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目的:探讨RNA干扰(RNAi)技术对骨肉瘤细胞株(MG-63)中HMGA1基因表达和侵袭力的影响。方法:采用单细胞克隆技术,从MG-63中分离培养出高表达HMGA1的骨肉瘤细胞株,将细胞分成3组,通过pU6mRFP载体进行转染:实验组,转染HMGA1的小干扰RNA(siRNA);阴性对照组,转染HMGA1的无关序列;细胞对照组为未转染的MG-63细胞。转染细胞株后,荧光检测转染效率;用实时荧光定量RT-PCR和蛋白质印迹法分别从mRNA和蛋白质水平检测siRNA对HM-GA1表达的影响;Transwell趋化侵袭实验观察转染后细胞株侵袭能力的变化。结果:荧光检测转染效率,实验组为(55.68±6.74)%,阴性对照组为(49.87±4.33)%;细胞对照组观察不到明显的荧光细胞。实验组HMGA1siRNA转染骨肉瘤细胞后,明显下调细胞中HMGA1mRNA及蛋白的表达,与转染时间和转染浓度相关;阴性对照组和细胞对照组在转染前后HMGA1基因的mRNA及蛋白质表达均无明显变化。实验组细胞株穿过侵袭膜的细胞数明显低于阴性对照组和细胞对照组,P<0.01。结论:HMGA1siRNA可以下调骨肉瘤细胞中HMGA1mRNA及其蛋白的表达,抑制骨肉瘤细胞侵袭能力。
Objective: To investigate the effect of RNA interference (RNAi) technology on HMGA1 gene expression and invasiveness in osteosarcoma cell line MG-63. Methods: The osteosarcoma cell lines with high expression of HMGA1 were isolated and cultured from MG-63 cells by single cell cloning technique. The cells were divided into 3 groups and transfected by pU6mRFP vector: experimental group, small interfering RNA (siRNA) transfected with HMGA1 ); Negative control group, transfected with HMGA1 irrelevant sequences; Cell control group was untransfected MG-63 cells. Transfection of cell lines, fluorescence detection of transfection efficiency; real-time fluorescent quantitative RT-PCR and Western blot were detected mRNA and protein levels of HM-GA1 expression; Transwell chemotactic invasion assay transfected cell lines Changes in invasive ability. Results: The transfection efficiency was detected by fluorescence assay (55.68 ± 6.74%) in the experimental group and (49.87 ± 4.33)% in the negative control group. No obvious fluorescent cells were observed in the cell control group. The expression of HMGA1 mRNA and protein in the experimental group was significantly decreased after transfected with osteosarcoma cells, which was related to the transfection time and transfection concentration. The mRNA and protein expression of HMGA1 in the negative control group and the cell control group were not obvious change. The number of cells in the experimental group crossing the invasion membrane was significantly lower than that in the negative control group and the cell control group (P <0.01). Conclusion: HMGA1 siRNA can down-regulate the expression of HMGA1 mRNA and protein in osteosarcoma cells and inhibit the invasion ability of osteosarcoma cells.