Glypican-5抑制肺腺癌淋巴管生成和淋巴结转移的机制研究

Phosphatidylinositol protein 5 (glypican-5, GPC5) is a tumor suppressor gene which had a significant negative correlation with lymph node metastasis of lung adenocarcinoma and validated by our previous key research. We previously found that: ①Lung adenocarcinoma cell line H1693 with overexpressed GPC5 gene was co-cultured with human lymphatic endothelial cells (hLECs), and the ability of lymphatic vessel formation and cell viability had dropped significantly; ②There was a significant negative correlation between GPC5 expression level and .lymphatic vessel density; ③The results of gene expression profiling, bioinformatics analysis and validation showed that overexpressing GPC5 could induce the downregulation of lymphangiogenesis factor-FGF2 expression. So we hypothesized that GPC5 was able to inhibit lymph node metastasis of lung adenocarcinoma by regulating FGF2 signaling pathway. In this proposal, firstly, we plan to clarify the functional role of GPC5 in lymphangiogenesis of lung adenocarcinoma. To achieve this goal, we design to over-express and knock-down GPC5 both in vivo and in vitro (in two separated high lymphatic metastatic lung adenocarcinoma cell lines and lymphatic endothelial cell line) and evaluate the impact of GPC5 on lymphangiogenesis and lymph node metastasis of lung adenocarcinoma. Secondly, we aim to explore how GPC5 exerts its regulatory function through FGF2 signaling pathway. Thirdly, combining with tissue microarray, we would validate the correlation between GPC5-FGF2 signaling pathway and lymph node metastasis. To date, no data is available about the function and mechanism of GPC5 inhibited lymph node metastasis of lung adenocarcinoma at home or abroad. Our study can provide new ideas for the prevention and treatment of lung adenocarcinoma.

GPC5是前期筛选获得的一条与肺腺癌淋巴结转移程度显著负相关的抑癌基因。已发现：①过表达GPC5的肺腺癌细胞株H1693与人淋巴管内皮细胞hLECs共培养后，淋巴管形成能力及细胞活力显著下降；②人肺腺癌免疫组化显示GPC5表达与淋巴管密度负相关；③表达谱芯片、生物信息学分析及验证提示：过表达GPC5可下调促淋巴管生成因子FGF2表达。故我们提出“GPC5 通过调控FGF2 信号通路抑制肺腺癌淋巴结转移”这一科学假说，并拟以两种高淋巴转移性肺腺癌细胞株和hLECs为对象，采用过表达/沉默策略，进行体内外实验，证实GPC5可抑制淋巴管生成及淋巴结转移；以FGF2为机制线索，阐明GPC5抑制肺腺癌淋巴结转移的分子机制；结合高通量组织芯片，临床大样本验证GPC5-FGF2通路与淋巴结转移的相关性。有关GPC5抑制肺腺癌淋巴结转移的功能和机制研究国内外尚未见报道，本研究将为肺腺癌防治提供新思路。

研究背景：.肺癌是常见的肿瘤，其死亡率在因癌症死亡的患者中位居首位。磷脂酰肌醇蛋白聚糖-5（glypican5，GPC5），属于硫酸类肝素蛋白多糖（heparan sulfate proteoglycans， HSPGs）家族，其生物功能在肺癌细胞的浸润和进展中的作用有待揭示。.研究方法：.采用慢病毒载体构建了两株GPC5过表达的肺癌稳转细胞株(H1693 and H2009)。利用ExoQuick-TCTM外泌体分离试剂盒，分离了肺癌细胞分泌的外泌体。通过透射电镜和WesternBlot对外泌体进行了表征。通过CCK8试剂盒检测了淋巴管内皮细胞（hLECs）和肺癌细胞的增殖水平。通过划痕时间检测hLECs的迁移能力。通过成管实验评价了hlECs的成管能力。利用miRNA高通量测序，分析了外泌体中的miRNAs含量差异。通过生信分析差异表达的miRNA及其调控靶点，并经过双荧光素酶进行验证。最后，通过裸鼠体内的成瘤和转移实验，验证了GPC5-CTDSP1-miR-26b信号轴对肺癌细胞作用。.研究结果：.本项目中，我们首次发现GPC5过表达组的肺癌细胞分泌的外泌体显著抑制淋巴管内皮细胞的成管和迁移。通过miRNA-Seq对比分析正常组和GPC5过表达组肺癌细胞分泌的外泌体中miRNAs的含量差异，结果发现miR-26b富集于GPC5过表达组肺癌细胞分泌的外泌体中。在进一步的机制研究中，我们发现GPC5可以转录水平激活miR-26b的宿主基因CTDSP1的表达，并且此激活方式依赖于ARH-ARNT信号通路。与此同时，GPC5转录激活的CTDSP1基因，显著抑制肺癌细胞本身的增殖水平。外泌体中含有的miR-26b通过靶向结合于PTK2基因的3’UTR区域，抑制PTK2基因的表达，进而抑制淋巴管内皮细胞的成管和迁移能力。.研究意义：.本研究揭示了GPC5-CTDSP1-miR-26b-PTK2信号通路在肺癌细胞淋巴管转移中的关键抑癌作用。此发现将为肺癌的靶向治疗提供新的思路和策略。.