SiRNA沉默Pim-1基因转染MDA-MB231细胞情况对放疗敏感性的影响

Effects of SiRNA silencing Pim-1 gene transfection on the sensitivity of MDA-MB231 cells to radiotherapy

目的使用SiRNA沉默Pim-1基因转染MDA-MB231细胞,分析转染情况对细胞活性及放疗敏感性的影响。方法使用SiRNA转染液对MDA-MB231细胞进行转染,采用Western blot法筛选,根据转染结果分为空白组(没有转染)、阴性组(转染后未测出MDA-MB231/siRNA序列)、阳性1组(转染后测出MDA-MB231/siRNA1序列)、阳性2组(转染后测出MDA-MB231/siRNA2序列)、阳性3组(转染后测出MDA-MB231/siRNA3序列),每组60孔。每组细胞又细分为3个小组,每个小组20孔板,分别接受3个梯度(2 mV、4 mV、8 mV)X线的单次照射。比较5组MDA-MB231细胞增殖率、凋亡率、放疗敏感性蛋白含量(p38、p-p38、Cleaved Caspase-3)的差异。结果5组MDA-MB231细胞增殖率和凋亡率差异均明显(P<0.05),两两比较,阳性1组、阳性2组、阳性3组与空白组(或阴性组)间差异有统计学意义(P<0.05);X线进行细胞照射的剂量越高,细胞增殖率越低(P<0.05)、凋亡率越高(P<0.05);3个阳性组分别与空白组(或阴性组)比较,p-p38、Cleaved Caspase-3蛋白表达含量的差异有统计学意义(P<0.05),阳性组细胞表达含量相对较高。结论SiRNA沉默Pim-1基因转染MDA-MB231细胞可降低细胞增殖率、提高细胞凋亡率,测出siRNA3序列的MDA-MB231细胞尤为明显;细胞放疗敏感性的差异可能与耐药蛋白的产生有关。

Objective To explore the effects of transfection on viability of MDA-MB231 cells and their sensitivity to radiotherapy after they were transfected with SiRNA silencing Pim1 gene.Methods MDA-MB231 cells were transfected with SiRNA transfection solution and screened by Western blot.According to the transfection results,they were divided into blank group(no transfection)and negative group(no MDA-MB231/siRNA sequence detected after transfection),positive group 1(MDA-MB231/siRNA1 sequence detected after transfection),positive group 2(MDA-MB231/siRNA2 sequence detected after transfection),positive group 3(MDA-MB231/siRNA3 sequence detected after transfection),with 60 holes in each group.Cells in each group were subdivided into 3 groups,with 20-well plates in each subgroup,which received a single X-ray of 3 gradients(2 mV,4 mV,and 8 mV).The proliferation rate,apoptosis rate and radiosensitivity protein content(p38,p-p38,Cleaved Caspase-3)of MDA-MB231 cells were compared among the five groups.Results The proliferation rate and apoptosis rate of MDA-MB231 cells in five groups were significantly different(P<0.05).Statistical differences between positive group 1,positive group 2,positive group 3 and the blank group(or the negative group)were found(P<0.05).The higher the dose of X-ray for cell irradiation,the lower the cell proliferation rate(P<0.05),and the higher the apoptosis rate(P<0.05).As compared with the blank group(or negative group),there were significant differences in the expression of p-p38 and Cleaved Caspase-3 protein in positive groups 1,2,and 3(P<0.05),and the expression levels were relatively high in the positive groups.Conclusion Transfection of silencing Pim-1 gene into MDA-MB231 cells can decrease the cell proliferation rate and increase the apoptosis rate,especially for MDA-MB231 cells that detect siRNA3 sequences.The differences in sensitivity of cells to radiotherapy may be related to the production of drug resistant protein.