双荧光素酶报告基因系统验证hsa-miR-1291对ARHGAP29基因的调控作用

doi:10.3969/j.issn.1006-7795.2021.01.009

首都医科大学学报 ›› 2021, Vol. 42 ›› Issue (1): 53-57.doi: 10.3969/j.issn.1006-7795.2021.01.009

双荧光素酶报告基因系统验证hsa-miR-1291对ARHGAP29基因的调控作用

徐倩^*, 汪沙, 刘麒薇, 吕承晓, 甘露, 柳鑫

首都医科大学附属北京妇产医院妇科微创中心,北京 100006

收稿日期:2020-03-25 出版日期:2021-02-21 发布日期:2021-02-02
基金资助:
国家自然科学基金青年基金(81801403),首都医科大学附属北京妇产医院中青年学科骨干培养专项(FCYY201823)。

Regulating role of has-miR-1291 on gene ARHGAP29 verified by dual luciferase reporter system

Xu Qian^*, Wang Sha, Liu Qiwei, Lyu Chengxiao, Gan Lu, Liu Xin

Department of Gynecology Minimally Invasive Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, China

Received:2020-03-25 Online:2021-02-21 Published:2021-02-02
Contact: ^*E-mail:qyxuqian1989@163.com
Supported by:
Youth Fund of Natural Science Foundation of China (81801403);Beijing Obstetrics and Gynecology Hospital,Capital Medical University(FCYY201823).

摘要/Abstract

摘要： 目的验证hsa-miR-1291对ARHGAP29基因的靶向调控作用。方法利用PCR方法,根据目的基因ARHGAP29的3'非编码区(3' untranslated region,3'UTR)序列信息设计扩增引物,以293T细胞基因组DNA为模板,扩增ARHGAP29基因的野生型3'UTR片段(ARHGAP29-WT 3'UTR)及突变型3'UTR片段(ARHGAP29-MUT 3'UTR),并将其分别克隆到双荧光素酶报告pmiR-RB-Report载体中,构建双荧光素酶基因报告载体,即野生型载体ARHGAP29-WT 3'UTR pmiR-RB-Report和突变型载体ARHGAP29-MUT 3'UTR pmiR-RB-Report。将hsa-miR-1291 mimic、阴性对照(non-target control,NC组)同野生型载体、突变型载体载体分别共转染于293T细胞中,进一步检测相对荧光值。结果成功构建ARHGAP29-WT 3'UTR pmiR-RB-Report载体(野生型载体)、ARHGAP29- MUT 3'UTR pmiR-RB-Report载体(突变型载体)。荧光检测结果显示,野生型载体转染hsa-miR-1291 mimic后,其荧光表达较NC组明显下降(0.67±0.04 vs 1.00±0.08, P=0.014);转染hsa-miR-1291 mimic后,突变型载体的荧光表达相对于野生型载体的荧光表达有所上升(1.20±0.05 vs 0.67±0.04, P<0.001)。结论初步证实hsa-miR-1291很可能对ARHGAP29 3'UTR上的该位点有靶向调控作用。

关键词: 双荧光素酶报告基因系统, hsa-miR-1291, ARHGAP29, 宫腔粘连

Abstract: Objective To verify the targeted-regulating effect of hsa-miR-1291 on ARHGAP29 gene. Methods PCR was used to amplify the 3'UTR sequence of the target gene ARHGAP29, using the genome DNA of 293T cells as template and the amplification primer designed based on the 3'UTR sequence information of the gene. After cloning the amplification of ARHGAP29-WT 3'UTR or ARHGAP29-MUT 3'UTR into the pmiR-RB-Report vector, dual-luciferase reporter system was conducted. For further detection of relative fluorescence values, hsa-miR-1291 mimic or Non-target Control were respectively co-transfected into 293T cells with ARHGAP29-WT 3'UTR pmiR-RB-Report(wild-type vector) or ARHGAP29-MUT 3'UTR pmiR-RB-Report(mutant vector). Results ARHGAP29-WT 3'UTR pmiR-RB-Report vector (wild-type vector) and ARHGAP29-MUT 3'UTR pmiR-RB-Report vector (mutant vector) were successfully constructed. Fluorescence test showed that after transfection with wild-type vector, fluorescence expression decreased significantly in hsa-miR-1291 mimic group compared with non-target control (NC) group (0.67±0.04 vs 1.00±0.08, P=0.014). After transfection with hsa-miR-1291 mimic, the fluorescence expression of the mutant vector increased compared with that of the wild-type vector (1.20±0.05 vs 0.67±0.04, P<0.001). Conclusion These data suggested that has-miR-1291 may have a targeted regulatory effect on ARHGAP29.

Key words: dual luciferase reporter system, hsa-miR-1291, ARHGAP29, intrauterine adhesions

中图分类号:

Q752

徐倩, 汪沙, 刘麒薇, 吕承晓, 甘露, 柳鑫. 双荧光素酶报告基因系统验证hsa-miR-1291对ARHGAP29基因的调控作用[J]. 首都医科大学学报, 2021, 42(1): 53-57.

Xu Qian, Wang Sha, Liu Qiwei, Lyu Chengxiao, Gan Lu, Liu Xin. Regulating role of has-miR-1291 on gene ARHGAP29 verified by dual luciferase reporter system[J]. Journal of Capital Medical University, 2021, 42(1): 53-57.

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双荧光素酶报告基因系统验证hsa-miR-1291对ARHGAP29基因的调控作用

Regulating role of has-miR-1291 on gene ARHGAP29 verified by dual luciferase reporter system

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