Circular RNA circNTRK2 facilitates the progression of esophageal squamous cell carcinoma through up-regulating NRIP1 expression via miR-140-3p
Circular RNA circNTRK2 facilitates the progression of esophageal squamous cell carcinoma through up-regulating NRIP1 expression via miR-140-3p
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环状RNA circNTRK2通过miR-140-3p上调NRIP1表达促进食管鳞状细胞癌的进展
DOI：
10.1186/s13046-020-01640-9
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发表时间：
2020-07-11
影响因子：
11.3
通讯作者：
Zheng, Yuling
中科院分区：
文献类型：
--
作者：
Chen, Xiaoqi;Jiang, Jing;Zheng, Yuling
食管鳞状细胞癌（Esophageal squamous cell carcinoma，ESCC）是最常见的胃肠道恶性肿瘤之一，死亡率高。环状RNA（CircRNA）因其在肿瘤发生发展中的重要作用而成为近年来的研究热点。本研究旨在阐明circNTRK 2在ESCC中的作用及其分子机制。方法采用qRT-PCR方法检测circNTRK 2、miR-140- 3 p和核受体相互作用蛋白1（NRIP 1）mRNA的表达水平。CCK-8法、EdU法和集落形成实验检测细胞增殖能力。用transwell法检测其侵袭能力。流式细胞仪检测细胞凋亡率。通过蛋白质印迹法测定裂解的PARP、裂解的caspase-3、E-钙粘蛋白、波形蛋白和NRIP 1的蛋白水平。通过桑格测序、趋异引物PCR和RNase R处理进行环状结构的验证。通过双荧光素酶报告基因、RIP和RNA pull-down试验观察circNTRK 2的ceRNA调控机制。结果CircNTRK 2在食管鳞癌组织和细胞中高表达。circNTRK 2的高表达与肿瘤的TNM分期、淋巴结转移及生存期相关。在体外，敲低circNTRK 2抑制ESCC细胞增殖、侵袭和上皮-间质转化（EMT），并加速凋亡。机制分析揭示，circNTRK 2可以充当miR-140- 3 p的海绵，以减轻其对靶NRIP 1表达的抑制。此外，miR-140- 3 p诱导的对ESCC细胞恶性表型的抑制作用通过circNTRK 2的过表达而减弱。此外，NRIP 1的缺失阻碍了细胞增殖、侵袭和EMT，同时增强了凋亡。此外，circNTRK 2的沉默通过调节NRIP 1表达来抑制细胞增殖和侵袭。结论CircNTRK 2通过调控miR-140- 3 p/NRIP 1通路促进食管鳞癌的进展。我们的研究结果有助于更好地理解circRNA作为miRNA海绵，并突出了ESCC中有希望的治疗靶点。
BackgroundEsophageal 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.MethodsThe 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.ResultsCircNTRK2 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.ConclusionCircNTRK2 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.