环状 RNA circNTRK2 通过上调 miR-140-3p 表达促进食管鳞癌细胞的进展。

Circular RNA circNTRK2 facilitates the progression of esophageal squamous cell carcinoma through up-regulating NRIP1 expression via miR-140-3p.

机构信息

Department of Gastroenterology, The First Affiliated Hospital of Henan University of Chinese Medicine, No. 19 Renmin Road, Jinshui District, Zhengzhou, 450000, China.

School of Mechanical and Power Engineering, Zhengzhou University, Zhengzhou, 450000, China.

出版信息

J Exp Clin Cancer Res. 2020 Jul 11;39(1):133. doi: 10.1186/s13046-020-01640-9.

DOI:10.1186/s13046-020-01640-9

PMID:32653032

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7353745/

Abstract

BACKGROUND

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent gastrointestinal malignancies with high mortality. Circular RNAs (CircRNAs) have become a research hotspot in recent years for their vital roles in cancer development and progression. This study aims to clarify the roles of circNTRK2 and its underlying molecular mechanisms in ESCC.

METHODS

The levels of circNTRK2, miR-140-3p, and nuclear receptor-interacting protein 1 (NRIP1) mRNA were examined by qRT-PCR. The cell proliferation ability was detected via CCK-8, EdU and colony formation assays. The invasion capacity was tested by using transwell assay. The apoptotic rate was evaluated through flow cytometry. The protein levels of cleaved PARP, cleaved caspase-3, E-cadherin, vimentin, and NRIP1 were measured by western blot assay. The validation of circular structure was performed by Sanger sequencing, divergent primer PCR, and RNase R treatments. The ceRNA regulatory mechanism of circNTRK2 was observed via dual-luciferase reporter, RIP and RNA pull-down assays. The mice xenograft models were constructed to confirm the oncogenicity of circNTRK2 in ESCC in vivo.

RESULTS

CircNTRK2 was highly expressed in ESCC tissues and cells. High expression of circNTRK2 was correlated with advanced TNM stage, lymph node metastasis and short survival. Knockdown of circNTRK2 inhibited ESCC cell proliferation, invasion and epithelial-mesenchymal transition (EMT), and accelerated apoptosis in vitro. Mechanistic assays disclosed that circNTRK2 could act as a sponge for miR-140-3p to abate its suppression on target NRIP1 expression. Moreover, miR-140-3p-induced inhibitory effects on ESCC cell malignant phenotypes were attenuated by the overexpression of circNTRK2. In addition, depletion of NRIP1 impeded cell proliferation, invasion and EMT, while enhanced apoptosis. Furthermore, silencing of circNTRK2 suppressed cell proliferation and invasion through regulating NRIP1 expression. Also, knockdown of circNTRK2 slowed ESCC tumor growth in vivo.

CONCLUSION

CircNTRK2 promoted ESCC progression by regulating miR-140-3p/NRIP1 pathway. Our findings contribute to a better understanding of circRNAs as miRNA sponges and highlight a promising therapy target in ESCC.

摘要

背景

食管鳞状细胞癌（ESCC）是最常见的胃肠道恶性肿瘤之一，死亡率很高。环状 RNA（CircRNAs）近年来因其在癌症发展和进展中的重要作用成为研究热点。本研究旨在阐明 circNTRK2 的作用及其在 ESCC 中的潜在分子机制。

方法

通过 qRT-PCR 检测 circNTRK2、miR-140-3p 和核受体相互作用蛋白 1（NRIP1）mRNA 的水平。通过 CCK-8、EdU 和集落形成实验检测细胞增殖能力。通过 Transwell 实验检测侵袭能力。通过流式细胞术评估细胞凋亡率。通过 Western blot 检测裂解的 PARP、裂解的 caspase-3、E-钙黏蛋白、波形蛋白和 NRIP1 的蛋白水平。通过 Sanger 测序、分歧引物 PCR 和 RNase R 处理验证环状结构。通过双荧光素酶报告基因、RIP 和 RNA 下拉实验观察 circNTRK2 的 ceRNA 调控机制。构建小鼠异种移植模型以在体内证实 circNTRK2 在 ESCC 中的致癌性。

结果

circNTRK2 在 ESCC 组织和细胞中高表达。circNTRK2 的高表达与晚期 TNM 分期、淋巴结转移和生存期短有关。体外敲低 circNTRK2 抑制 ESCC 细胞增殖、侵袭和上皮-间充质转化（EMT），并加速细胞凋亡。机制研究表明，circNTRK2 可作为 miR-140-3p 的海绵，减弱其对靶基因 NRIP1 表达的抑制作用。此外，circNTRK2 的过表达减弱了 miR-140-3p 对 ESCC 细胞恶性表型的抑制作用。此外，敲低 NRIP1 抑制细胞增殖、侵袭和 EMT，同时促进凋亡。此外，沉默 circNTRK2 通过调节 NRIP1 表达抑制细胞增殖和侵袭。此外，体内敲低 circNTRK2 可抑制 ESCC 肿瘤生长。