沉默Ki-67基因对乳腺癌MCF-7/DOX细胞多柔比星耐药性的影响  

Effects of silencing Ki-67 gene on doxorubicin resistance of breast cancer MCF-7/DOX cells

作　　者：董军 崔凤鸣 刘军 DONG Jun;CUI Fengming;LIU Jun(Department of Thyroid and Breast Surgery,Nanjing Jiangbei Hospital,Jiangsu Nanjing 210048,China)

机构地区：[1]南京江北医院甲乳外科,江苏南京210048

出　　处：《外科理论与实践》2023年第3期254-259,共6页Journal of Surgery Concepts & Practice

摘　　要：目的:研究沉默Ki-67基因对乳腺癌细胞多柔比星(doxorubicin,DOX)耐药性的影响。方法:研究采用MCF-7/DOX细胞和脂质体瞬时转染法,用小干扰RNA(small interfering RNA,siRNA)转染和阴性对照(negative control,NC)siRNA转染体外培养乳腺癌MCF-7细胞。转染48 h后采用反转录PCR(RT-PCR)检测Ki-67 mRNA的表达。后续实验用干扰效率最高的Ki-67 siRNA。共分为3个组:MCF-7空白对照(CON组)、MCF-7 NC siRNA转染(NC组)和MCF-7 siRNA转染(siRNA组)。用RT-PCR和蛋白质印迹法检测3组细胞的Ki-67 mRNA表达和相关蛋白质表达。用不同浓度的DOX处理3组MCF-7/DOX细胞,MTT法检测细胞增殖。结果:特异性siRNA成功转染MCF-7/DOX细胞后,荧光显微镜下观察,见siRNA均匀分布在细胞质内,转染效率达85%以上。转染MCF-7 siRNA后,不同程度降低了MCF-7/DOX细胞Ki-67 mRNA表达,Ki-67蛋白的表达水平显著降低,抑制率为78.51%,与NC组和CON组比较,差异具有统计学意义(P<0.05)。MTT检测结果示siRNA干扰Ki-67mRNA表达后,MCF-7/DOX细胞的体外增殖能力被显著抑制。siRNA组MCF-7/DOX细胞对DOX的半抑制浓度为(7.45±0.18)μmol/L,显著低于NC组(55.19±2.86)μmol/L和CON组(56.43±4.22)μmol/L,相对于CON组的耐药逆转倍数达7.69倍(P<0.05)。siRNA组不同浓度DOX对细胞的抑制作用显著高于NC组和CON组(P<0.05)。结论:siKi-67能有效抑制Ki-67基因的表达,抑制乳腺癌MCF-7/DOX细胞增殖,以及部分逆转乳腺癌MCF-7/DOX细胞对DOX的耐药性。ObjectiveTo study the effect of Ki-67 gene silencing on doxorubicin(DOX) resistance in breast cancer MCF-7 cells.MethodsLiposome transient transfection method and breast cancer MCF-7 cells cultured in vitro were used combined with the transfection of small interfering RNA(siRNA) and negative control(NC) siRNA.The expression of Ki-67mRNA was detected by RT-PCR after 48 h of transfection.Ki-67 siRNA with the highest interference efficiency was used in subsequent experiments including three groups:control(CON) group without transfection,NC group transfected with MCF-7 negative control siRNA and siRNA group with MCF-7 transfected with Ki-67 siRNA.The expression of Ki-67mRNA and related proteins in three groups were detected by RT-PCR and Western blot.MCF-7/DOX cells were treated with DOX at different concentrations.Proliferation of MCF-7/DOX cells was detected by MTT assay.ResultsAfter the specific siRNA was successfully transfected into MCF-7/DOX cells,it was observed under fluorescence microscope that siRNA was evenly distributed in the cytoplasm,and transfection efficiency was more than 85%.Results of transfection of MCF-7 siRNA cells showed that Ki-67 mRNA expression in MCF-7 cells reduced Ki-67 at varying degrees.The expression of Ki-67 protein reduced significantly with the inhibition of 78.51% and the statistically significant difference compared with that in NC group and CON group(P<0.05).MTT showed that the proliferation ability of MCF-7/DOX cells in vitro was inhibited significantly after Ki-67 mRNA expression interfered by siRNA.The half maximal inhibitory concentration(IC50)of DOX to MCF-7/DOX cells in siRNA group was(7.45±0.18) μmol/L,lower significantly than(55.19±2.86) μmol/L in NC group and(56.43±4.22) μmol/L in CON group.The reversal of drug resistance was 7.69 times compared with that in CON group(P<0.05).The inhibitory effect of DOX at different concentrations on the cells in siRNA group was significantly higher than that in NC group and CON group with statistically significant difference(P<0

关 键 词：乳腺癌 KI-67 多柔比星 凋亡 耐药性 

分 类 号：R737.9[医药卫生—肿瘤]