结直肠癌分泌的外泌体 circ_001422 通过靶向 miR-195-5p 调控内皮细胞中 KDR 的表达并激活 mTOR 信号通路。

Colorectal cancer-secreted exosomal circ_001422 plays a role in regulating KDR expression and activating mTOR signaling in endothelial cells by targeting miR-195-5p.

机构信息

Department of Cellular and Molecular Biology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.

Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

出版信息

J Cancer Res Clin Oncol. 2023 Oct;149(13):12227-12240. doi: 10.1007/s00432-023-05095-1. Epub 2023 Jul 11.

DOI:10.1007/s00432-023-05095-1

PMID:37432457

Abstract

BACKGROUND

As non-coding RNAs, exosomal circular RNAs (circRNAs) regulate colorectal cancer (CRC) progression, although the functional mechanisms by which such molecules affect the tumor microenvironment are still elusive. Herein, we aimed to explore the potential clinical significance of a signature of five serum-derived circRNAs in CRC and investigated the mechanisms underlying endothelial cell angiogenesis mediated by CRC-secreted exosomal circ_001422.

METHODS

The expression of a signature of five serum-derived circRNAs (circ_0004771, circ_0101802, circ_0082333, circ_0072309, and circ_001422) were measured by RT-qPCR, and their associations with tumor staging and lymph node metastasis were further evaluated in CRC patients. In silico analysis was used to show the relationship between circ_001422, miR-195-5p, and KDR, validated by dual-luciferase reporter and Western blotting assays. CRC cell-derived exosomes were isolated and characterized by scanning electron microscopy and Western blotting. Endothelial cell uptake of PKH26-labeled exosomes was demonstrated using a spectral confocal microscope. In vitro genetic strategies were used to exogenously alter the expression level of circ_001422 and miR-195-5p expression. Cell proliferation assay, transwell migration assay, and capillary tube formation assay were conducted to explore the role of CRC-secreted exosomal circ_001422 in endothelial cell function in vitro.

RESULTS

The expression levels of serum-derived circ_0004771, circ_0101802, circ_0082333, and circ_001422 were significantly higher in CRC and were positively correlated with the lymph node metastasis status. However, circ_0072309 showed a significant down-regulation in CRC than in healthy individuals. Furthermore, a higher expression level of circ_001422 in both cellular and exosomal fractions was found in HCT-116 CRC cells. We found that HCT-116 exosomes considerably enhanced proliferation and migration of endothelial cells through shuttling of circ_001422. We also observed that exosomes derived from HCT-116 cell, but not non-aggressive Caco-2 CRC cells, increased in vitro tubulogenesis of endothelial cells. Importantly, knockdown of circ_001422 impaired the capability of endothelial cells to form the capillary-like tube structures. CRC-secreted circ_001422 acted as an endogenous miR-195-5p sponge to inhibit miR-195-5p activity, which led to increased KDR expression and mTOR signaling activation in endothelial cells. Importantly, ectopic expression of miR-195-5p mimicked the effect of circ_001422 silencing on KDR/mTOR signaling in endothelial cells.

CONCLUSION

This study attributed a biomarker role for circ_001422 in CRC diagnosis and proposed a novel mechanism whereby circ_001422 up-regulates KDR through sponging miR-195-5p. These interactions may give rise to the activation of mTOR signaling and may be a possible clarification for the pro-angiogenesis effects of CRC-secreted exosomal circ_001422 on endothelial cells.

摘要

背景

作为非编码 RNA，外泌体环状 RNA（circRNA）调节结直肠癌（CRC）的进展，尽管这些分子影响肿瘤微环境的功能机制仍不清楚。在此，我们旨在探索 CRC 患者血清来源的 circRNA 特征的潜在临床意义，并研究 CRC 分泌的外泌体 circ_001422 介导的内皮细胞血管生成的机制。

方法

通过 RT-qPCR 测量了一组五个血清衍生 circRNA（circ_0004771、circ_0101802、circ_0082333、circ_0072309 和 circ_001422）的表达，并进一步评估了它们与肿瘤分期和淋巴结转移的相关性。通过双荧光素酶报告和 Western blot 实验进行了生物信息学分析，显示了 circ_001422、miR-195-5p 和 KDR 之间的关系。用扫描电子显微镜和 Western blot 对 CRC 细胞来源的外泌体进行了分离和鉴定。用光谱共聚焦显微镜显示 PKH26 标记的外泌体被内皮细胞摄取。用体外遗传策略改变 circ_001422 和 miR-195-5p 的表达水平。用细胞增殖实验、transwell 迁移实验和毛细管形成实验来研究 CRC 分泌的外泌体 circ_001422 在体外对内皮细胞功能的影响。

结果

circ_0004771、circ_0101802、circ_0082333 和 circ_001422 的血清表达水平在 CRC 中显著升高，并与淋巴结转移状态呈正相关。然而，circ_0072309 在 CRC 中的表达水平明显低于健康个体。此外，在 HCT-116 CRC 细胞中，细胞和外泌体部分的 circ_001422 表达水平均升高。我们发现 HCT-116 外泌体通过 circ_001422 的穿梭显著增强了内皮细胞的增殖和迁移。我们还观察到 HCT-116 细胞来源的外泌体而非非侵袭性 Caco-2 CRC 细胞能增加内皮细胞的体外管形成。重要的是，circ_001422 的敲低削弱了内皮细胞形成毛细血管样管结构的能力。CRC 分泌的 circ_001422 作为内源性 miR-195-5p 的海绵，抑制 miR-195-5p 的活性，导致内皮细胞中 KDR 表达和 mTOR 信号通路的激活。重要的是，外源性表达 miR-195-5p 模拟了 circ_001422 沉默对内皮细胞中 KDR/mTOR 信号通路的影响。

结论

本研究将 circ_001422 作为 CRC 诊断的生物标志物，并提出了一种新的机制，即 circ_001422 通过海绵 miR-195-5p 来上调 KDR。这些相互作用可能导致 mTOR 信号通路的激活，可能为 CRC 分泌的外泌体 circ_001422 对内皮细胞的促血管生成作用提供了一种可能的解释。