摘要
目的探讨微小RNA-374a-5p(miR-374a-5p)对SRC激酶信号抑制剂1(SRCIN1)的靶向调控作用及对骨肉瘤(OS)细胞迁移、侵袭和Wnt/β-连环蛋白(β-catenin)信号通路的影响。方法采用实时荧光定量PCR(qPCR)检测人正常成骨细胞hFOB 1.19及OS细胞(HOS、Saos-2、MG-63和U2OS)的miR-374a-5p水平。脂质体法向U2OS细胞转染miR-374a-5p抑制物(Inhibitor组)和无关对照序列(NC组),并设未转染细胞为对照组,qPCR、MTT比色法、划痕实验和Transwell小室实验检测miR-374a-5p水平、细胞增殖活力、划痕愈合率和穿膜细胞数,qPCR和Western blotting检测SRCIN1、基质金属蛋白酶9(MMP-9)、Wnt1和β-catenin水平。通过生物信息学和双荧光素酶报告实验鉴定miR-374a-5p的靶基因。结果OS细胞的miR-374a-5p水平均高于hFOB 1.19细胞(P<0.05)。Inhibitor组转染24、48 h后的增殖活力均低于其余两组(P<0.05)。Inhibitor组的miR-374a-5p水平、划痕愈合率和穿膜细胞数分别为0.327±0.062、(30.893±4.287)%和(140.390±13.598)个,均低于对照组的1.021±0.116、(77.339±5.130)%和(363.922±24.411)个及NC组的1.028±0.142、(75.019±3.670)%和(351.381±19.872)个(P<0.05)。与其余两组相比,Inhibitor组的SRCIN1水平升高,而MMP-9、Wnt1和β-catenin水平降低(P<0.05)。在线TargetScan预测显示,miR-374a-5p能够与SRCIN1的3’端非翻译区结合,miR-374a-5p模拟物和野生型SRCIN1质粒共转染细胞的相对荧光强度较其他转染组合降低(P<0.05)。结论异常高水平的miR-374a-5p在OS中发挥致癌作用,miR-374a-5p可能通过靶向SRCIN1激活Wnt/β-catenin通路,进而促进OS细胞的迁移和侵袭,miR-374a-5p/SRCIN1/Wnt/β-catenin轴有望阐明OS的发病机制,为其抗肿瘤治疗提供新候选靶点。
Objective To investigate the targeted regulation of microRNA-374a-5p(miR-374a-5p)on SRC kinase signaling inhibitor 1(SRCIN1)and migration,invasion and Wnt/β-catenin signaling pathway of osteosarcoma(OS)cells.Methods Real-time quantitative PCR(qPCR)was used to detect miR-374a-5p levels in human normal osteoblasts hFOB 1.19 and OS cells including HOS,Saos-2,MG-63 and U2OS.U2OS cells were transfected with miR-374a-5p inhibitor(Inhibitor group)and unrelated control sequence(NC group)by liposome method,and untransfected cells were set as Control group.QPCR,MTT colorimetry,scratch test and Transwell chamber test were used to detect miR-374a-5p level,cell proliferation activity,scratch healing rate and number of penetrating cells.Levels of SRCIN1,matrix metalloproteinase 9(MMP-9),Wnt1 andβ-catenin were detected by qPCR and Western blotting,respectively.The target gene of miR-374a-5p was identified by bioinformatics analysis and double luciferase report analysis.Results MiR-374a-5p level in OS cells was higher than that in hFOB 1.19 cells(P<0.05).The proliferation activity of Inhibitor group was lower than that of the other two groups at 24 and 48 h after transfection(P<0.05).MiR-374a-5p level,wound healing rate and number of penetrating cells in Inhibitor group were 0.327±0.062,(30.893±4.287)%and 140.390±13.598,lower than 1.021±0.116,(77.339±5.130)%and 363.922±24.411 in Control group and 1.028±0.142,(75.019±3.670)%and 351.381±19.872 in NC group(P<0.05).Compared with other two groups,there were elevated level of SRCIN1 but decreased levels of MMP-9,Wnt1 andβ-catenin in Inhibitor group(P<0.05).Online TargetScan prediction showed that miR-374a-5p could bind to the 3'untranslated region of SRCIN1,and the relative fluorescence intensity of cells co-transfected with miR-374a-5p mimics and wild-type SRCIN1 plasmid was lower than that of other transfection combinations(P<0.05).Conclusion The abnormally high level of miR-374a-5p plays a carcinogenic role in OS.MiR-374a-5p may activate Wnt/β-catenin pathway by targeting SRCIN1,thus promoting the proliferation,migration and invasion of OS cells.The miR-374a-5p/SRCIN1/Wnt/β-catenin axis is expected to elucidate the pathogenesis of OS and provide a new candidate target for its anti-tumor therapy.
作者
龚敏勇
熊超
廖立潇
GONG Minyong;XIONG Chao;LIAO Lixiao(Department of Oncology, Jiujiang Hospital of Traditional Chinese Medicine, Jiujiang 332000, China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2020年第11期975-981,共7页
Chinese Clinical Oncology
