摘要
目的探究肿瘤蛋白翻译调控1反义RNA1(TPT1-AS1)靶向miR-324/核转录因子扭曲相关蛋白1(TWIST1)轴调控上皮性卵巢癌(EOC)细胞的增殖、侵袭、迁移及血管生成从而影响卵巢癌(OC)发展的作用机制。方法实时定量PCR检测29例OC灶及癌旁组织样本中TPT1-AS1和miR-324的表达;构建“TPT1-AS1过表达”与“miRNA324敲除”的两种OC细胞模型,并采用CCK-8、Transwell TM和划痕试验法检测细胞增殖、侵袭和迁移能力;Western blot法检测TWIST1、上皮钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、血管内皮生长因子A(VEGF-A)的蛋白表达;荧光原位杂交实验、RNA下拉实验验证TPT1-AS1与miR-324之间的相互作用;免疫组织化学染色法检测、Targetscan生物信息学分析验证miR-324的表达对上皮-间质转化(EMT)过程及血管生成有负调控作用。结果TPT1-AS1在OC组织中的表达水平显著高于癌旁组织,而miR-324的表达水平在OC组织中则显著低于癌旁组织。在TPT1-AS1过表达的人卵巢癌细胞株SKOV3细胞中,miR-324的表达水平明显下降,TPT1-AS1与miR-324呈显著负相关。实验还发现,TPT1-AS1与miR-324在OC细胞中共同表达,并且二者之间存在直接结合关系。下调miR-324显著促进了SKOV3细胞的增殖、侵袭和迁移能力。进一步研究发现miR-324与EMT关键转录因子TWIST13′-UTR端有结合位点,抑制miR-324表达可以上调TWIST1的转录水平,从而导致E-cadherin蛋白表达的下降和Vimentin蛋白表达的上升。此外,miR-324下调时,VEGF-A蛋白表达水平上升。结论本研究揭示了TPT1-AS1通过负调控miR-324/TWIST1轴促进EOC细胞增殖、侵袭、迁移以及血管生成,从而促进OC的发展,这些发现为OC的诊断和治疗提供了新的潜在靶点。
Objective To explore the mechanism of TPT1-AS1 targeting miR-324/TWIST1 axis to regulate the proliferation,invasion,migration and angiogenesis of epithelial ovarian cancer(EOC)cells,thereby affecting ovarian cancer(OC)progression.Methods RT-qPCR was used to detect the expression of TPT1-AS1 and miR-324 in 29 OC lesions and adjacent tissue samples.The two OC cell models of TPT1-AS1 overexpression and miRNA324 knockdown were constructed,and the cell proliferation,invasion and migration abilities were detected by CCK-8,Transwell TM and scratch test.Western blot analysis was used to detect the protein expression levels of TWIST1,epithelial cadherin(E-cadherin),Vimentin,and vascular endothelial growth factor A(VEGF-A)in OC cells.Fluorescence in situ hybridization(FISH)and RNA pull-down experiments were used to verify the interaction between TPT1-AS1 and miR-324.Immunohistochemistry and Targetscan bioinformatics analysis were used to verify the negative regulatory role of miR-324 in the epithelial-mesenchymal transition(EMT)process.Results The TPT1-AS1 expression was significantly higher in OC tissues than that in para-cancerous tissues,while the miR-324 expression was significantly lower.In SKOV3 cells with TPT1-AS1 overexpression,the miR-324 expression decreased significantly,and TPT1-AS1 was negatively correlated with miR-324.It was also found that TPT1-AS1 and miR-324 were co-expressed in OC cells,and there was a direct binding relationship between them.Down-regulation of miR-324 significantly promoted the proliferation,invasion and migration of SKOV3 cells.Further studies revealed that miR-324 had a binding site at the 3′-UTR end of the TWIST1,a key transcription factor for EMT.Inhibiting miR-324 expression increased the transcription level of TWIST1,leading to a decrease in E-cadherin protein expression and an increase in Vimentin protein expression.Additionally,the downregulation of miR-324 resulted in an increased expression level of VEGF-A protein,which in turn enhanced angiogenesis of OC.Conclusion TPT1-AS1 promotes EOC cell proliferation,invasion,migration and angiogenesis by negatively regulating the miR-324/TWIST1 axis,thus promoting the development of OC.These findings provide new potential targets for the diagnosis and treatment of OC.
作者
王爱萍
戚莹莹
WANG Aiping;QI Yingying(Department of Gynecology,The Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou 510700,China)
出处
《细胞与分子免疫学杂志》
北大核心
2025年第6期536-543,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
广州医科大学附属第六医院开放课题资助项目(202011-308)
广东省基础与应用基础研究基金企业联合基金(2021A1515220055)
广州市卫生健康科技项目-一般引导项目(20211A011107)。
