长链非编码RNA REG1CP上调TNF-α激活TGF-β通路介导喉鳞癌侵袭转移的机制研究

Invasion and metastasis are critical risk factors hindering the prognosis of laryngeal squamous cell carcinoma（LSCC）. The epithelial mesenchymal transformation (EMT) is the major mechanism of tumor invasion and metastasis, and TGF-β pathway plays a key role in EMT process. It has been reported that TNF-α enhances the EMT process by activating the TGF-β pathway, but the upstream regulatory mechanism is unclear. Whole transcriptomic sequencing of LSCC tissues and paired adjacent normal mucosa tissues revealed that the long non-coding RNA REG1CP was upregulated significantly. Knockdown of REG1CP inhibited the migration and invasion of LSCC cells. Meanwhile, knockdown of REG1CP decreased expression of TNF-α at transcription and translation level, and the TGF-β signaling pathway was blocked. Therefore, we hypothesized that REG1CP promotes invasion and metastasis of LSCC through the activation of the TGF-β pathway mediated by upregulating TNF-α. This project will explore the mechanism of REG1CP-mediated invasion and migration in LSCC through combination of various biochemical and molecular biological experiments, and examine the role of REG1CP in LSCC using in vitro and in vivo model. Moreover, the correlation between REG1CP and clinical features of LSCC patients will be analyzed. Overall, results of this project will provide new insight and candidate targets for molecular targeting therapy of LSCC.

易侵袭及颈淋巴结转移是严重阻碍喉鳞癌预后水平提升的重要风险因素。上皮间质转化（EMT）是肿瘤侵袭和转移重要机制之一，而TGF-β通路发挥关键作用。文献报道TNF-α可增强TGF-β通路介导的EMT过程，但上游调控机制不明。课题组经喉鳞癌转录组测序发现长链非编码RNA REG1CP显著上调，敲降后可遏制喉鳞癌细胞迁移及侵袭能力，同时TNF-α转录与翻译水平下调, TGF-β信号通路受阻。提示REG1CP可通过TNF-α激活TGF-β通路介导的EMT进而促进喉鳞癌侵袭转移。本项目拟利用RNA pull-down、ChIP、gain/loss of function体内外实验，以REG1CP为核心揭示TNF-α/TGF-β上游调控机制，观察EMT变化及对喉鳞癌细胞体外迁移侵袭及体内成瘤与转移能力的影响，并分析REG1CP与喉鳞癌患者临床病理参数及预后关系，为喉鳞癌靶向治疗提供新靶标与理论支撑。

喉鳞癌（laryngeal squamous cell carcinoma，LSCC）是头颈部发病率第二位的恶性肿瘤，预后较差，过去30年LSCC患者5年生存率无明显提升。易发生局部侵袭和颈淋巴结转移是导致LSCC预后不良的主要危险因素。长链非编码RNA（long non-coding RNA，lncRNA）在肿瘤发生发展过程具有重要调控作用。但lncRNA在LSCC中的功能和机制还知之甚少。本研究利用实时荧光定量PCR（qPCR）、增殖分析、Transwell assay、基因表达谱芯片、免疫组化、双荧光素酶报告分析、RNAi等技术，研究了lncRNA REG1CP在LSCC中的表达模式、生物学功能和分子机制。临床样品分析结果表明REG1CP在LSCC组织中表达水平显著上调，而且REG1CP高表达与LSCC恶性进展显著相关。对TCGA数据库的数据进行分析显示REG1CP高表达与头颈鳞癌患者预后不良相关。体内外功能研究发现，敲降REG1CP抑制LSCC细胞增殖、迁移、侵袭及成瘤能力，表明REG1CP在LSCC中发挥促癌基因功能。机制研究揭示REG1CP通过作为竞争性内源RNA（competing endogenous RNA, ceRNA）特异性结合miR-181c-5p，上调miR-181c-5p靶基因TNF的水平，进而增强TGFβ信号通路活性，从而促进LSCC增殖、迁移、侵袭等恶性表型。综上所述，本研究结果从non-coding RNA角度拓展了对LSCC发生发展机制的认知，将为LSCC的诊断、预后评估和治疗提供新的潜在标志物和治疗靶点。