肉鸡(P<0.05);靶基因CHPT1在高脂肉鸡腹脂组织中的表达量显著高于低脂肉鸡(P<0.05)。上述实验结果提示,miR-33与鸡脂类代谢和脂肪沉积相关,CHPT1基因是其潜在的靶基因。本研究为揭示鸡脂类代谢调控机理提供了新的线索。
In addition to the regulation of lipid metabolism, miR-33 is also reported to be involved in glucose metabolism, inflammatory response, and cell cycle. However, the role of the chicken(Gallus gallus) miR-33 and its relationship with the predicted target gene choline phosphotransferase 1(CHPT1) are still unclarified.The present study was conducted to investigate whether CHPT1 was the target gene of miR-33. The bioinformatics methods were used to predict the target genes of miR-33, and 378 genes from 3'-UTR database were predicted and CHPT1 gene was included. Then, the miR-33 overexpression vector and CHPT1 luciferase reporter vector were constructed and co-transfected into the mouse(Mus musculus) C2 C12 myoblast cell, dualluciferase reporter assay showed that the expression of luciferase reporter gene linked to the 3'-untranslated region of CHPT1 mRNA was down-regulated by miR-33 overexpression in C2 C12 cells(P<0.01).Furthermore, the down-regulation was completely abolished when the predicted miR-33 target site in CHPT13'-UTR was mutated. Then mir-33 antagonists LNA(locked nucleic acid)-antimiR-33 was designed and synthetised, after transfecting chicken primary liver cells, the expression level of miR-33 decreased by 44%,while CHPT1 mRNA increased with a certain degree. By q RT-PCR, it was found that miR-33 highly expressed in muscular stomach and heart muscle, and the miR-33 expression in muscular stomach was significantly different from that in spleen, kidney, brain and glandular stomach(P<0.05), and very significantly different from that in liver and thigh muscle(P<0.01). In liver and abdomen fat tissue of 4 week lean and fat line chicken, miR-33 was significantly higher in the fat line than that in the lean line(P<0.05). The CHPT1 expression in the abdominal fat tissues was significantly higher in fat line chicken than that in lean line chicken(P<0.05). The above data indicate that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of CHPT1. The present study provides new clues for lipid synthesis.
In addition to the regulation of lipid metabolism, miR-33 is also reported to be involved in glucose metabolism, inflammatory response, and cell cycle. However, the role of the chicken(Gallus gallus) miR-33 and its relationship with the predicted target gene choline phosphotransferase 1(CHPT1) are still unclarified.The present study was conducted to investigate whether CHPT1 was the target gene of miR-33. The bioinformatics methods were used to predict the target genes of miR-33, and 378 genes from 3'-UTR database were predicted and CHPT1 gene was included. Then, the miR-33 overexpression vector and CHPT1 luciferase reporter vector were constructed and co-transfected into the mouse(Mus musculus) C2 C12 myoblast cell, dualluciferase reporter assay showed that the expression of luciferase reporter gene linked to the 3'-untranslated region of CHPT1 mRNA was down-regulated by miR-33 overexpression in C2 C12 cells(P<0.01).Furthermore, the down-regulation was completely abolished when the predicted miR-33 target site in CHPT13'-UTR was mutated. Then mir-33 antagonists LNA(locked nucleic acid)-antimiR-33 was designed and synthetised, after transfecting chicken primary liver cells, the expression level of miR-33 decreased by 44%,while CHPT1 mRNA increased with a certain degree. By q RT-PCR, it was found that miR-33 highly expressed in muscular stomach and heart muscle, and the miR-33 expression in muscular stomach was significantly different from that in spleen, kidney, brain and glandular stomach(P<0.05), and very significantly different from that in liver and thigh muscle(P<0.01). In liver and abdomen fat tissue of 4 week lean and fat line chicken, miR-33 was significantly higher in the fat line than that in the lean line(P<0.05). The CHPT1 expression in the abdominal fat tissues was significantly higher in fat line chicken than that in lean line chicken(P<0.05). The above data indicate that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of CHPT1. The present study provides new clues for lipid synthesis.
引文
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Iliopoulos D,Drosatos K,Hiyama Y,et al.2010.MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism[J].Journal of Lipid Research,51(6):1513-1523.
Kent C.2005.Regulatory enzymes of phosphatidylcholine biosynthesis:A personal perspective[J].Biochimica et Biophysica Acta,1733(1):53-66.
Kim J,Yoon H,Ramirez CM,et al.2012.MiR-106b impairs cholesterol efflux and increases Abeta levels by repressing ABCA1 expression[J].Experimental Neurology,235(2):476-483.
Marquart T J,Allen R M,Ory D S,et al.2010.miR-33 links SREBP-2 induction to repression of sterol transporters[J].Proceedings of the National Academy of Sciences of the USA,107(27):12228-12232.
Poritsanos N J,Lew P S,Mizuno T M.2010.Relationship between blood glucose levels and hepatic Fto mRNA expression in mice[J].Biochemical and Biophysical Research Communications,400(4):713-717.
Rayner K J,Esau C C,Hussain F N,et al.2011.Inhibition of miR-33a/b in non-human primates raises plasma HDLand lowers VLDL triglycerides[J].Nature,478:404-407.
Rayner K J,Sheedy F J,Esau C C,et al.2011.Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis[J].The Journal of Clinical Investigation,121(7):2921-2931.
Rayner K J,Suarez Y,Davalos A,et al.2010.MiR-33 contributes to the regulation of cholesterol homeostasis[J].Science,328(5985):1570-1573.
Sanchez-Pulido L,Andrade-Navarro M A.2007.The FTO(fat mass and obesity associated)gene codes for a novel member of the non-heme dioxygenase superfamily[J].BMC Biochemistry,8:23.
Shao F,Wang X G,Yu J F,et al.2014.Expression of miR-33from an SREBF2 intron targets the FTO gene in the chicken[J].PloS One,9(3):e91236.
Xie H,Sun L,Lodish H F.2009.Targeting microRNAs in obesity[J].Expert Opinion on Therapeutic Targets,13(10):1227-1238.
Boumann H A,de Kruijff B,Heck A J,et al.2004.The selective utilization of substrates in vivo by the phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes Ept1p and Cpt1p in yeast[J].FEBS letters,569(1-3):173-177.
Cirera-Salinas D,Pauta M,Allen R M,et al.2012.Mir-33 regulates cell proliferation and cell cycle progression[J].Cell cycle,11(5):922-933.
Davalos A,Goedeke L,Smibert P,et al.2011.miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling[J].Proceedings of the National Academy of Sciences of the USA,108(22):9232-9237.
Elmen J,Lindow M,Schutz S,et al.2008.LNA-mediated microRNA silencing in non-human primates[J].Nature452(7189):896-899.
Elmen J,Lindow M,Silahtaroglu A,et al.2008.Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target m RNAs in the liver[J].Nucleic Acids Research,36(4):1153-1162.
Gerin I,Bommer G T,McCoin C S,et al.2010.Roles for miRNA-378/378*in adipocyte gene expression and lipogenesis[J].American Journal of Physiology-Endocrinology and Metabolism,299(2):E198-E206.
Gerin I,Clerbaux L A,Haumont O,et al..2010.Expression of miR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation[J].The Journal of Biological Chemistry,285(44):33652-33661.
Heneghan H M,Miller N,and Kerin MJ.2010.Role of microRNAs in obesity and the metabolic syndrome[J]Obesity reviews:An official Journal of the Internationa Association for the Study of Obesity,11(5):354-361.
Hilton C,Neville M J,Karpe F.2013.MicroRNAs in adipose tissue:Their role in adipogenesis and obesity[J].International Journal of Obesity,37(3):325-332.
Hjelmstad R H,Bell R M.1987.Mutants of Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase.Isolation,characterization,and cloning of the CPT1 gene[J].The Journal of Biological Chemistry,262(8):3909-3917.
Horie T,Ono K,Horiguchi M,et al.2010.MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2(Srebp2)regulates HDL in vivo[J].Proceedings of the National Academy of Sciences of the USA107(40):17321-17326.
Hoekstra M,van der Sluis R J,Kuiper J,et al.2012.Nonalcoholic fatty liver disease is associated with an altered hepatocyte microRNA profile in LDL receptor knockou mice[J].Journal of Nutritional Biochemistry,23(6):622-628.
Iliopoulos D,Drosatos K,Hiyama Y,et al.2010.MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism[J].Journal of Lipid Research,51(6):1513-1523.
Kent C.2005.Regulatory enzymes of phosphatidylcholine biosynthesis:A personal perspective[J].Biochimica et Biophysica Acta,1733(1):53-66.
Kim J,Yoon H,Ramirez CM,et al.2012.MiR-106b impairs cholesterol efflux and increases Abeta levels by repressing ABCA1 expression[J].Experimental Neurology,235(2):476-483.
Marquart T J,Allen R M,Ory D S,et al.2010.miR-33 links SREBP-2 induction to repression of sterol transporters[J].Proceedings of the National Academy of Sciences of the USA,107(27):12228-12232.
Poritsanos N J,Lew P S,Mizuno T M.2010.Relationship between blood glucose levels and hepatic Fto mRNA expression in mice[J].Biochemical and Biophysical Research Communications,400(4):713-717.
Rayner K J,Esau C C,Hussain F N,et al.2011.Inhibition of miR-33a/b in non-human primates raises plasma HDLand lowers VLDL triglycerides[J].Nature,478:404-407.
Rayner K J,Sheedy F J,Esau C C,et al.2011.Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis[J].The Journal of Clinical Investigation,121(7):2921-2931.
Rayner K J,Suarez Y,Davalos A,et al.2010.MiR-33 contributes to the regulation of cholesterol homeostasis[J].Science,328(5985):1570-1573.
Sanchez-Pulido L,Andrade-Navarro M A.2007.The FTO(fat mass and obesity associated)gene codes for a novel member of the non-heme dioxygenase superfamily[J].BMC Biochemistry,8:23.
Shao F,Wang X G,Yu J F,et al.2014.Expression of miR-33from an SREBF2 intron targets the FTO gene in the chicken[J].PloS One,9(3):e91236.
Xie H,Sun L,Lodish H F.2009.Targeting microRNAs in obesity[J].Expert Opinion on Therapeutic Targets,13(10):1227-1238.