miR-506-3p对PIK3CA基因的靶向调控作用

摘要

Objective To investigate the effect of miR-506-3p via targeting PIK3CA of PI3K-Akt pathway. Methods By using commercially available miR-506-3p mimic and inhibitor,we constructed PIK3CA 3'UTR wild-type and mutant into luciferase reporter plasmid. Then,the sequenced and positive recombinants were transfected into HEK-293T cells with miR-506-3p mimic or inhibitor and pRL-TK. The targeting effect of miR-506-3p on PIK3CA was verified by the dual-luciferase reporter assay system and Western blot. Results The recombinant plasmids were constructed successfully and Western blot texts confirmed that the overexpression of miR-506-3p suppressed the protein level of PIK3CA significantly(P<0.05),whereas the protein level of PIK3CA was increased by suppression of miR-506-3p accordingly(P<0.05).Conclusion MiR-506-3p can directly target at the gene of PIK3CA. It regulates at the protein level,and lays the foundation of miR-506-3p for further research on the signaling pathways.%目的 研究miR-506-3p对PI3K-Akt信号通路中关键基因PIK3CA的靶向调控机制.方法 化学合成miR-506-3p过表达模拟物mimic及miR-506-3p抑制剂inhibitor;构建含有PIK3CA基因3'-UTR野生型及突变体到荧光素酶报告载体中;将测序结果正确的pMIR-Report-PIK3CA-Wt和pMIR-Report-PIK3CA-Mut重组质粒,与miR-506-3p的mimic/inhibitor及pRL-TK共转染至HEK-293T细胞,利用双荧光素酶报告系统和Western blot验证miR-506-3p与PIK3CA的靶向关系.结果 测序结果显示pMIR-Report-PIK3CA-Wt和pMIR-Report-PIK3CA-Mut两个重组质粒构建成功,Western blot结果显示转染miR-506-3p mimic的293T细胞中PIK3CA蛋白表达下调(P<0.05);而转染miR-506-3p inhibitors的PIK3CA蛋白表达上调(P<0.05).结论 miR-506-3p可作用于PI3K-AKt信号通路,通过靶向PIK3CA发挥其负性调控作用.