miR-505对骨肉瘤细胞增殖、侵袭的影响及相关机制研究

Effect of miR-505 on proliferation and metastasis of osteosarcoma cells and its related mechanisms

摘要:

目的：探讨miR-505对骨肉瘤细胞增殖、侵袭的影响及相关机制。方法：采用q-PCR测定人成骨细胞hFOB1.19及骨肉瘤细胞MG63中的miR-505水平。分别设置miR-505 mimics组与阴性对照 （miR-NC）组，采用LipofectamineTM 2000将miR-505 mimics及miR-NC转染至MG63细胞中，比较２组细胞增殖速率及侵袭细胞数，进一步采用Western blot及q-PCR考察转染miR-505的MG63细胞中MMP-9、CDK4、Cyclin-D1、HMGB-1及β-联蛋白（β-catenin）、磷酸糖原合成酶激酶-3β（GSK-3β）的蛋白及mRNA水平。结果：骨肉瘤细胞MG63中miR-505水平为0.57±0.06，明显低于人成骨细胞hFOB1.19的1.03±0.12（P<0.01）。转染miR-505后，miR-505 mimics组的表达量为3.59±0.37，明显高于miR-NC组的1.09±0.12（P<0.01）。miR-505 mimics组转染48 h和72 h后的OD450 nm值分别为0.84±0.09和1.29±0.13，均明显低于miR-NC组（P<0.05）。miR-505 mimics组侵袭细胞数为（42.84±4.35）个，明显低于miR-NC组的（66.84±6.85）个（P<0.05）。miR-505 mimics组MMP-9、CDK4、Cyclin-D1及HMGB-1蛋白水平均低于miR-NC组（P<0.05）。miR-505 mimics组MMP-9、CDK4、Cyclin-D1及HMGB-1 mRNA水平均低于miR-NC组（P<0.05）。同时，miR-505 mimics组Wnt信号通路中关键靶点β-catenin和GSK-3β蛋白与基因表达均明显低于miR-NC组（P<0.05）。结论：miR-505可通过抑制Wnt信号通路，下调MMP-9、CDK4、Cyclin-D1及HMGB-1表达，从而抑制骨肉瘤细胞的增殖与侵袭。

Abstract:

Objective：To investigate the effect of miR-505 on proliferation and metastasis of osteosarcoma cells and its mechanisms. Methods：The miR-505 level in human osteoblast of hFOB1.19 and osteosarcoma of cell MG63 was determined by q-PCR. The miR-505 mimics group and the negative control group（miR-NC） were set. Both miR-505 mimics and miR-NC were transfected into MG63 cells with Lipofectamine TM 2000 and cell proliferation rate and invasive cells were compared between two groups. Proteins and mRNA levels of MMP-9，CDK4，Cyclin-D1，HMGB-1，β-catenin and GSK-3β in MG63 cells transfected with miR-505 was further detected via Western blot and q-PCR technologies. Results：The expression level of miR-505 in osteosarcoma cell of MG63 was 0.57±0.06，which was significantly lower than that of 1.03±0.12 in human osteoblast of hFOB1.19（P<0.01）. After transfecting miR-505，expression level in the miR-505 mimics group was 3.59±0.37，which was significantly higher than that of 1.09±0.12 in the miR-NC group（P<0.01）. After transfecting 48 h and 72 h，A450 nm values in the miR-505 mimics group were 0.84±0.09 and 1.29±0.13，respectively，which were significantly lower than those in the miR-NC group（P<0.05）. Invasive cells in the miR-505 mimics group was （42.84±4.35），which was significantly lower than that of （66.84±6.85） in miR-NC group（P<0.05）. Protein lev－els of MMP-9，CDK4，Cyclin-D1 and HMGB-1 in the miR-505 mimics group were lower than those in the miR-NC group（P<0.05）. The mRNA levels of MMP-9，CDK4，Cyclin-D1 and HMGB-1 in the miR-505 mimics group were lower than those in the miR-NC group（P<0.05）. Meanwhile，protein and gene expressions of β-catenin and GSK-3β in Wnt signaling pathway in the miR-505 mim－ics group were significantly lower than those in the miR-NC group（P<0.05）. Conclusion：The miR-505 can down-regulate the expres－sion of MMP-9，CDK4，Cyclin-D1 and HMGB-1 by inhibiting Wnt signaling pathway，so as to inhibit the proliferation and metastasis of osteosarcoma cells.