RORα miRNA真核表达载体构建及对人胃癌细胞增殖的影响

Construction of human ROR alpha miRNA eukaryotic expression vector and its effects on proliferation in gastric cancer cells

目的构建RORα(Retinoid acid receptor related orphan receptorα,维甲酸相关孤核受体α)miRNA真核表达载体,分析其对人胃癌MGC803细胞RORα蛋白表达及细胞增殖的影响。方法构建针对设计RORαmiRNA表达的3对miRNA干扰序列,克隆入pcDNATM6.2-GW/EmGFP miRNA真核表达载体,在测序鉴定无误后将含RORαmiRNA转染人胃癌MGC803细胞,用Western blo检测MGC803细胞转染前后RORα蛋白的表达水平变化。MTT法检测对转染MGC803细胞增殖的影响。结果 Western blot结果证实,MGC803细胞转染RORαmiRNA后24h RORα蛋白表达水平明显低于转染阴性组(P<0.05)。MTT结果显示,转染RORαmiRNA后细胞增殖率(光吸收值)高于转染阴性组(P<0.05)。结论 miRNA靶向抑制RORα表达后,可促进人胃癌MGC803细胞增殖,提示RORα是一个潜在的胃癌基因及药物治疗的靶点。

Objective To construct a ROR alpha miRNA expression vector,and evaluate its effects on ROR alpha protein expressions and proliferation in MGC803 gastric cancer cells in vitro. Methods Three pairs of RNA interference sequences of ROR alpha targetting RORα miRNA were cloned into the eukaryotic expression vector pcDNATM6.2-GW/EmGFP.After sequencing,the vector was then transfected into MGC803 cells. The change of ROR alpha protein expression in transfected cells were demonstrated by Western blot.The impact on proliferation in transfected MGC803 cells was investigated by MTT method. Results Western blot showed that the level of ROR alpha protein in gastric cancer MGC803 cells was distinctly decreased after transfection with ROR alpha miRNA for 24h.The OD570 value of miR-ROR alpha 24h transfection group was higher than negative control group showed by MTT method （P0.05）. Conclusion In MGC803 gastric cancer cells,ROR alpha is involved in the regulation of proliferation,suggesting ROR alpha is a potential target for treating gastric cancer with genes and drugs.