摘要： 目的 探讨长链非编码RNA （lncRNA） H19靶向微RNA 194 （miR-194）对骨肉瘤细胞增殖和侵袭的影响及其作用机制。方法 收集2018年1月至2019年6月大连医科大学附属大连市第五人民医院骨外二科行骨肉瘤切除术患者的骨肉瘤组织及对应癌旁组织标本各30例。采用实时荧光定量PCR （qRT-PCR）检测30例骨肉瘤组织、对应的癌旁组织以及人正常成骨细胞hFOB1.19和骨肉瘤细胞SAOS-2、U2OS中lncRNA H19和miR-194的表达水平。利用脂质体转染法将lncRNA H19小干扰RNA （si-H19组）、RNA干扰无意义序列（si-NC组）、miR-194模拟物（miR-194 mimics组）、miRNA阴性对照（miR-NC组）分别转染SAOS-2细胞，MTT法检测各组细胞活力，细胞侵袭实验检测细胞侵袭能力，Western blotting检测细胞N-钙黏蛋白（CDH2）和1型胰岛素样生长因子受体（IGF1R）蛋白的表达。生物信息学分析、双荧光素酶报告基因实验和qRT-PCR验证lncRNA H19和miR-194的靶向调控关系。结果 与癌旁组织比较，骨肉瘤组织中lncRNA H19的表达水平升高，miR-194的表达水平降低（均P<0.05）；与人正常成骨细胞hFOB1.19比较，骨肉瘤细胞SAOS-2、U2OS中lncRNA H19表达水平升高（P<0.05），miR-194表达水平下降（P<0.05）。与si-NC组比较，si-H19组SAOS-2细胞活力降低，侵袭能力降低，CDH2和IGF1R蛋白表达水平降低（均P<0.05）；与miR-NC组比较，miR-194 mimics组SAOS-2细胞的活力降低，侵袭能力降低，CDH2和IGF1R蛋白表达水平降低（P<0.05）。生物信息学分析及双荧光素酶报告基因实验结果显示，lncRNA H19可以靶向调控miR-194。干扰miR-194的表达逆转了抑制lncRNA H19的表达对骨肉瘤SAOS-2细胞增殖和侵袭的影响。结论 lncRNA H19在骨肉瘤组织和细胞系中的表达升高，miR-194在骨肉瘤组织和细胞系中的表达下降，沉默骨肉瘤细胞SAOS-2中lncRNA H19的表达，能够抑制骨肉瘤细胞的增殖和侵袭，其机制可能与上调SAOS-2细胞中miR-194表达，进一步下调CDH2和IGF1R的表达有关。

lncRNA H19靶向miR-194对骨肉瘤细胞增殖和侵袭的影响及其作用机制

李盈义¹, 王勇², 邱冠臻², 李洪敬³

1. 大连医科大学附属大连市第五人民医院骨外二科, 辽宁 大连 116021;
2. 沈阳医学院附属中心医院骨科, 沈阳 110075;
3. 大连医科大学附属第一医院关节外科, 辽宁 大连 116011

收稿日期:2022-07-27出版日期:2023-03-30发布日期:2023-03-21
通讯作者:李洪敬E-mail:hongjingl@hotmail.com
作者简介:李盈义(1988-),男,主治医师,硕士.
基金资助:国家自然科学基金（81972522）


关键词: 长链非编码RNA, H19, 微RNA 194, 骨肉瘤细胞, 增殖, 侵袭
Abstract: Objective To investigate the effect of long non-coding RNA （lncRNA） H19 targeting microRNA 194 （miR-194） on the proliferation and invasion of osteosarcoma cells and its molecular mechanism. Methods Thirty osteosarcoma tissue specimens and 30 corresponding adjacent cancer tissue specimens were collected from patients who underwent osteosarcoma resection in the Second Department of Orthopedics，the affiliated Dalian Fifth People’s Hospital of Dalian Medical University，from January 2018 to June 2019. The expressions of lncRNA H19 and miR-194 in 30 osteosarcoma tissues and corresponding adjacent tissues，human normal osteoblasts hFOB1.19 and osteosarcoma cells SAOS-2 and U2OS，were detected using qRT-PCR. lncRNA H19 small interfering RNA （si-H19），small interfering RNA negative control （si-NC），miR-194 mimics （miR-194 mimics），and miRNA negative control （miR-NC） were transfected into SAOS-2 cells by liposome transfection. Cell viability was detected by MTT assay，the invasive ability was detected by Transwell assay，and the expressions of cadherin 2 （CDH2） and insulin-like growth factor 1 receptor （IGF1R） were detected by western blotting. The relationship between lncRNA H19 and miR-194 were verified using bioinformatics，dual-luciferase reporter gene experiments，and qRT-PCR. Results The expression level of lncRNA H19 in osteosarcoma tissues was increased，and the expression level of miR-194 was decreased （all P<0.05） compared with those of adjacent tissues. The expression level of lncRNA H19 in SAOS-2 and U2OS osteosarcoma cells was increased （P<0.05），and the expression level of miR-194 was decreased （P<0.05） compared with those of hFOB1.19. The viability of SAOS-2 cells in the si-H19 group decreased，invasion ability decreased，and expression levels of CDH2 and IGF1R proteins decreased （P<0.05） compared with those of the si-NC group. The cell activity of SAOS-2 cells in the miR-194 mimic group decreased，invasion ability decreased，and expression levels of CDH2 and IGF1R proteins decreased （P<0.05） compared with those of the miR-NC group. Bioinformatic analysis and double luciferase assay results showed that lncRNA H19 could target miR-194. Knockdown of miR-194 reversed the effect of inhibiting lncRNA H19 expression on the proliferation and invasion of osteosarcoma SAOS-2 cells. Conclusion The expression of lncRNA H19 was increased in osteosarcoma tissues and cell lines，while the expression of miR-194 was decreased. Silencing the expression of lncRNA H19 in osteosarcoma cells SAOS-2 inhibited proliferation and invasion. The mechanism may be related to the upregulation of miR-194 expression in SAOS-2 cells and further downregulation of CDH2 and IGF1R expression.
Key words: long non-coding RNA, H19, microRNA 194, osteosarcoma cells, proliferation, invasion
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