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目的:探讨胃癌细胞中膜型黏蛋白1(mucin1,MUC1)的表达对曲妥珠单抗耐药的影响。方法:应用CCK-8(cell counting kit-8)法检测胃癌MKN45和NCI-N87细胞对曲妥珠单抗的敏感性差异,筛选出耐药细胞株。蛋白质印迹法检测胃癌细胞中表皮生长因子受体-2(human epidermal growth factor receptor2,HER2)和MUC1蛋白的表达水平,实时荧光定量PCR检测胃癌细胞中HER2和MUC1mRNA的表达。采用小干扰RNA(small interfering RNA,siRNA)转染技术特异性沉默耐药细胞中MUC1基因的表达,CCK-8法检测MUC1基因沉默前后耐药细胞株对曲妥珠单抗的敏感性变化。结果:不同质量浓度的曲妥珠单抗(0.01、0.03、0.1、0.3和1μg/mL)处理胃癌细胞MKN45和NCI-N87后,随着药物浓度的增加,NCI-N87细胞的生长抑制率相应增高(P<0.05),而MKN45细胞则无明显抑制(P>0.05)。两株胃癌细胞之间HER2mRNA及蛋白表达水平比较的差异均无统计学意义(P>0.05),而MKN45细胞的MUC1mRNA及蛋白表达水平显著高于NCI-N87细胞(P<0.05)。与空白对照组和阴性转染组比较,转染MUC1-siRNA后,MKN45细胞中MUC1的蛋白及mRNA表达水平显著下降,而细胞增殖未受到抑制,但细胞对曲妥珠单抗的敏感性显著增加(P<0.05)。结论:HER2阳性的胃癌MKN45细胞对曲妥珠单抗耐药,而沉默MUC1表达可使其对曲妥珠单抗的耐药性得到逆转,提示MUC1过表达可能促进或诱导了胃癌细胞对曲妥珠单抗的耐药性。6
Objective: To investigate the effect of mucin1 (MUC1) expression on trastuzumab resistance in gastric cancer cells. Methods: The sensitivity of MKN45 and NCI-N87 cells to trastuzumab was detected by CCK-8 (cell counting kit-8), and drug-resistant cell lines were screened out. The expression of HER2 and MUC1 protein in gastric cancer cells was detected by Western blotting. The expression of HER2 and MUC1 mRNA in gastric cancer cells was detected by real-time fluorescence quantitative PCR. The expression of MUC1 gene was detected by using small interfering RNA (siRNA) transfection technique. The sensitivity of cell line to MUC1 gene silencing to trastuzumab was detected by CCK-8 assay. Results: After treated with different concentrations of trastuzumab (0.01,0.03,0.1,0.3 and 1μg / mL), the growth inhibition rate of NCI-N87 cells increased with the increase of the drug concentration in gastric cancer cells MKN45 and NCI-N87 (P <0.05), while MKN45 cells had no significant inhibition (P> 0.05). There was no significant difference in the expression of HER2 mRNA and protein between the two gastric cancer cells (P> 0.05), while the expression of MUC1 mRNA and protein in MKN45 cells was significantly higher than that of NCI-N87 cells (P <0.05). Compared with the blank control group and the negative transfection group, the expression of MUC1 protein and mRNA in MKN45 cells decreased significantly after transfection of MUC1-siRNA, but the cell proliferation was not inhibited, but the sensitivity of the cells to trastuzumab was significant Increase (P <0.05). CONCLUSIONS: HER2-positive gastric cancer MKN45 cells are resistant to trastuzumab, whereas silencing MUC1 expression reverses its resistance to trastuzumab, suggesting that overexpression of MUC1 may promote or induce gastric cancer cell dysfunction Resistant to trastuzumab. 6