PERK信号通路介导结肠癌细胞对5-氟尿嘧啶耐药机制的研究

PERK signaling pathway mediates 5-fluorouracil resistance in colon cancer cells

目的:观察PERK蛋白对结肠癌细胞药物敏感性的影响,并进一步探讨其相关作用机制。方法:结肠癌细胞系HCT116分为三组:空白对照(Control)组、下调PERK表达(si-PERK)组、阴性对照(si-NC)组;采用免疫荧光及RT-PCR验证转染效率;利用CCK-8实验检测下调PERK表达后结肠癌细胞对化疗药物5-FU的敏感性变化;Annexin V-FITC凋亡实验检测下调PERK表达对结肠癌细胞凋亡的影响;利用RT-PCR及Western Blot实验检测PERK信号通路中关键分子eIF2α、ATF4、CHOP、XIAP的mRNA及蛋白表达变化。结果:RT-PCR实验表明:与正常对照组相比,si-PERK组mRNA的表达显著下降(P<0.05),免疫荧光提示转染效率达80%以上;CCK-8实验发现与si-NC组相比,5-FU对si-PERK组细胞的半数抑制浓度(IC 50)明显降低(P<0.01);Annexin V-FITC凋亡实验发现与si-NC组相比,si-PERK组细胞的凋亡发生率显著升高(P<0.05);RT-PCR及Western Blot实验发现与si-NC组相比,si-PERK组细胞中PERK信号通路下游关键分子eIF2α、ATF4、CHOP的mRNA及蛋白表达均明显降低(P<0.05)。结论:在结肠癌细胞中,抑制PERK表达后,其可能通过下调eIF2α、ATF4、CHOP的表达促进细胞发生凋亡,从而促进细胞对化疗药物5-FU的敏感性。

Objective:To observe the effect of PERK protein on the chemotherapeutic resistance of colon cancer cells and to further explore its related mechanism.Methods:Colon cancer cell line HCT116 was divided into three groups:Blank control group(Control),down-regulation of PERK expression(si-PERK)group and negative control group(si-NC).Immunofluorescence and RT-PCR were used to verify the transfection efficiency.CCK-8 assay was used to detect the sensitivity of colon cancer cells to 5-FU after down-regulation of PERK expression.Annexin V-FITC apoptosis test was used to detect the effect of down-regulation of PERK expression on apoptosis of colon cancer cells.RT-PCR and Western Blot were used to detect the changes of the expression of key molecules eIF2alpha,ATF4,CHOP and XIAP in PERK signaling pathway.Results:RT-PCR assay showed that compared with the control group,the expression of mRNA in the si-PERK group decreased significantly(P<0.05),and the transfection efficiency of immunofluorescence was more than 80%.CCK-8 assay showed that compared with the si-NC group,the half inhibitory concentration(IC 50)of 5-FU on the cells of si-PERK group was significantly lower(P<0.01).Annexin V-FITC apoptotic experiment showed that compared with the si-NC group,the apoptotic rate of cells in the si-PERK group was significantly higher(P<0.05).RT-PCR and Western Blot experiments showed that compared with the si-NC group,the expressions of eIF2alpha,ATF4 and CHOP,the downstream key molecules of PERK signaling pathway,were significantly lower in the si-PERK group(P<0.05).Conclusion:After inhibiting the expression of PERK in colon cancer cells,it may promote cell apoptosis by down-regulating the expression of eIF2alpha,ATF4 and CHOP,thus promoting cell sensitivity to 5-FU.