Glypican5调控肺腺癌转移的分子机制研究

GPC5 is a differently expressing gene associated with lung cancer metastasis identified by the gene expression chip screening and validated by the real-time RT-PCR. We previously found that: 1) In a large cohort of clinical non small cell lung cancer samples, the relative expression of GPC5 in lung cancer tissue was negtively correlated with the lymph node metastasis. The GPC5 expression was also positively correlated with the metastasis suppressing gene E-cadherin expression however negatively correlated with the metastasis promoting gene MMP9 expression; 2) In A549 adenocarcinoma cell line, cell invasion was significantly suppressed by overexpressing GPC5; 3) Previous study demonstrated that GPC3, another sub-family member of GPC5, decreases the invasive capability of breast cancer cells by inhibiting the canonical Wnt signal pathway. Also in A549 cell line, overexpression of GPC5 inhibited the expression of the Wnt signal pathway related genes. Therefore, the present project intended to hypothesize that GPC5 might suppress the lung adenocarcinoma metastasis by regulating canonical Wnt pathway. We will engage human lung adenocarcinoma cell line A549 and H1975 as our research objects, and plan to perform cell invasion assay, canonical Wnt pathway expression assay, co-immunoprecipitations, TOP/FOPflash report gene assay, and the in-site lung cancer metastasis model in nude mice so as to validate our hypothesis in vitro and in vivo. Since there is no similar researches regarding GPC5 regulating lung cancer metastasis found until now, our investigation might provide a potential novel target for the treatment of lung cancer metastasis.

GPC5是课题组采用表达谱芯片筛选并验证与肺癌转移相关的差异表达基因。前期发现：①人非小细胞肺癌组织GPC5表达与有无淋巴结转移负相关，与转移抑制基因E-cad表达正相关，与促转移基因MMP9表达负相关②人肺腺癌细胞株A549过表达GPC5显著降低侵袭能力③文献表明与GPC5同源亚家族成员GPC3可抑制经典Wnt通路从而抑制乳腺癌细胞侵袭，课题组发现A549过表达GPC5亦可显著抑制经典Wnt通路关键基因表达。本项目提出调节经典Wnt通路是GPC5抑制肺腺癌转移重要机制这一假说，拟以A549、H1975两种肺腺癌细胞株为对象，结合过表达/沉默策略，通过观察细胞侵袭能力、经典Wnt通路基因表达、免疫共沉淀、TOP/FOPflash报告基因系统验证、裸鼠原位肺癌自发转移模型等体外、体内研究系统验证该假说。GPC5调控肺癌转移机制研究尚未见报道，本研究有源头创新性，可为肺癌转移防治提供潜在靶标。

背景和目的：非小细胞肺癌（NSCLC）是全世界范围内死亡率最高的恶性肿瘤。磷脂酰肌醇蛋白聚糖家族成员5（Glypican5，GPC5）是项目组前期结合表达谱芯片筛选出的潜在新型转移抑制基因，迄今为止还无任何研究深入阐释GPC5在NSCLC中的功能和分子机制。本研究拟从临床样本、细胞、动物转移模型三个层次全面评价NSCLC患者中GPC5的表达特征及其生物学功能，并结合生物信息学及分子生物学实验系统探讨其分子机制。.研究内容：①结合临床NSCLC患者肿瘤及瘤旁正常配对的新鲜组织及组织芯片样本，系统评价GPC5 mRNA及蛋白水平的表达特征及其与各临床信息、预后之间的关系；②以NSCLC细胞株A549、H1975为研究对象，在体外、体内分别探讨GPC5影响肺癌侵袭迁移、细胞周期、增殖等恶性行为的生物学功能；③结合转录组测序、生物信息学分析，运用沉默/过表达策略，结合TOP/FOP荧光素酶报告基因、免疫共沉淀（Co-IP）及拯救实验系统验证GPC5是否能通过调控Wnt信号通路从而抑制NSCLC侵袭转移的分子机制。.主要结果：①GPC5在NSCLC肿瘤组织中较癌旁正常组织显著低表达（p<0.001），其表达水平与淋巴结转移程度显著负相关（p=0.002），在肺腺癌中GPC5表达水平越高，患者预后越好（p=0.036）；②GPC5可在体外实验中抑制NSCLC细胞株侵袭迁移、诱导G1/S期细胞阻滞并抑制细胞增殖，在NO-SCID小鼠体内被动转移模型中可显著抑制肿瘤肝脏及肺转移；③过表达GPC5可通过上调E-cadherin表达显著抑制NSCLC细胞上皮-间质转化（EMT）进程；④TOP/FOP报告基因、Co-IP及拯救实验证实GPC5可竞争性结合Wnt3a，抑制下游经典Wnt/β-catenin信号通路，从而抑制EMT进程。⑤临床样本验证证实GPC5与E-cadherin的表达显著正相关，而与Wnt/β-catenin通路下游基因Twist1、MMP2的表达显著负相关。.结论及意义：首次系统探索GPC5在NSCLC中的表达特征及生物学功能，证实其是一个新型NSCLC转移抑制基因；证实GPC5可竞争性结合Wnt3a，通过调控经典Wnt3a/β-catenin通路、进而抑制EMT进程从而抑制肺癌侵袭转移这一分子机制。本研究具有源头创新性，为临床肺癌防治提供了潜在新靶标。