HIF-1α上调毒蕈碱受体3促进非小细胞肺癌侵袭转移的分子机制

In our previous study, we found that muscarinic receptor 3(M3R) was overexpressed in NSCLC tissues. The expression intensity was strongly correlated to metastasis status of NSCLC patients, but conversely correlated to the 5-year survival rate. We found that M3R promoted invasion and metastasis of NSCLC via PI3K/Akt/MMP9 pathway. However, why M3R is highly expressed in NSCLC patients is still unclear. Further analysis revealed that M3R expression level was conversely related to airway obstruction status and NSCLC tissues with chronic pulmonary obstructive disease (COPD) showed higher intensity compared with those without COPD. COPD, a common disease that increases the risk of lung cancer and predicts worse prognosis of lung cancer, is also characterized with hypoxia. We performed bioinformatics analysis and found hypoxia response element in the promoter of M3R gene. We also found that NSCLC cell lines under hypoxia showed upregulation of M3R and expression level of M3R was positively related to hypoxia-inducible factor-1α (HIF-1α) in NSCLC tissues. We propose a hypothesis that hypoxia upregulates M3R via hypoxia-inducible HIF-1α, and this promotes invasion and metastasis of NSCLC. In this project, we aim to investigate the role of HIF-1α in M3R expression as well as invasion and metastasis of NSCLC. NSCLC cell lines with HIF-1α or M3R upregulated or downregulated, as well as M3R agonist and antagonist, would be used. The invasion potential of NSCLC cell lines would be evaluated. Furthermore, the interaction between HIF-1α and M3R would be studied using Dual Luciferase reporter, Ch-IP and two hybrid system. We hope to find out the mechanism of hypoxia in modulating the expression of M3R and thus propose that M3R could be another therapy target in treating lung cancer.

申请者前期研究发现毒蕈碱受体3（M3R）在非小细胞肺癌（NSCLC）组织中高表达，M3R通过PI3K/Akt/MMP9通路促进NSCLC侵袭转移。但M3R高表达机制不清。进一步研究发现NSCLC组织M3R表达与患者气流受限程度呈正相关，合并慢性阻塞性肺疾病（COPD）者M3R水平显著高于不合并COPD者。低氧是COPD的重要特征，提示低氧可能是M3R高表达的原因。初步实验发现：M3R启动子存在低氧反应元件，低氧条件下NSCLC细胞株M3R表达上调，NSCLC组织中M3R与HIF-1α表达呈正相关。低氧是否通过HIF-1α上调M3R，进而促进NSCLC侵袭转移未见报道。本课题拟在低氧、常氧环境，采用双荧光素酶报告系统、Ch-IP、哺乳动物双杂交系统等方法，阐明低氧通过HIF-1α上调M3R进而促进NSCLC侵袭转移的作用及机制，为NSCLC发生发展机制提供理论和实验依据，并为其防治提供新靶点

非小细胞肺癌（NSCLC）是最常见的肺癌类型，转移是其治疗失败的重要原因。前期研究发现毒蕈碱受体3（M3R）在NSCLC组织中高表达，M3R通过PI3K/Akt/MMP9通路促进NSCLC侵袭转移。但M3R高表达机制不清。进一步研究发现NSCLC组织M3R表达与患者气流受限程度呈正相关，合并慢性阻塞性肺疾病（COPD）者M3R水平显著高于不合并COPD者。低氧是COPD的重要特征，提示低氧可能是M3R高表达的原因。本研究主要为明确低氧如何上调M3R，进而促进NSCLC侵袭转移。.我们研究发现，HIF-1α在NSCLC中高表达，并与NSCLC肿瘤大小、远处转移及不良预后相关；HIF-1α 与 M3R 表达水平呈正相关。低氧培养条件下，NSCLC细胞株L78和A549 细胞的 M3R 均发现有不同水平的表达。低氧条件下，L78和A549细胞侵袭转移能力增加。而应用HIF-1α shRNA处理后，细胞的迁移、侵袭能力均得到明显抑制，提示低氧通过HIF-1α促进NSCLC细胞侵袭转移。而在低氧培养条件下，使用M3R shRNA下调M3R表达，L78和A549细胞迁移侵袭能力得到抑制。以上提示：HIF-1α通过上调M3R表达促进L78和A549细胞迁移侵袭。通过生物信息学分析显示， M3R上游2000bp以内启动子区域存在2个低氧反应原件（HREs），分别位于-328bp（HRE-1）和-1247bp（HRE-2），进一步双荧光素酶实验显示：HIF-1α可通过与HRE-1结合并促进M3R转录。低氧培养下，L78和A549细胞p-Akt活性上调；下调M3R表达后，p-Akt活性下调。.综上，我们研究发现，HIF-1α通过上调M3R，通过PI3K/Akt通路促进NSCLC细胞侵袭转移。