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目的构建特异AT序列结合蛋白2(SATB2)的3′非翻译区(3′UTR)荧光素酶报告基因载体行微小RNA-31(miR-31)靶基因验证。方法应用生物信息学预测软件TargetScan、MirBase和PicTar对miR-31靶基因进行预测,选定靶基因SATB2的3′UTR克隆到带有萤火虫荧光素酶的真核表达载体(PGL3-control)下游,通过双荧光素酶报告基因检测miR-31对靶基因SATB2的3′UTR的调控能力。Western blot检测miR-31模拟物/抑制物瞬时转染人高转移肺腺癌细胞95D对SATB2蛋白表达的影响。结果成功构建SATB2的3′UTR表达载体PGL3-SATB2。双荧光素酶报告基因分析表明,miR-31能够作用于SATB2的3′UTR。Western blot检测结果显示,与空白组相比,转染miR-31模拟物组的SATB2的蛋白表达降低(P<0.01),而转染miR-31抑制物组的SATB2的蛋白表达升高(P<0.01)。结论 miR-31作用于SATB2的3′UTR可在转录后水平上调SATB2蛋白的表达。SATB2是miR-31的靶基因,有望成为肿瘤生物治疗的靶点。
Objective To construct a microRNA-31 (miR-31) target gene by constructing a 3 ’untranslated region (3’UTR) luciferase reporter vector with specific AT sequence binding protein 2 (SATB2). Methods The miR-31 target gene was predicted by bioinformatics prediction software TargetScan, MirBase and PicTar. The 3’UTR of the target gene SATB2 was cloned downstream of the PGL3-control with firefly luciferase. The dual luciferase reporter gene was used to detect the regulation of miR-31 on the 3’UTR of the target gene SATB2. Western blot was used to detect the effect of miR-31 mimics / inhibitor transient transfection on the expression of SATB2 protein in human highly metastatic lung adenocarcinoma cell line 95D. Results The 3’UTR expression vector PGL3-SATB2 was successfully constructed. Dual luciferase reporter gene analysis showed that miR-31 can act on the 3’UTR of SATB2. Western blot results showed that compared with the blank control group, the protein expression of SATB2 in transfected miR-31 mimics group was decreased (P <0.01), while the expression of SATB2 protein in transfected miR-31 inhibitor group was increased <0.01). Conclusion The 3’UTR acting on SATB2 by miR-31 can up-regulate the expression of SATB2 protein at the post-transcriptional level. SATB2 is the target gene of miR-31 and is expected to be the target of tumor biotherapy.