摘要
目的观察miR-495在骨肉瘤细胞中的表达变化及其对骨肉瘤MG-63细胞增殖的影响,并探讨可能的分子机制。方法采用real-time PCR法检测人骨肉瘤细胞系Saos-2、U20S、MG-63及人成骨细胞h FOB1.19中的miR-495。将MG-63细胞分为阴性对照组、miR-495 mimics组、miR-495 mimics+HMGA2组,分别转染mimics-NC、miR-495 mimics、miR-495 mimics+HMGA2表达质粒,转染24、48、72、96 h后,采用CCK-8法检测MG-63细胞增殖能力。应用靶基因在线预测数据库miRanda并结合基因的功能分析进行miR-495的靶基因预测。采用双荧光素酶报告实验验证miR-495与靶基因的结合位点。采用Western blotting法验证miR-495对高迁移率族蛋白A2(HMGA2)表达的靶向调控作用。结果 Saos-2、MG-63、U20S细胞中miR-495的相对表达量低于h FOB1.19细胞(P均<0.01)。转染48、72、96 h后miR-495 mimics组MG-63细胞OD值低于阴性对照组组,miR-495 mimics+HMGA2组MG-63细胞OD值高于miR-495 mimics组(P均<0.01)。双荧光素酶报告实验结果提示miR-495可与HMGA2的3'UTR区特异性结合,从而显著抑制荧光素酶活性。miR-495 mimics组MG-63细胞中HMGA2蛋白的相对表达量低于mimics-NC组,miR-495 mimics+HMGA2组MG-63细胞中HMGA2蛋白的相对表达量高于miR-495 mimics组(P均<0.01)。结论骨肉瘤细胞中miR-495的表达水平低于正常成骨细胞;上调miR-495表达后骨肉瘤细胞增殖受到抑制;miR-495可能通过靶向调控HMGA2表达从而抑制骨肉瘤细胞的增殖。
Objective To explore the changes in the miR-495 expression of the osteosarcoma cells,and its effect on the cell proliferation of osteosarcoma MG-63 cells as wells as the underlying molecular mechanism. Methods The miR-495 expression in the human osteosarcoma Saos-2,U20 S,and MG-63 cells,and human osteoblast h FOB1. 19 cells was detected by using real-time PCR. The MG-63 cells were divided into three groups： negative control group（ transfected with mimics-NC）,miR-495 mimics group（ transfected with miR-495 mimics）,miR-495 mimics ＋ HMGA2 group（ co-transfected with miR-495 mimics and HMGA2 expression plasmid）. The proliferation of MG-63 cell was detected by using CCK-8 at24,48,72 and 96 h after transfection. Target gene online prediction database miRanda combined with gene functional analysis were used to predict the target genes of miR-495. The binding sites of miR-495 and target genes were confirmed by double luciferase reporter assay. Western blotting was used to verify the regulatory effect of miR-495 on high mobility group protein A2（ HMGA2） expression. Results The relative expression of miR-495 in Saos-2,MG-63 and U20 S cells was lower than that in h FOB1. 19 cells（ all P〈0. 01）. The OD value of MG-63 cells in the miR-495 mimics group was lower than that in the negative control group,and the OD value of MG-63 cells in the miR-495 mimics ＋ HMGA2 group was higher than that in miR-495 mimics group at 24,48,72 and 96 h after transfection（ all P〈0. 01）. Moreover,dual-luciferase reporter assay suggested that miR-495 directly bound with the 3＇-UTR of HMGA2,and thus inhibited the luciferase activity. The relative expression of HMGA2 protein in MG-63 cells of the miR-495 mimics group was lower than that in the mimics-NC group,and the relative expression of HMGA2 protein in MG-63 cells of the miR-495 mimics ＋ HMGA2 group was higher than that in miR-495 mimics group（ all P〈0. 01）. Conclusions The expression level of miR-495 in the osteosarcoma cells is lower than that in normal osteoblasts cells. Up-regulating the miR-495 expression inhibits the proliferation of osteosarcoma cells,and miR-495 may inhibit the proliferation of osteosarcoma cells by targeting HMGA2 expression.
作者
王美凤
邢国英
宫锦蕾
孙英华
夏海
王复超
张兆卿
WANG Meifeng;XING Guoying;GONG Jinlei;SUN Yinghua;XIA Hai;WANG Fuchao;ZHANG Zhaoqing(Yidu Central Hospital Affiliated to Weifang Medical University,Weifang 262500,China)
出处
《山东医药》
CAS
2018年第23期18-21,共4页
Shandong Medical Journal
基金
山东省医药卫生科技发展计划面上项目(2017WS174)
作者简介
王美凤（1979-）,女,本科,主要研究方向：肿瘤的分子靶向治疗.E-mail：wangmeifengwf@yeah.net;通信作者：孙英华（1976-）,男,博士,主要研究方向为骨肉瘤的分子靶向治疗.E-mail：qzydsyh@163.com
