姜黄素通过miR-181a调控KLF6表达抑制骨肉瘤细胞的生长  

Curcumin inhibits the growth of osteosarcoma cells by regulating the expression of KLF6 through miR-181a

作　　者：白洋 黄纬[2] 张菲斐[1] 李向辉[1] 金杰[3] BAI Yang;HUANG Wei;ZHANG Feifei;LI Xianghui;JIN Jie(Dept.of Traditional Chinese Medicine,Nanyang Central Hospital,Nanyang 473005,Henan,China;Nanyang Medical College,Nanyang 473004,Henan,China;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450099,Henan,China)

机构地区：[1]南阳市中心医院中医科,河南南阳473005 [2]南阳医学高等专科学校,河南南阳473004 [3]河南中医药大学第一附属医院,河南郑州450099

出　　处：《武汉大学学报（医学版）》2023年第3期298-303,315,共7页Medical Journal of Wuhan University

基　　金：国家中医药管理局第四批全国中医(临床、基础)优秀人才研修项目(编号:国中医药办人教函[2017]24号);河南省中医药科学研究专项课题(编号:2019JDZX2080)。

摘　　要：目的:探讨姜黄素通过微小RNA-181a(miR-181a)调控锌指蛋白Kruppel样因子6(KLF6)表达对骨肉瘤细胞生长的影响。方法:体外培养骨肉瘤细胞系U2OS细胞,将U2OS细胞分为对照组(不作处理)、姜黄素组(终浓度分别为5, 10, 20μmol/L姜黄素)、miR-181a阴性对照(miR-NC)组、miR-181a模拟物(miR-181a mimics)组、姜黄素(20μmol/L)+NC组、姜黄素(20μmol/L)+miR-181a mimics组,细胞计数试剂盒-8(CCK-8)法与平板克隆形成实验检测各组U2OS细胞增殖情况;实时荧光定量PCR(RT-qPCR)检测miR-181a与KLF6 mRNA表达情况;蛋白印迹分析法检测各组U2OS细胞KLF6、细胞周期蛋白B1(Cyclin B1)、Cyclin D1表达变化;双荧光素酶报告实验验证miR-181a与KLF6的靶向关系。结果:与对照组相比,随着姜黄素浓度升高,U2OS细胞存活率、细胞平板克隆形成率、Cyclin D1、Cyclin B1蛋白、miR-181a表达水平逐渐降低,KLF6表达水平逐渐升高,并呈浓度依赖性(P<0.05);经targetscan数据库预测显示,miR-181a与KLF6 mRNA 3’UTR区有结合位点,与KLF6-3’UTR-WT+miR-181a NC组比较,KLF6-3’UTR-WT+miR-181a mimics组荧光素酶活性降低(P<0.05);与对照组及miR-NC组相比,miR-181a mimics组U2OS细胞存活率、细胞平板克隆形成率、Cyclin D1、Cyclin B1蛋白表达水平显著升高(P<0.05),KLF6蛋白表达水平显著降低(P<0.05);过表达miR-181a显著降低姜黄素对U2OS细胞增殖抑制作用。结论:姜黄素可抑制骨肉瘤细胞的生长,其作用可能是通过抑制miR-181a表达而调控KLF6表达实现。Objective: To investigate the effect of curcumin on the growth of osteosarcoma cells by regulating the expression of zinc finger protein Kruppel-like factor 6(KLF6) through microRNA-181a(miR-181a). Methods: U2OS cells of the osteosarcoma cell line were cultured in vitro and divided into control group(none treatment), curcumin group(curcumin with final concentration of 5, 10, and 20μmol/L), miR-181a negative control(miR-NC) group, miR-181a mimic group, curcumin(20 μmol/L)+NC group, and curcumin(20 μmol/L)+miR-181a mimics group. Cell counting kit-8(CCK-8)method and plate clone formation experiment were used to detect the proliferation of U2OS cells in each group;Real-time fluorescent quantitative PCR(RT-qPCR) was used to detect the expression of miR-181a and KLF6 mRNA;Western Blot was used to detect the expression levels of KLF6, Cyclin B1(Cyclin B1) and Cyclin D1 in U2OS cells of each group;the dual luciferase reporter experiment was used to verify the targeting relationship between miR-181a and KLF6. Results: Compared with those of control cells, with the increase of curcumin concentration, U2OS cells’ survival rate, cell plate clone formation rate, Cyclin D1, Cyclin B1 protein, and miR-181a expression levels gradually decreased, the KLF6 expression level gradually increased and was concentration-dependent(P<0. 05);the targetscan database prediction showed that miR-181a had a binding site with the 3’UTR region of KLF6 mRNA, compared with the KLF6-3’UTR-WT+miR-181a NC group, the KLF6-3’UTR-WT+miR-181a mimics group had lower luciferase activity(P<0. 05);compared with those in control group and miR-NC group, the U2OS cell survival rate, cell plate clone formation rate, Cyclin D1 and Cyclin B1 protein expression levels in the miR-181a mimics group were significantly increased(P<0. 05), the KLF6 protein expression level was significantly reduced(P<0. 05);overexpression of miR-181a significantly reduced the inhibitory effect of curcumin on U2OS cell proliferation. Conclusion: Curcumin can inhibit the growth

关 键 词：姜黄素 微小RNA-181a 锌指蛋白Kruppel样因子6 骨肉瘤细胞 增殖 

分 类 号：R730.5[医药卫生—肿瘤]