《中国康复理论与实践》

中国康复理论与实践 >

2017 , Vol. 23 >Issue 2: 166 - 172

DOI: https://doi.org/10.3969/j.issn.1006-9771.2017.02.010

基础研究

大鼠细胞外信号调节激酶1基因3ʹ非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响

  • 罗汉将 ,
  • 徐云峰 ,
  • 李晓晓 ,
  • 杨予涛 ,
  • 徐志卿
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  • 1.首都医科大学基础医学院,北京市 100069;
    2. 黄岛出入境检验检疫局,山东青岛市 266555
罗汉将(1990-),男,汉族,浙江余姚市人,硕士研究生,主要研究方向：miR-15b参与抑郁症发生的机制研究。

收稿日期: 2016-09-05

  修回日期: 2016-09-18

  网络出版日期: 2017-03-06

基金资助

1.北京市自然科学基金面上项目(No.7162016); 2.国家自然科学基金面上项目(No.31271154; No.31171032)

收起

Construction of Rat Extracellular Signal-regulated Kinase 1 Gene 3ʹ Untranslated Regions Dual-luciferase Reporter Plasmids and Effect of rno-miR-15b-5p on Its Activitiy

  • LUO Han-jiang ,
  • XU Yun-feng ,
  • LI Xiao-xiao ,
  • YANG Yu-tao ,
  • XU Zhi-qing
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  • 1. Captial Medical University School of Basic Medical Sciences, Beijing 100069, China;
    2. Huangdao Entry-Exit Inspection and Quarantine Bureau, Qingdao, Shandong 266555, China

Received date: 2016-09-05

  Revised date: 2016-09-18

  Online published: 2017-03-06

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摘要

目的 以大鼠细胞外信号调节激酶1(ERK1)基因3ʹ非编码区(UTR)为研究对象,构建含有ERK1基因3ʹ UTR区的野生型以及突变型重组双荧光素酶报告载体,通过分析双荧光素酶报告载体的活性,明确rno-miR-15b-5p对报告载体的调节作用。方法 采用miRNeasy Mini Kit提取大鼠肾上腺嗜铬细胞瘤PC12细胞的总RNA,以PC12细胞的cDNA为模板,利用聚合酶链式反应(PCR)将ERK1基因3ʹ UTR克隆到pmiR-RB-ReportTM vector 中。利用重叠延伸PCR,将ERK1基因3ʹ UTR中的rno-miR-15b-5p潜在的靶序列TGCTGCT分别突变成CGAACGT和GTACACG,并将突变的ERK1基因3ʹ UTR克隆到pmiR-RB-ReportTM vector中。结果 成功构建了包含ERK1基因3ʹ UTR区的野生型报告载体pmiR-ERK1 3ʹ UTR和突变型报告载体pmiR-ERK1-mut 3ʹ UTR。利用荧光素酶活性检测系统,发现rno-miR-15b-5p mimic可以降低野生型报告载体的活性(P<0.001),但不影响突变型报告载体的活性。结论 成功构建ERK1基因3ʹ UTR区野生型和突变型双荧光素酶报告载体,初步证明ERK1基因3ʹ UTR区是rno-miR-15b-5p的结合靶点。

关键词： rno-miR-15b-5p; 细胞外信号调节激酶1; 重叠延伸聚合酶链式反应; pmiR-ERK1 3'UTR; pmiR-ERK1-mut 3'UTR; 荧光素酶活性检测

本文引用格式

罗汉将 , 徐云峰 , 李晓晓 , 杨予涛 , 徐志卿 . 大鼠细胞外信号调节激酶1基因3ʹ非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响[J]. 中国康复理论与实践, 2017 , 23(2) : 166 -172 . DOI: 10.3969/j.issn.1006-9771.2017.02.010

Abstract

Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulated kinase 1 (ERK1) gene 3ʹ untranslated regions (UTR) which were used to detect rno-miR-15b-5p's putative target gene. Methods The rat ERK1 gene 3ʹ UTR fragment was amplified by polymerase chain reaction (PCR) from PC12 cell cDNA and cloned into pmiR-RB-ReportTM vector. The mutant rat ERK1 gene 3ʹ UTR fragment was obtained by overlap PCR and inserted into pmiR-RB-ReportTM vector. Successful wild type and mutant recombinant plasmids were confirmed by DNA sequencing. PC12 cells were co-transfected with rno-miR-15b-5p mimic and pmiR-ERK1 3ʹ UTR or pmiR-ERK1-mut 3ʹ UTR and then analyzed by dual-luciferase reporter assay system. The achieved mutation sequence of the target site TGCTGCT was mutated to CGAACGT and GTACACG, respectively. Results The wild-type reporter vector pmiR-ERK1 3ʹ UTR and the mutant reporter vector pmiR-ERK1-mut 3ʹ UTR were successfully constructed. The rno-miR-15b-5p mimic decreased the activity of pmiR-ERK1 3ʹ UTR plasmid (P<0.001) but did not decrease the activity of pmiR-ERK1-mut 3ʹ UTR plasmid. Conclusion The recombinant pmiR-ERK1 3ʹ UTR and pmiR-ERK1-mut 3ʹ UTR plasmids were constructed successfully, and luciferase activities demonstrated that the 3ʹ UTR of ERK1 gene might be a potential target of rno-miR-15b-5p.

Key words： rno-miR-15b-5p; extracellular signal-regulated kinase 1; overlap polymerase chain reaction; pmiR-ERK1 3'UTR; pmiR-ERK1-mut 3'UTR; luciferase reporter assay

参考文献

[1] Dwivedi Y. Evidence demonstrating role of microRNAs in the etiopathology of major depression [J]. J Chem Neuroanat, 2011, 42(2): 142-156.
[2] Friedlander MR, Lizano E, Houben AJ, et al. Evidence for the biogenesis of more than 1,000 novel human microRNAs [J]. Genome Biol, 2014, 15(4): R57.
[3] Bunkar N, Pathak N, Lohiya NK, et al. Epigenetics: a key paradigm in reproductive health [J]. Clin Exp Reprod Med, 2016, 43(2): 59-81.
[4] Friedman RC, Farh KK, Burge CB, et al. Most mammalian mRNAs are conserved targets of microRNAs [J]. Genome Res, 2009, 19(1): 92-105.
[5] Hausser J, Zavolan M. Identification and consequences of miRNA-target interactions–beyond repression of gene expression [J]. Nat Rev Genet, 2014, 15(9): 599-612.
[6] Jonas S, Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing [J]. Nat Rev Genet, 2015, 16(7): 421-433.
[7] Tonevitsky AG, Maltseva DV, Abbasi A, et al. Dynamically regulated miRNA-mRNA networks revealed by exercise [J]. BMC Physiol, 2013, 13: 9.
[8] Makarova JA, Maltseva DV, Galatenko VV, et al. Exercise immunology meets MiRNAs [J]. Exerc Immunol Rev, 2014, 20: 135-164.
[9] Lu J, Liang J, Wang JR, et al. Acupuncture activates ERK-CREB pathway in rats exposed to chronic unpredictable mild stress [J]. Evid Based Complement Alternat Med, 2013, 2013: 469765.
[10] Palanichamy JK, Rao DS. miRNA dysregulation in cancer: towards a mechanistic understanding [J]. Front Genet, 2014, 5: 54.
[11] Papageorgiou N, Zacharia E, Tousoulis D. Association between microRNAs and coronary collateral circulation: is there a new role for the small non-coding RNAs? [J]. Ann Transl Med, 2016, 4(11): 223.
[12] Vimalraj S, Partridge NC, Selvamurugan N. A positive role of microRNA-15b on regulation of osteoblast differentiation [J]. J Cell Physiol, 2014, 229(9): 1236-1244.
[13] Xia H, Qi Y, Ng SS, et al. MicroRNA-15b regulates cell cycle progression by targeting cyclins in glioma cells [J]. Biochem Biophys Res Commun, 2009, 380(2): 205-210.
[14] Busca R, Pouyssegur J, Lenormand P. ERK1 and ERK2 MAP kinases: specific roles or functional redundancy? [J]. Front Cell Dev Biol, 2016, 4: 53.
[15] Roskoski R Jr. ERK1/2 MAP kinases: structure, function, and regulation [J]. Pharmacol Res, 2012, 66(2): 105-143.
[16] Lei YY, Wang WJ, Mei JH, et al. Mitogen-activated protein kinase signal transduction in solid tumors [J]. Asian Pac J Cancer Prev, 2014, 15(20): 8539-8548.
[17] Gaestel M. MAPK-activated protein kinases (MKs): novel insights and challenges [J]. Front Cell Dev Biol, 2015, 3: 88.
[18] Pages G, Guerin S, Grall D, et al. Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice [J]. Science, 1999, 286(5443): 1374-1377.
[19] Szulwach KE, Li X, Smrt RD, et al. Cross talk between microRNA and epigenetic regulation in adult neurogenesis [J]. J Cell Biol, 2010, 189(1): 127-141.
[20] Cheng LC, Pastrana E, Tavazoie M, et al. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche [J]. Nat Neurosci, 2009, 12(4): 399-408.
[21] Marsden WN. Synaptic plasticity in depression: molecular, cellular and functional correlates [J]. Prog Neuropsychopharmacol Biol Psychiatry, 2013, 43: 168-184.
[22] Berditchevski F, Odintsova E. ErbB receptors and tetraspanins: casting the net wider [J]. Int J Biochem Cell Biol, 2016, 77 (Pt A): 68-71.
[23] Alvaro CG, Thorner J. Heterotrimeric G protein-coupled receptor signaling in yeast mating pheromone response [J]. J Biol Chem, 2016, 291(15): 7788-7795.
[24] Buraei Z, Lumen E, Kaur S, et al. RGK regulation of voltage-gated calcium channels [J]. Sci China Life Sci, 2015, 58 (1): 28-38.
[25] Kyriakis JM, Force TL, Rapp UR, et al. Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase [J]. J Biol Chem, 1993, 268(21): 16009-16019.
[26] Rauch N, Rukhlenko OS, Kolch W, et al. MAPK kinase signalling dynamics regulate cell fate decisions and drug resistance [J]. Curr Opin Struct Biol, 2016, 41: 151-158.
[27] Marais R, Wynne J, Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain [J]. Cell, 1993, 73(2): 381-393.
[28] Shankar E, Song K, Corum SL, et al. A signaling network controlling androgenic repression of c-Fos protein in prostate adenocarcinoma cells [J]. J Biol Chem, 2016, 291(11): 5512-5526.
[29] Morton S, Davis RJ, McLaren A, et al. A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun [J]. EMBO J, 2003, 22(15): 3876-3886.
[30] Ashabi G, Alamdary SZ, Ramin M, et al. Reduction of hippocampal apoptosis by intracerebroventricular administration of extracellular signal-regulated protein kinase and/or p38 inhibitors in amyloid beta rat model of Alzheimer's disease: involvement of nuclear-related factor-2 and nuclear factor-kappaB [J]. Basic Clin Pharmacol Toxicol, 2013, 112(3): 145-155.
[31] Borges G, Berrocoso E, Mico JA, et al. ERK1/2: function, signaling and implication in pain and pain-related anxio-depressive disorders [J]. Prog Neuropsychopharmacol Biol Psychiatry, 2015, 60: 77-92.
[32] Qi X, Lin W, Li J, et al. The depressive-like behaviors are correlated with decreased phosphorylation of mitogen-activated protein kinases in rat brain following chronic forced swim stress [J]. Behav Brain Res, 2006, 175(2): 233-240.
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