下调METTL3基因表达影响人骨肉瘤细胞增殖、凋亡和成骨分化的作用及机制探讨

Study on the effect and mechanism of down-regulating METTL3 gene expression on human osteosarcoma cell proliferation,apoptosis and osteogenic differentiation

目的:检测甲基转移酶样3(methyltransferase-like 3,METTL3)基因在骨肉瘤组织中的表达及其对骨肉瘤MG63细胞增殖、凋亡和成骨分化的影响,并初步探讨其作用机制。方法:免疫组织化学染色与实时荧光定量PCR(qRT-PCR)检测骨肉瘤组织与癌旁组织中METTL3表达水平;RNA干扰技术抑制人骨肉瘤MG63细胞中METTL3表达,qRT-PCR和蛋白质印迹法(Western blot)检测METTL3 mRNA和蛋白表达水平来检测抑制效果;CCK-8法和Annexin V-FITC双染法分别检测细胞增殖活性与凋亡情况;碱性磷酸酶染色和茜素红染色检测细胞成骨分化作用;Western blot检测骨钙蛋白(osteocalcin, OCN)、骨桥蛋白(osteopontin, OPN)及Runt相关转录因子2(Runt related transcription factor 2,RUNX2)蛋白表达水平,以及PI3K、Akt和p-Akt蛋白表达水平。结果:骨肉瘤组织中METTL3表达较癌旁组织明显升高(P<0.05),抑制MG63细胞中METTL3表达后,细胞活性显著下降(P<0.05),细胞凋亡率显著增高(P<0.05),成骨诱导MG63细胞后碱性磷酸酶染色和茜素红染色阳性增加,钙盐结节明显增多,OCN、OPN、RUNX2蛋白相对表达量显著升高(P <0.05),PI3K、Akt和p-Akt蛋白相对表达量均显著降低(P <0.05)。结论:METTL3在骨肉瘤组织中高表达,下调METTL3表达可抑制人骨肉瘤细胞增殖,促进凋亡与成骨分化,这一作用可能与抑制PI3K/Akt信号通路有关。

Objective:To detect the expression of methyltransferase-like 3(METTL3)in osteosarcoma tissues and the effect on proliferation,apoptosis and osteogenic differentiation of osteosarcoma MG63 cells,and to preliminarily explore its mechanism of action.Methods:Immunohistochemical staining and real-time fluorescence quantitative PCR(qRT-PCR)were used to detect the expression level of METTL3 in osteosarcoma tissues and adjacent tissues.Inhibit METTL3 expression in human osteosarcoma MG63 cells by RNA interference technology,and detect the expression level of METTL3 mRNA and protein by qRT-PCR and Western blot to detect the inhibitory effect.CCK-8 method and Annexin V-FITC double staining method were used to detect cell proliferation activity and apoptosis.Alkaline phosphatase staining and alizarin red staining were used to detect the osteogenic differentiation of cells.Western blot detected osteocalcin(OCN),osteopontin(OPN)and Runt related transcription factor 2(RUNX2)protein expression levels,as well as PI3K,Akt and p-Akt protein expression levels.Results:The expression of METTL3 in osteosarcoma tissues was significantly increased compared with adjacent tissues(P<0.05).After inhibiting the expression of METTL3 in MG63 cells,the cell activity decreased significantly(P<0.05),and the apoptosis rate increased significantly(P<0.05).After osteogenic induction of MG63 cells,alkaline phosphatase staining and alizarin red staining increased,calcium nodules increased significantly,and relative expression levels of OCN,OPN,and RUNX2 proteins increased significantly(P<0.05).The relative expression of PI3K,Akt,p-Akt proteins were significantly down-regulated(P<0.05).Conclusion:METTL3 is highly expressed in osteosarcoma tissues.Down-regulating the expression of METTL3 can inhibit the proliferation of human osteosarcoma cells and promote apoptosis and osteogenic differentiation.This effect may be related to the inhibition of PI3K/Akt signaling pathway.