miRNA let-7a靶向抑制ACVR1B表达

doi:10.3969/j.issn.2095-9400.2020.01.002

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摘要目的：探讨miRNA let-7a对ACVR1B表达的影响。方法：生物信息学分析表明activin receptor type 1B（ACVR1B）是一个let-7a的潜在靶基因。构建ACVR1B 3’UTR野生型（ACVR1B 3’UTR-WT）和ACVR1B 3’UTR突变型（ACVR1B 3’UTR-MUT）双荧光素酶报告载体，分别与let-7a模拟物（let-7a mimic）或let-7a阴性对照（let-7a NC）共同转染HEK293T细胞，通过双荧光素酶报告系统检测各组细胞的荧光素酶活性。分别转染let-7a mimic、let-7a inhibitor和let-7a NC至HEK293T细胞，采用qRT-PCR和Western blot分别检测ACVR1B的mRNA和蛋白表达水平。结果：ACVR1B 3’UTR含有let-7a潜在的高度保守的结合靶点（77～83位点）。在HEK293T细胞中共转染ACVR1B 3’UTR-WT报告载体的let-7a mimic组比let-7a NC组荧光素酶活性组明显降低（P＜0.05）；而共转染ACVR1B 3’UTR-MUT报告载体的let-7a mimic与let-7a NC组其荧光素酶活性差异无统计学意义（P＞0.05）。在HEK293T细胞中let-7a mimic转染组中ACVR1B的mRNA和蛋白水平均低于let-7a NC组和let-7a inhibitor组（P＜0.05）。结论：ACVR1B是let-7a潜在调控的靶基因，miRNA let-7a可以直接抑制HEK293T细胞中ACVR1B基因的表达。

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关键词 ： miRNA let-7a, ACVR1B, 转染

Abstract：Objective: To investigate the effect of miRNA let-7a on ACVR1B expression. Methods: Bioinformatics analysis predicted that activin receptor type 1B (ACVR1B) is a potential target gene for let-7a. By constructing ACVR1B 3’UTR wild-type and ACVR1B 3’UTR mutant dual-luciferase reporter vector (pmirGLO-ACVR1B 3’UTR-WT and pmirGLO-ACVR1B 3’UTR-MUT), let-7a mimic or let-7a negative control (let-7a NC) were co-transfected into HEK293T cells respectively and the luciferase activity of each group was detected by dual-luciferase reporter system after the cells were collected. qRT-PCR and Western blot were used respectively to detect the ACVR1B mRNA and protein levels after transfection of let-7a mimics, let-7a inhibitor and let-7a NC respectively. Results: The bioinformatics analysis revealed that the human ACVR1B mRNA 3’UTR region (position 77-83) contains perfect complementarity region with a seed sequence of let-7a. The luciferase activity of the let-7a mimic and pmirGLO-ACVR1B 3’UTR-WT co-transfected group was significantly lower than the let-7a NC and pmirGLO-ACVR1B 3’UTR-WT co-transfected group (P<0.05), while the luciferase activities of pmirGLO-ACVR1B 3’UTR-MUT transfected groups were no statistical significance (P>0.05). In HEK293T cells, mRNA and protein levels of ACVR1B in let-7a mimic transfected group were significantly lower than let-7a NC group and let-7a inhibitor group (P<0.05), and the expression levels decreased by 70% and 50% respectively. Conclusion: ACVR1B was a potential target gene directly regulated by let-7a and let-7a might suppress the expression of ACVR1B gene in HEK293T cells.

Key words： miRNA let-7a activin receptor type 1B transfection

收稿日期: 2019-02-15

基金资助:国家自然科学基金资助项目（31501164）；浙江省科技计划项目（2017C37127）；浙江省大学生科技创新活动计划项目（2018R413185）；温州市医药卫生科研项目（2018A09，2018A10）；温州市科技计划项目（Y20180505，Y20180489，Y20170254）。

通讯作者: 陈肖鸣，主任医师，教授，硕士生导师，Email：cxm@wmu.edu.cn。

作者简介: 洪子璞（1993-），男，浙江温州人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/10.3969/j.issn.2095-9400.2020.01.002 或 https://xb.wmu.edu.cn/CN/Y2020/V50/I1/7

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