RNA干扰技术抑制Galectin-3表达对三阴性乳腺癌细胞 MDA-MB-231增殖和凋亡的影响

摘要

Objective To observe the relationship of expression of Galectin-3 with the proliferation and apoptosis of tumor cells and explore the RNA interference technique for anticancer gene therapy . Methods Galectin-3 small interfering RNA ( siRNA ) was constructed by chemistry synthesis. Effective siRNA was transfected into breast cancer cell MDA-MB-231( as Galectin-3siRNA group ). Non-transfected cells were used as blank control and negative RNA as negative control. Inverted fluorescence microscope and RT-PCR were used to verify the interference result. Cellproliferation ( absorbance value ) was observed by MTT after interference for 24, 48, 72 and 96 hours. Cell apoptosis was measured with flow cytomter. Wound healing test and transwell test were performed for cell mo-tility and invasion assay. Results ①After transfected 24 hours,the transfection efficiency rate by inverted fluorescence microscope was ( 80. 60 ± 5. 62 )% . RT-PCR showed that the expression of Galectin-3 was markedly decreased in Galectin-3 siRNA group after transfected ( P < 0. 01 ).② Cells proliferated still in the Galectin-3 siRNA group after 24, 48 , 72 and 96 hours, but very slow, and the inhibition rate was lower than that in the normal and blank control group( P <0. 05 ). ③The apoptosis rate by flow cytomter was slight higher in the Galectin-3 siRNA group than that in the normal and blank control group ( P <0. 01 ). ④The results of Transwell experiment indicated that the number of cells in down-pore of micro-membrane in the transfected group was less than that in the control group. Wound-healing assay shows that cell motility could be effectively suppressed by Galectin-3siRNA( P < 0. 01 ). Conclusion The interference of RNA inhibits the expression of the Galectin-3 and the proliferation of MDA-MB-231 cells significantly, while it promotes the apoptosis of tumor cells and impedes tumour cell invasion.%目的 观察半乳凝集素-3(Galectin-3)表达与肿瘤细胞增殖和凋亡的关系,探讨利用RNA干扰技术作为抗肿瘤基因治疗的方法.方法 设计并化学合成Galectin-3小干扰片段并瞬时转染乳腺癌细胞系MDA-MB-231(Galectin-3siRNA组),以未转染细胞为空白对照组,以转染无关siRNA为阴性对照组.荧光倒置显微镜和实时定量PCR检测干扰结果,MTT法观察干扰后24、48、72、96 h各组细胞增殖吸光度(A)值,流式细胞仪检测各组细胞凋亡情况,划痕实验和Transwell实验比较不同组间细胞迁移和侵袭情况.结果 ①转染后24 h,荧光倒置显微镜显示转染效率为(80.60±5.62)%,实时定量PCR提示Galectin-3siRNA转染后相应Galectin-3的表达明显下降(P<0.01);②干扰后24、48、72、96 h Galectin-3siRNA组细胞继续生长,但细胞生长减慢,抑制率低于空白对照组和阴性对照组(P＜0.05) ;③ 流式细胞术分析发现,Galectin-3siRNA组比空白对照组和阴性对照组凋亡率高(P<0.01);④ Transwell侵袭实验显示Galectin-3siRNA组穿膜细胞数少于空白对照组和阴性对照组.细胞划痕实验显示Galectin-3siRNA组细胞迁移能力明显低于空白对照组和阴性对照组(P＜0.01).结论 RNA干扰能抑制Galectin-3的表达以及MDA-MB-231细胞的增殖,并诱导细胞凋亡、降低其迁移侵袭能力.