微小RNA-490-3p靶向FK506相关蛋白14表达抑制骨肉瘤MG63细胞侵袭和迁移的实验研究  

Inhibitory effect of miR-490-3p targeting FKBP14 on migration and invasion of osteosarcoma MG63 cells

作　　者：陈夫圆 孙利平 朱远见 孟猛 CHEN Fuyuan;SUN Liping;ZHU Yuanjian;MENG Meng(Department of Hand and Foot Surgery,Zaozhuang Mining Group Central Hospital,Zaozhuang,Shandong 277000,China)

机构地区：[1]枣庄矿业集团中心医院手足外科,山东枣庄277000

出　　处：《安徽医药》2022年第10期2063-2067,I0005,共6页Anhui Medical and Pharmaceutical Journal

摘　　要：目的探讨微小RNA-490-3p(miR-490-3p)对骨肉瘤MG63细胞侵袭、迁移的影响及其机制。方法2019年9月至2020年4月,从中科院上海细胞库购买人骨肉瘤细胞株MG63进行体外培养,分为对照组(未转染)、miR-NC组(转染miR-NC)、miR-490-3p组(转染miR-490-3p mimics),miR-490-3p+pcDNA组(共转染miR-490-3p mimics与空载体)和miR-490-3p+FKBP14组(共转染miR-490-3p mimics与FKBP14过表达载体),采用RT-PCR检测miR-490-3p表达,Transwell小室法检测细胞侵袭、迁移,蛋白质印迹法检测钙黏蛋白E(E-cadherin)、基质金属蛋白酶-2(MMP-2)、上皮间质转化因子Twist转录因子(Twist)和FK506相关蛋白14(FKBP14)蛋白表达,双荧光素酶报告基因实验检测miR-490-3p和FKBP14的靶向关系。结果与对照组比较,miR-490-3p组细胞中miR-490-3p表达水平明显升高(3.68±0.37比1.02±0.10,P<0.05),而侵袭细胞数(33.25±2.02比76.52±6.00,P<0.05)、迁移细胞数(45.56±3.38比97.85±6.45,P<0.05)及MMP-2、Twist和FKBP14蛋白的表达水平明显降低,E-cadherin蛋白表达水平明显升高;而miR-NC组和对照组之间差异无统计学意义(P>0.05)。双荧光素酶报告基因实验结果证实FKBP14是miR-490-3p的潜在靶基因。与miR-490-3p+pcDNA组比较,miR-490-3p+FKBP14组MG63细胞侵袭数、迁移数及MMP-2、Twist、FKBP14蛋白的表达水平明显升高,而E-cadherin蛋白的表达水平明显降低(均P<0.05)。结论miR-490-3p可通过靶向抑制FKBP14表达降低骨肉瘤MG63细胞的侵袭和迁移能力,可作为骨肉瘤治疗的分子靶点。Objective To investigate the effect of miR-490-3p on the invasion and migration of osteosarcoma MG63 cells and its mechanism.Methods From September 2019 to April 2020,human osteosarcoma cell line MG63 was purchased from Shanghai cell bank,Chinese Academy of Sciences,cultured in vitro and assigned into control group(untransfected),miR-NC group(transfected with miR-NC),miR-490-3p group(transfected with miR-490-3p mimics),miR-490-3p+pcDNA group(co-transfected miR-490-3p mimics and empty vector)and miR-490-3p+FKBP14 group(co-transfected miR-490-3p mimics and FKBP14 overexpression vector).The expression of miR-490-3p was detected by RT-PCR;the invasion and migration of MG63 cells were detected by Transwell;the protein expressions of E-cadherin,Matrix metalloproteinase-2(MMP-2),epithelial-mesenchymal transforming factor Twist transcription factor(Twist)and FK506-associated protein 14(FKBP14)were detected by Western blotting.The targeting relationship of miR-490-3p and FKBP14 was determined by double luciferase reporter gene assay.Results Compared with the control group,the expression level of miR-490-3p in the cells of the miR-490-3p group was significantly increased[(3.68±0.37)vs.(1.02±0.10),P<0.05],while the number of invaded cells[(33.25±2.02)vs.(76.52±6.00),P<0.05],number of migrating cells[(45.56±3.38)vs.(97.85±6.45),P<0.05]and the protein expression levels of MMP-2,Twist and FKBP14 reduced,and the protein expression level of E-cadherin increased,while there was no significant difference between the miR-NC group and the control group(P>0.05).The results of double luciferase reporter gene experiment confirmed that FKBP14 was a potential target gene of miR-490-3p.Compared with the miR-490-3p+pcDNA group,the number of invaded cells,number of migrating cells and the protein expression levels of MMP-2,Twist and FKBP14 in the miR-490-3p+FKBP14 group increased,while the protein expression level of E-cadherin reduced(P<0.05).Conclusion miR-490-3p could inhibit targeted FKBP14 expression to reduce the invasion and mi

关 键 词：骨肉瘤 骨肉瘤MG63 微小RNA-490-3p 细胞侵袭 迁移 FK506相关蛋白14 

分 类 号：R738.1[医药卫生—肿瘤]