下调RRM1基因增强胃癌AGS细胞对奥沙利铂敏感性的实验研究

目的 探讨下调RRM1基因对人胃癌AGS细胞奥沙利铂敏感性的影响。方法 人工构建RRM1 siRNA1、RRM1 siRNA2、RRM1siRNA3 3条特异性干扰片段转染至胃癌AGS细胞，同时设空白对照组和阴性对照组（非特异性干扰片段siRNA）；实时荧光定量PCR及蛋白印迹法检测转染后胃癌AGS细胞RRM1 mRNA及蛋白表达，并筛选出转染效果最佳的干扰片段；CCK-8检测不同浓度奥沙利铂处理后各组胃癌细胞的增殖抑制率并计算半数抑制浓度（IC50）；流式细胞仪检测奥沙利铂处理后各组胃癌细胞的凋亡率。结果 RRM1 siRNA1、RRM1 siRNA2、RRM1 siRNA3 3组胃癌AGS细胞RRM1 mRNA表达水平较空白对照组分别下调28％、40％和82％。干扰效果最佳的RRM1 siRNA3组AGS细胞RRM1蛋白相对表达量为0.135±0.017，明显低于空白对照组及阴性对照组的0.641±0.003、0.636±0.056（P＜0.05），故选择siRNA3干扰片段用于后续实验。不同浓度奥沙利铂作用于各组AGS细胞24 h，RRM1 siRNA3组的细胞增殖抑制率均显著低于空白对照组及阴性对照组（P＜0.05），3组胃癌AGS细胞的IC50分别为3.34、5.85和5.13 μmol／L。经3 μmol／L奥沙利铂处理上述3组细胞24 h后，RRM1 siRNA3组的凋亡率为（35.84±2.0）％，显著高于空白对照组和阴性对照组的（27.32±2.6）％和（28.26±1.5）％，差异具有统计学意义（P＜0.05）。结论 下调RRM1基因可增加胃癌细胞对奥沙利铂敏感性，为预测胃癌以奥沙利铂为基础的化疗方案的疗效及制订个体化治疗方案提供了一定的理论依据。

An experimental study on RRM1 silencing increases the sensitivity of gastric cancer AGS cells to oxaliplatin

Objective To determine the inhibitory effect of the synthetic RRM1 siRNA on the expression of RRM1 in human gastric cancer AGS cell lines and investigate the effect of RRM1 siRNA on chemosensitivity of AGS cells to oxaliplatin. Methods Three specific RRM1 interference fragment RRM1 siRNA1， RRM1 siRNA2 and RRM1 siRNA3 were constructed and transfected into gastric cancer AGS cells；meanwhile， blank control group and negative control group were set up. The expression of RRM1 mRNA and protein in the AGS cells were detected by qPCR and Western blotting， and the interfering fragment with best transfection effect was selected. CCK-8 and flow cytometry assay were used to determine cell proliferation and apoptosis of AGS cells treated by different concentrations of oxaliplatin. Results With the transfection of specific RRM1 interference fragment， compared with blank control group， the expression level of RRM1 mRNA for three groups of AGS cells（RRM1 siRNA1， RRM1 siRNA2 and RRM1 siRNA3） were reduced by 28％， 40％ and 82％， respectively. The relative protein expression of AGS cells in RRM1 siRNA3 group was 0.135±0.017， lower than 0.641±0.003 and 0.636±0.056 of blank control group and negative control group（P＜0.05）. siRNA3 interfering fragment was used in the following experiments. With different concentrations of oxaliplatin treated in each group for 24 h， the proliferation inhibited rate in RRM1 siRNA3 group was higher than those of the other two groups. The IC50 of oxaliplatin to RRM1 siRNA3 group， blank control group and negative control group were 3.34， 5.85 and 5.13 μmol／L. Gastric cells in RRM1 siRNA3 group treated by 3 μmol／L oxaliplatin for 24 h， the rate of apoptosis was（35.84±2.0）％， higher than（28.26±1.5）％ and（27.32±2.6）％ of negative control group and blank control group（P＜0.05）. Conclusion Interfering the expression of RRM1 can effectively enhance the sensitivity of gastric cancer cells to oxaliplatin. It provides a theoretical support for efficacy prediction of oxaliplatin-based regimen and individualized regimen design.