目的 探究siRNA下调含SET结构域(赖氨酸甲基转移酶)8(SETD8)基因表达对骨肉瘤MG-63细胞增殖、侵袭和迁移的影响及其可能的机制.方法 采用脂质体法将特异性针对SETD8基因的siRNA转入骨肉瘤MG-63细胞中,得到关于SETD8基因沉默表达的MG-63稳定的细胞系即实验组,将杂序siRNA用脂质体法同时转染骨肉瘤MG-63细胞,得到关于Control siRNA 的MG-63细胞系即对照组,未经转染的骨肉瘤MG-63的稳定的细胞系即空白组.以上三组细胞系通过应用细胞毒性活性检测法(CCK-8法)、平板克隆法同时检测被SETD8基因沉默后骨肉瘤MG-63细胞的增殖能力,侵袭迁移小室即(Transwell小室)法检测骨肉瘤MG-63细胞的侵袭和迁移能力,实时荧光定量法即(PCR法)以及蛋白质印迹法即(Western Blot法)检测SETD8基因沉默表达效果及细胞内SETD8,β- catenin,cyclinD1,vimentin、MMP3 的mRNA和相应蛋白的表达水平.结果 SETD8 siRNA转染后,骨肉瘤MG-63细胞的增殖能力显著低于对照组(control siRNA转染MG-63细胞)和空白组(未经转染的MG-63细胞)(P ＜ 0.05);Transwell小室结果 显示,沉默SETD8基因组的侵袭和迁移能力明显下降;实验组(SETD8 siRNA转染MG-63细胞)中SETD8、β - catenin、cyclinD1、vimentin、MMP3 的mRNA 及蛋白表达水平显著低于阴性对照组和空白对照组(P ＜ 0.05).结论 siRNA下调SETD8基因表达可以抑制骨肉瘤MG-63细胞的增殖、侵袭和迁移能力,Wnt/β - catenin信号通路可能参与并调控这一过程.%Objective To investigate the effect of siRNA - induced down - regulation of lysine methyltransferase 8 gene on proliferation of human osteosarcoma MG - 63 cells and the regulation of this process by Wnt / β - catenin signaling pathway. Methods The siRNA specific for SETD8 gene was transferred into MG - 63 cells of osteosarcoma by liposome method,and the MG - 63 cell line with silencing expression of SETD8 gene was obtained as experimental group. The heterologous siRNA was transfected into MG - 63 cells of osteosarcoma by liposome method at the same time. The MG - 63 cell line about Control siRNA was obtained as the control group. The stable cell line of MG - 63 in the untransfected osteosarcoma was obtained as the blank group. The above three cell lines passed through by CCK - 8 method and plate clone method to detect the proliferative ability of MG - 63 cells in osteosarcoma. Detection of invasion and migration of osteosarcoma MG - 63 cells by Transwell chamber assay. The expression level of SETD8,β - catenin D1,cyclinD1,vimentin,MMP3 mRNA and protein were detected by real - time fluorescent quantitative - PCR and Western blotting,respectively. Results After transfection of SETD8 siRNA,The proliferative ability of osteosarcoma MG - 63 cells was significantly lower than control group (control siRNA transfected MG - 63 cells) and blank group (untransfected MG - 63 cells) (P ＜ 0. 05). The results of Transwell chamber showed that the ability of invasiveness and migration of silencing SETD8 genome was significantly decreased. The mRNA and protein expression levels of SETD8,β - catenin,cyclinD1,vimentin,MMP3 in SETD8 siRNA transfection group were lower than those of the negative control and blank control groups (P ＜ 0. 05). Conclusion Down - regulation of SETD8 gene expression by siRNA may inhibit the proliferation,invasion and migration of osteosarcoma MG - 63 cells. Wnt /β - catenin signaling pathway may be involved in the regulation of this process.
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