miR-181b慢病毒载体的构建及其对HEK-293T细胞MAPK1基因的靶向调控作用

基础医学与临床 ›› 2016, Vol. 36 ›› Issue (8): 1124-1129.

miR-181b慢病毒载体的构建及其对HEK-293T细胞MAPK1基因的靶向调控作用

王筝,邢译文,朱凤英,王静波

航天中心医院

收稿日期:2015-12-22 修回日期:2016-05-27 出版日期:2016-08-05 发布日期:2016-07-13
通讯作者: 王筝 E-mail:13718881814@163.com

Construction of miR-181b lentiviral vector and its targeting effect on MAPK1 gene

Received:2015-12-22 Revised:2016-05-27 Online:2016-08-05 Published:2016-07-13

摘要/Abstract

摘要： 目的构建miR-181b慢病毒过表达载体，探讨其对HEK-293T细胞MAPK1基因的靶向调控作用。方法分别构建pSicoR-miR-181b及pMIR-Report-MAPK1过表达载体，转染HEK-293T细胞后，用实时荧光定量PCR（RT-qPCR）法检测MAPK1mRNA的表达、用Western blot检测MAPK1蛋白的表达及用双荧光素酶报告基因活性验证miR-181b与MAPK1的靶向调控关系。结果成功构建了pSicoR-miR-181b及pMIR-Report-MAPK1重组质粒，miR-181b过表达明显抑制HEK-293T细胞MAPK1蛋白及mRNA表达水平（P<0.05）,抑制miR-181b的表达后，MAPK1的蛋白及mRNA的表达水平上调（P<0.05）。结论成功构建pSicoR-miR-181b慢病毒载体，并证实通过靶向作用MAPK1-3’UTR的特异序列直接抑制MAPK1基因的表达。

关键词: microRNA, miR-181b, MAPK1

Abstract: Objective To construct alentiviral vector of miR-181b andverify its targeted effect on MAPK1 gene. MethodspSicoR-miR-181b and pMIR-Report-MAPK1 over-expression vector were construsted.ThemRNA of MAPK1 was verified by real-time PCR testand the protein level of MAPK1 was verified by Western blot .To validate the targeted regulatory relationship between miR-181band MAPK1 3’-UTRthrough relative luciferase activity.ResultsTherecombinant plasmids of pSicoR-miR-181b and pMIR-Report-MAPK13'-UTR were constructedsuccessfully. And it was showed that over-expression of miR-181b suppressed the mRNA and protein expression level of MAPK1significantly(p<0.05),suppression of miR-181b expression increased the expression level of MAPK1 mRNA and protein significantly(p<0.05). ConclusionsThelentiviral vector containing miR-181b gene has successfully constructed.MiR-181b can suppress MAPK1 gene expression by targeting the specific sequence ofMAPK1-3'UTR.

Key words: microRNA, miR-181b, MAPK1

中图分类号:

R392

王筝邢译文朱凤英王静波. miR-181b慢病毒载体的构建及其对HEK-293T细胞MAPK1基因的靶向调控作用[J]. 基础医学与临床, 2016, 36(8): 1124-1129.

[1]	翟新颖郝文慧陈茜. miR-203a-5p通过抑制丝裂原活化蛋白激酶1的表达调控人牙周膜干细胞的炎性反应[J]. 基础医学与临床, 2019, 39(6): 846-850.
[2]	李静李娜庄乾宇赵春华. miR-181b抑制骨髓间充质干细胞成骨分化可能参与青少年特发性脊柱侧弯骨密度降低[J]. 基础医学与临床, 2019, 39(6): 832-839.
[3]	古丽尼格尔·雪合拉提王延蛟祖力皮喀尔·阿卜杜热合曼娜孜拉木·玉苏甫江斯坎德尔·白克力. 食管癌患者外周血中miR-181b-5p表达水平下降[J]. 基础医学与临床, 2018, 38(5): 600-603.
[4]	邓资翔左中. ABCA1在脂质沉积中的研究进展[J]. 基础医学与临床, 2017, 37(6): 870-873.
[5]	崔铭萱李伟马良坤平凡刘俊涛伍学焱茅江峰王曦聂敏. ABCG5/8基因microRNA结合位点多态性与妊娠期糖脂代谢相关[J]. 基础医学与临床, 2017, 37(5): 682-686.
[6]	崔洁洁龚梦嘉何昀毕杨. 全反式维甲酸对小鼠肝癌细胞Hepa1-6增殖、迁移、侵袭的影响及其机制[J]. 基础医学与临床, 2016, 36(2): 211-217.
[7]	刘凤霞牛淑亮李建勇郑树涛卢晓梅刘文亚. ERK1/2 MAPK 通路在Eca109细胞体外增殖和迁移中的作用及其机制[J]. 基础医学与临床, 2014, 34(3): 345-349.
[8]	刘大全李东华刘洪斌. MiR-141增强人血管内皮细胞增殖及迁移能力[J]. 基础医学与临床, 2010, 30(8): 801-806.
[9]	余佳付红艺汪晓艳刘倩王芳冯涛张俊武. MicroRNA-205对人皮肤上皮细胞迁移能力的调控[J]. 基础医学与临床, 2010, 30(6): 613-618.
[10]	苏金梅金晔曲强张奉春唐福林. 骨髓间充质干细胞向软骨细胞分化过程中miR130a的表达[J]. 基础医学与临床, 2010, 30(5): 520-523.
[11]	宋宝宋现让魏玲魏玲. miRNAs及其靶基因的识别[J]. 基础医学与临床, 2007, 27(9): 1063-1065.
[12]	宋宝. microRNA与人类肿瘤[J]. 基础医学与临床, 2007, 27(10): 1181-1184.