背景:抗血管生成疗法已成为肿瘤的有效治疗方法之一。长链非编码RNA(lncRNA)正在成为EC中肿瘤发生和血管生成的重要调节因子。然而,lncRNATRPM2-AS在EC中的潜在机制仍不清楚。
方法:我们通过生物信息学分析筛选了与EC的不良预后和血管生成高度相关的不同表达的lncRNAs,并构建了基于预后lncRNAs的ceRNA网络。TRPM2-AS的亚细胞定位通过荧光原位杂交(FISH)和核胞质分级分离测定来确定。CCK-8,EdU,transwell,westernblot,qRT-PCR和内皮管形成试验评价TRPM2-AS对细胞增殖的影响,入侵,EC细胞的迁移和血管生成。通过生物信息学方法预测TRPM2-AS的靶向微小RNA(miRNA)。TRPM2-AS与miR497-5p的相互作用,通过RNA免疫沉淀和双荧光素酶报告基因测定分析miR497-5p和SPP1。使用皮下肿瘤模型来探索TRPM2-AS的体内功能。应用CIBERSORT分析TRPM2-AS与EC中免疫细胞浸润的相关性。
结果:我们发现TRPM2-AS和SPP1的表达异常上调,而miR-497-5p在EC组织和细胞中表达显著下调。TRPM2-AS与EC患者的血管生成和不良预后密切相关。机械上,TRPM2-AS可以海绵化miR-497-5p释放SPP1,从而促进细胞增殖,EC细胞的侵袭和迁移以及HUVECs的血管生成。在异种移植小鼠模型中敲除TRPM2-AS抑制体内肿瘤增殖和血管生成。此外,TRPM2-AS在调节EC的肿瘤免疫微环境中起着至关重要的作用,TRPM2-AS在EC细胞中的过表达通过分泌富含SPP1的外泌体刺激M2巨噬细胞的极化和血管生成。
结论:TRPM2-AS的缺失通过靶向miR-497-5p/SPP1轴抑制EC的致癌作用。这项研究提供了更好地理解TRPM2-AS在调节血管生成中的作用,并为EC治疗提供了新的靶标。
BACKGROUND: Anti-angiogenic therapy has become one of the effective treatment methods for tumors. Long noncoding RNAs (lncRNAs) are emerging as important regulators of tumorigenesis and angiogenesis in EC. However, the underlying mechanisms of lncRNA TRPM2-AS in EC are still not clear.
METHODS: We screened the differently expressed lncRNAs that were highly associated with poor prognosis and angiogenesis of EC by bioinformatics analysis, and constructed a ceRNA network based on the prognostic lncRNAs. The subcellular localization of TRPM2-AS was determined by fluorescence in situ hybridization (FISH) and nuclear cytoplasmic fractionation assay. CCK-8, EdU, transwell, western blot, qRT-PCR and endothelial tube formation assay were used to evaluate the effects of TRPM2-AS on the proliferation, invasion, migration of EC cells and angiogenesis. The targeted microRNA (miRNA) of TRPM2-AS was predicted by bioinformatic methods. The interaction between TRPM2-AS and miR497-5p, miR497-5p and SPP1 were analyzed by RNA immunoprecipitation and dual-luciferase reporter assay. A subcutaneous tumor model was used to explore TRPM2-AS\'s function in vivo. CIBERSORT was used to analyze the correlation between TRPM2-AS and immune cell immersion in EC.
RESULTS: We found that the expression of TRPM2-AS and SPP1 was aberrantly upregulated, while miR-497-5p expression was significantly downregulated in EC tissues and cells. TRPM2-AS was closely correlated with the angiogenesis and poor prognosis in EC patients. Mechanistically, TRPM2-AS could sponge miR-497-5p to release SPP1, thus promoting the proliferation, invasion and migration of EC cells and angiogenesis of HUVECs. Knockdown of TRPM2-AS in xenograft mouse model inhibited tumor proliferation and angiogenesis in vivo. In addition, TRPM2-AS plays a vital role in regulating the tumor immune microenvironment of EC, overexpression of TRPM2-AS in EC cells stimulated the polarization of M2 macrophages and angiogenesis through secreting SPP1 enriched exosomes.
CONCLUSIONS: The depletion of TRPM2-AS inhibits the oncogenicity of EC by targeting the miR-497-5p/SPP1 axis. This study offers a better understanding of TRPM2-AS\'s role in regulating angiogenesis and provides a novel target for EC treatment.