微小RNA-22通过靶向调控异黏蛋白表达影响骨肉瘤细胞化疗敏感性  

MicroRNA-22 influenced on chemosensitivity of osteosarcoma cells by targeting metadherin

作　　者：孙亮[1] 郭世炳[1] 娜日松 赵振群[1] 赵伟[1] 王玉鑫[1] Sun Liang;Guo Shibing;Na Risong;Zhao Zhenqun;Zhao Wei;Wang Yuxin(Department of Orthopedics,the Second Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010030,China;Health Center of Cadre,Inner Mongolia People’s Hospital,Hohhot 010020,China)

机构地区：[1]内蒙古医科大学第二附属医院骨科,呼和浩特010030 [2]内蒙古自治区人民医院干部保健所,呼和浩特010020

出　　处：《中华实验外科杂志》2019年第11期1959-1962,共4页Chinese Journal of Experimental Surgery

基　　金：内蒙古自治区自然科学基金(2018MS08031);国家自然科学基金(81660440)。

摘　　要：目的探讨微小RNA(miRNA,miR)-22通过靶向调控异黏蛋白(MTDH)表达影响骨肉瘤细胞化疗敏感性。方法通过体外培养骨肉瘤细胞株MG-63进行实验研究,利用噻唑蓝(MTT)法检测MG-63细胞增殖的变化,定量聚合酶链反应(PCR)法检测MTDH的mRNA表达变化,蛋白质印迹法(Western blot)定量检测MTDH的蛋白表达。荧光素酶报告检测MTDH是否为miR-22的直接靶点,Western blot和定量PCR检测miR-22过表达和抑制时MTDH表达变化。单因素方差分析比较多组间差异,t检验比较两组间差异。结果(1)MTT法结果表明顺铂可抑制MG-63细胞增殖,而加入miR-22协同顺铂抑制MG-63细胞增殖的更加明显(F=841.919,P<0.01)。(2)顺铂可促进MG-63细胞中MTDH的表达(t=20.430,P<0.01),miR-22过表达可以减弱顺铂对MTDH表达的促进作用(t=11.570,P<0.01)。miR-22过表达时,MTDH mRNA表达明显减低(t=9.532,P<0.01),miR-22表达受到抑制时,MTDH mRNA表达明显升高(t=9.637,P<0.01)。Western blot实验证实细胞内MTDH蛋白具有相同的变化趋势。(3)MTDH靶点1突变,可以显著减弱miR-22抑制MTDH表达的作用(t=6.754,P<0.01),MTDH靶点2突变时也减弱了miR-22对MTDH的调节作用(t=9.225,P<0.01)。MTDH1+2靶点均突变,miR-22对MTDH无明显调节作用(t=2.079,P>0.05)。证实miR-22可以直接靶向调节MTDH的表达,且两者有两个结合位点。结论miR-22通过靶向调控MTDH的表达来影响骨肉瘤细胞化疗敏感性。Objective To investigate effect of microRNA(miRNA,miR)-22 on the chemosensitivity of osteosarcoma cells by targeting metadherin(MTDH),so as to provide new ideas for targeted therapy of osteosarcoma.Methods The osteoblastoma cell line MG-63 was cultured in vitro,thiazolyl Blue tetrazolium bromide(MTT)assay were used to detect the proliferation of MG-63 cells.The mRNA expression of MTDH was detected by quantitative polymerasechain reactio(PCR).The protein expression of MTDH was detected by Western blotting.The Luciferase assay(luciferase)detects whether MTDH is a direct target of miR-22,and the expression of MTDH is detected by Western blotting and quantitative PCR when miR-22 is overexpressed and inhibited.One-way analysis of variance was used to compare differences between groups,and t-test was used to compare differences between the two groups.Results(1)The results of MTT assay showed that cisplatin inhibited the proliferation of MG-63 cells,while the addition of miR-22 and cisplatin inhibited the proliferation of MG-63 cells(F=841.919,P<0.01).(2)Cisplatin promoted the expression of MTDH in MG-63 cells(t=20.430,P<0.01).Overexpression of miR-22 attenuated the promotion of MTDH expression by cisplatin(t=11.570,P<0.01).When miR-22 was overexpressed,the expression of MTDH mRNA was significantly decreased(t=9.532,P<0.01).When the expression of miR-22 was inhibited,the expression of MTDH mRNA was significantly increased(t=9.637,P<0.01).Western blotting analysis confirmed that the intracellular MTDH protein had the same trend.(3)MTDH target 1 mutation can significantly attenuate the effect of miR-22 on MTDH expression(t=6.754,P<0.01),and MTDH target 2 mutation also attenuated the regulation of miR-22 on MTDH(t=9.225,P<0.01).MTDH1+2 targets were all mutated,and miR-22 had no significant regulatory effect on MTDH(t=2.079,P>0.05).It was confirmed that miR-22 can directly target the regulation of MTDH expression,and both have two binding sites.Conclusion MicroRNA-22 influenced on chemosensitivity of osteosarcoma cells by t

关 键 词：骨肉瘤 化疗敏感性 微小RNA-22 

分 类 号：R[医药卫生]