miR-132通过降低HMGA2的表达抑制骨肉瘤细胞迁移、侵袭及上皮间质转化

doi:10.3969/j.issn.2095-9400.2019.08.005

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摘要目的：研究微小RNA-132（miR-132）对骨肉瘤细胞迁移和侵袭能力的影响及其机制。方法：利用实时定量PCR（qRT-PCR）检测骨肉瘤细胞系U2OS和HOS及人成骨细胞系hFOB1.19中miR-132的表达水平。分别向HOS及U2OS细胞转染miR-132模拟物和抑制物，以过表达或敲低细胞内miR-132的表达，利用qRT-PCR验证转染效率。采用Transwell实验、qRT-PCR及Western blot检测过表达和敲低miR-132对骨肉瘤细胞迁移、侵袭及上皮间质转化的影响。通过对Targetscan网站进行检索并利用荧光素酶报告基因实验探究HMGA2为miR-132的靶基因。qRT-PCR和Western blot检测miR-132对HMGA2表达的调控作用。结果：与人成骨细胞系hFOB1.19相比，miR-132在骨肉瘤细胞中的表达水平显著降低（P＜0.05）；向U2OS细胞转染miR-132模拟物后，细胞miR-132的表达水平显著增加（P＜0.01）；过表达miR-132后，U2OS细胞的迁移和侵袭能力显著降低，E-cadherin表达增加，Vimentin表达降低（P＜0.05）；向HOS细胞转染miR-132抑制物后，细胞miR-132的表达水平显著降低（P＜0.01）；敲低miR-132后，HOS细胞的迁移和侵袭能力显著增强，E-cadherin表达降低，Vimentin表达增加（P＜0.05）；生物信息学检索及荧光素酶报告基因证明HMGA2基因为miR-132的靶基因。过表达miR-132可明显降低骨肉瘤细胞中HMGA2的表达，而敲低miR-132可明显增加骨肉瘤细胞中HMGA2的表达（P＜0.05）。结论：miR-132在骨肉瘤细胞中低表达，miR-132可通过抑制HMGA2的表达抑制骨肉瘤细胞的迁移、侵袭及上皮间质转化。

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	蒋臻
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关键词 ： miR-132, 骨肉瘤, HMGA2, 肿瘤转移, 上皮间质转化

Abstract：Objective: To investigate the effect of miR-132 on the migration, invasion and epithelial-mesenchymal transition (EMT) of osteosarcoma (OS) cells and explore the potential molecular mechanisms. Methods: The expression levels of miR-132 in human OS cells (U2OS and HOS) and human osteoblast hFOB1.19 were evaluated by quantitative real-time quantitative PCR (qRT-PCR). miR-132 mimic and miR-132 inhibitor were transfected into U2OS and HOS for the overexpression and knockdown of miR-132, respectively. qRT-PCR was employed to examine the efficacy of transfection. The effect of miR-132 on the migration, invasion and EMT of OS cells was detected by Transwell assay, qRT-PCR and Western blot. Bioinformatics website TargetScan was searched to identify the potential targets of miR-132 and dual luciferase reporter assay was performed to validate the interaction between miR-132 and HMGA2.Real-time quantitative PCR and Western blot were used to confirm the regulatory effect of miR-132 on HMGA2 expression. Results: The expression of miR-132 in OS cells was significantly decreased compared with that in hFOB1.19 cells (P<0.05). Transfection of miR-132 mimics significantly increased the expression level of miR-132 in U2OS cells (P<0.01). miR-132 overexpression led to decreased ability of cell migration and invasion, and increased E-cadherin level and decreased Vimentin level (P<0.05). Knockdown of miR-132 by transfection of miR-132 inhibitor resulted in enhanced metastatic ability, decreased E-cadherin expression and increased Vimentin expression (P<0.05). Data mining of bioinformatics website Targetscan suggested HMGA2 was a potential downstream target of miR-132. Dual luciferase reporter gene assay confirmed miR-132 could interact with HMGA2 3’-UTR. Overexpression of miR-132 significantly decreased HMGA2 expression while miR-132 knockdown led to increased expression of HMGA2 (P<0.05). Conclusion: The expression of miR-132 is decreased in OS cells, and miR-132 inhibits the migration, invasion and EMT of HCC cells by down-regulating the expression of HMGA2.

Key words： microRNA-132 osteosarcoma HMGA2 metastasis epithelial-mesenchymal transition

基金资助:浙江省医药卫生科技计划项目（2019KY281）。

作者简介: 蒋臻（1974-），男，浙江杭州人，副主任医师。

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https://xb.wmu.edu.cn/CN/10.3969/j.issn.2095-9400.2019.08.005 或 https://xb.wmu.edu.cn/CN/Y2019/V49/I8/568

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