| 谭芳,胡娟,李擎,杨晓燕,雷小勇.miR-1290与RASAL2基因靶向关系的探索.[J].中南医学科学杂志.,2021,(1):46-50. |
| miR-1290与RASAL2基因靶向关系的探索 |
| Exploration of the targeting relationship between miR-1290 and RASAL2 gene |
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| DOI：10.15972/j.cnki.43-1509/r.2021.01.009 |
| 中文关键词: RASAL2 3′-非翻译区 双荧光素酶报告基因 miR-1290 |
| 英文关键词:RASAL2 3′-untranslated region dual luciferase reporter vector miR-1290 |
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| 中文摘要: |
| 目的通过构建RASAL2 3′-非翻译区(3′-UTR)双荧光素酶报告载体,探索RASAL2与非编码核糖核酸(ncRNA)miR-1290的靶向调控情况。方法借助在线数据库Targetscan预测miR-1290与RASAL2结合位点；利用聚合酶链反应(PCR)技术扩增RASAL2基因3′-UTR序列,并以GV272为载体将其连接,分别构建野生型GV272-RASAL2-wt 3′-UTR和突变型GV272-RASAL2-mut 3′-UTR荧光素酶报告基因重组质粒；并将miR-1290及相应阴性对照分别与这两种重组质粒共转染到293T细胞中,通过检测各组荧光素酶活性,探索miR-1290与RASAL2基因的结合情况。结果酶切和测序数据表明:野生型GV272-RASAL2-wt 3′-UTR及突变型GV272-RASAL2-mut 3′-UTR双荧光素酶报告载体构建成功；相较于miR-1290与突变型GV272-RASAL2-mut 3′-UTR共转染组,miR-1290与野生型GV272-RASAL2-wt 3′-UTR共转染组的荧光素酶活性无明显变化(P＞0.05)。结论成功构建了RASAL2 3′-UTR的双荧光素酶报告基因载体,但miR-1290不能与RASAL2结合,不能抑制其表达。 |
| 英文摘要: |
| To construct RASAL2 3′-untranslated region (3′-UTR) dual luciferase reporter vector, and explore the targeted relationship between non-coding ribonucleic acid (ncRNA) miR-1290 and RASAL2. MethodsOnline database Targetscan was used to predict the binding sites between miR-1290 and RASAL2. Polymerase Chain Reaction (PCR) was used to amplify RASAL2 3′-UTR sequence,which was then cloned into GV272 vector to construct wild type GV272-RASAL2-wt 3′-UTR and mutant GV272-RASAL2-mut 3′-UTR dual luciferase report plasmid. The miR-1290 or negative control was co-transfected into 293T cells with wild-type GV272-RASAL2-wt 3′-UTR or mutant GV272-RASAL2-mut 3′-UTR dual luciferase reporter plasmid, respectively. Cotransfection of miR-1290 or negative control with wild type GV272-RASAL2-wt 3′-UTR or mutant GV272-RASAL2-mut 3′-UTR dual luciferase report plasmid to 293T cells was conducted, and the luciferase activity was detected via dual luciferase reporter system to explore the combine relationship between miR-1290 and RASAL2 gene. ResultsThe enzyme digestion and sequencing data showed that the wild type GV272-RASAL2-wt 3′-UTR and mutant GV272-RASAL2-mut 3′-UTR dual luciferase report vector were constructed successfully. Compared with the miR-1290 co-transfected with mutant GV272-RASAL2-mut 3′-UTR, the luciferase activity of miR-1290 co-transfected with wild-type GV272-RASAL2-wt 3′-UTR did not change significantly. ConclusionThe RASAL2 3′-UTR dual luciferase reporter vector was constructed successfully. miR-1290 could not combine with RASAL2 3′-UTR, and inhibit its expression. |
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