HNRNPK/CLCN3/p-AKT正反馈回路对肺腺癌发生发展的影响及机制研究

The high expression of CLCN3 is closely related with lung adenocarcinoma development. It is of great significance to discover the tumor-specific factors which regulate CLCN3 expression. In our preliminary study, we pulled down and identified transcription factor HNRNPK as a CLCN3 promoter-binding protein in lung adenocarcinoma cells by DNA probes and proteomics techniques. We also showed that HNRNPK regulated CLCN3 transcription, translation and formed feedback loop with p-AKT, thus influencing cell growth and migration. However, so far it is unclear about its molecular mechanisms as well as the clinical implications. In this study, we will knockdown or overexpress HNRNPK in human lung adenocarcinoma cell lines and animal models, and then study the effects of HNRNPK on CLCN3 expression, promoter activity, p-AKT feedback loop, and cell proliferation or migration, thereby revealing the mechanisms of action of the HNRNPK/CLCN3/p-AKT positive feedback loop in lung adenocarcinoma carcinogenesis. Moreover, we will use clinical data, to analyze the expression levels and the correlation of HNRNPK/CLCN3/p-AKT in tumor tissues from lung adenocarcinoma patients, and to evaluate their relationship with lung adenocarcinoma growth, metastasis and patient prognosis, thereby identifying the molecular diagnostic significance of HNRNPK/CLCN3/p-AKT. The results from this study will provide theoretical basis for the identification of new therapeutic targets for lung adenocarcinoma treatment.

氯通道蛋白CLCN3高表达与肺腺癌发生发展密切相关，因此发现肺腺癌中特异性CLCN3表达调控因子意义重大。前期研究中，我们利用DNA探针垂钓和蛋白质组学方法，在肺腺癌细胞中鉴定出转录因子HNRNPK为CLCN3启动子DNA结合蛋白，同时验证HNRNPK可调控CLCN3转录和翻译并与p-AKT形成反馈回路共同影响细胞生长和迁移，但具体分子机制及临床意义，目前尚不清楚。本研究拟在肺腺癌细胞和动物模型中敲低或过表达HNRNPK，研究其对CLCN3表达、启动子活性、p-AKT反馈回路的调控以及对肿瘤生长迁移等的影响，揭示HNRNPK/CLCN3/p-AKT正反馈回路在肺腺癌发展中的作用和分子机制；并结合临床资料，检测肺腺癌患者组织中HNRNPK/CLCN3/p-AKT的表达水平并分析相关性，评估其与肺腺癌生长、转移及预后的关系，明确其分子诊断意义。本项目将为确立新型的肺腺癌治疗靶点提供理论依据。

氯通道蛋白CLCN3高表达与肺腺癌发生发展密切相关，因此发现肺腺癌中特异性CLCN3表达调控因子意义重大。在本研究中，我们利用DNA探针垂钓和蛋白质组学方法，在肺腺癌细胞中鉴定出转录因子HNRNPK为CLCN3启动子DNA结合蛋白，并发现结合位点和motif序列可能为-538/-248bp和GCGAGG，同时证实了HNRNPK可调控CLCN3的启动子活性和基因转录。体内外实验中，我们通过敲低或过表达HNRNPK，发现HNRNPK可直接调控CLCN3的表达和肿瘤生长侵袭，同时证实HNRNPK/CLCN3轴与CAF细胞中的PI3K/Akt信号形成反馈回路，通过肿瘤-CAF相互作用促进肺腺癌进展。在临床样本中，我们发现CLCN3和HNRNPK在人肺腺癌组织中高表达并呈正相关，且CLCN3高表达和HNRNPK高表达的患者预后均较差。因此本课题揭示了HNRNPK/CLCN3轴在肺腺癌发生发展中的作用，发现了HNRNPK通过调控CLCN3表达及与CAF形成反馈回路促进肺腺癌生长侵袭的分子机制，明确了HNRNPK/CLCN3在肺腺癌分子诊断、预后评估中的临床意义。本项目将为确立新型的肺腺癌治疗靶点提供理论依据。