miRNA-1对胃癌细胞体外EGFR-TKI敏感性影响的实验研究

Effect of miRNA-1 on EGFR-TKI sensitivity of gastric cancer cells in vitro

目的探讨上调或沉默miRNA-1(miR-1)表达对胃癌细胞体外表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)敏感性影响的研究。方法选择人胃癌细胞系MKN-45作为受试对象,慢病毒转染miR-1或miR-1 shRNA,分别作为miR-1组和miR-1 shRNA组,并采用实时荧光定量PCR(qPCR)法检测GES-1和MKN-45细胞中miR-1表达。另外设置空白对照组和阴性对照组。采用MTT法检测MKN-45细胞对不同浓度AZD9291(奥西替尼)的敏感性;流式细胞术检测1μmol/L AZD9291作用48 h后MKN-45细胞凋亡活力和细胞周期。采用Western blotting法检测各组细胞中PI3K/AKT通路相关蛋白的表达。结果qPCR证实miR-1在MKN-45细胞系中低表达。MTT法检测结果显示,与空白对照组比较,miR-1组细胞对AZD9291敏感性明显增加,而miR-1 shRNA组细胞对AZD9291敏感性明显降低。Annexin V/PI双染法和BrdU法检测,miR-1组细胞凋亡率为(44.65±5.18)%,高于空白对照组的(15.40±2.13)%和阴性对照组的(17.92±3.14)%,差异有统计学意义(P<0.05)。miR-1 shRNA组细胞凋亡率为(11.20±2.48)%,低于空白对照组和阴性对照组,差异有统计学意义(P<0.05)。与空白对照组和阴性对照组比较,miR-1组细胞IGF-1R、p-IGF-1R、p-AKT、p-p70S6K蛋白表达量明显降低,miR-1 shRNA组细胞IGF-1R、p-IGF-1R、p-AKT、p-p70S6K蛋白表达量明显升高,差异有统计学意义(P<0.05)。结论上调miR-1表达可增加MKN-45胃癌细胞系对AZD9291的敏感性,其作用机制可能与抑制IGF-1R/PI3K/AKT信号通路的激活有关。

Objective To investigate the effect of up-regulating or silencing of miRNA-1(miR-1)expression on the sensitivity of epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)in gastric cancer cells in vitro.Methods Human gastric cancer cell line MKN-45 was selected as the test object.Lentivirus was transfected with miR-1 or miR-1 shRNA as miR-1 group and miR-1 shRNA group respectively.The miR-1 expression of GES-1 and MKN-45 cells was detected by real-time fluorescence quantitative PCR(qPCR).In addition,blank control group and negative control group were set.MTT assay was used to detect the sensitivity of MKN-45 cells to different concentrations of AZD9291(Osimertinib);Flow cytometry detection 1μmol/L AZD9291 on apoptosis activity and cell cycle of MKN-45 cells after 48 hours.The effects of PI3K/AKT pathway related proteins in cells of each group were detected by Western blotting.Results qPCR confirmed that miR-1 was low expressed in MKN-45 cell line.MTT assay showed that the sensitivity of miR-1 group cells to AZD9291 increased significantly,while the sensitivity of miR-1 shRNA group cells to AZD9291 decreased significantly.Annexin V/PI double staining method and BrdU method showed that the apoptosis rate of miR-1 group was(44.65±5.18)%,which was higher than that of blank control group(15.40±2.13)%and negative control group(17.92±3.14)%(P<0.05).The apoptosis rate of miR-1 shRNA group was(11.20±2.48)%,which was significantly lower than that of blank control group and negative control group(P<0.05).Compared with the blank control group and negative control group,the expression of IGF-1R,p-IGF-1R,p-AKT and p-p70S6K protein in miR-1 shRNA group decreased significantly,and the expression of IGF-1R,p-IGF-1R,p-AKT and p-p70S6K protein in miR-1 shRNA group increased significantly(P<0.05).Conclusion Up-regulating miR-1 expression can increase the sensitivity of MKN-45 gastric cancer cell line to AZD9291,and its possible mechanism is related to inhibiting the activation of IGF-1R/PI3K/AKT signaling pathway.