益气养肺方干预bFGF/FGFR1通路抑制肺癌侵袭转移的作用及分子机制

Invasion and metastasis of elderly lung cancer patients could suffer from deficiency of the healthy qi and yin，which indicated poor clinical prognosis. Basic fibroblast growth factor bFGF/FGFR1 signal transduction pathway is the major signal passageway of lung cancer invasion and metastasis. According to our previous research, supplement energy and nourish lung herbs (SENL) could inhibit metastasis of lung cancer and improve quality of life. SENL could increase efficiency and reduce toxicity during lung cancer chemotherapy, and also inhibit basic fibroblast growth factor (bFGF)-induced drug resistance. Recent research indicated that the combination of SENL and anti-bFGF mAbs ccould inhibit AKt/mTOR phosphorylation in lung cancer. Thus, bFGF/FGFR1 signaling pathway mediated by SENL might play an important regulation role in the inhibition of invasion and metastasis in lung cancer. In this research, we focus on personal treatment based on differentiation of symptoms and signs in lung cancer patients, clinical experience in Traditional Chinese Medicine (TCM). We select high metastasis lung cancer as research objective, and establish transplanted lung cancer cells in nude mice, adopt co-cultured lung cancer cells with migration and invasion research platform, employ biochips, flow cytometry, quantitative real-time RT-PCR and Western bolt methods by multiple molecular biological techniques. Therefore, from animal models to cells level, genes to protein moleculars level, we investigate the function of SENL on inhibition of invasion and search for precise molecular mechanism of suppressing lung cancer by interfering bFGF/FGFR1 signaling pathway. Key driver targets would be confirmed through the function of SENL research. Further systematical investigation on SENL of inhibiting lung cancer metastasis, might produce considerable strategy of effective combination therapy both on TCM theory and experimental research.

老年肺癌倂转移在中医临床多表现为气阴两亏证，与预后不良密切相关。碱性成纤维生长因子bFGF/FGFR1信号通路是参与肺癌侵袭转移重要途径。前期研究发现，益气养肺方具有明确抑制肺癌转移及改善患者生存质量作用；益气养肺方联合化疗增效减毒，拮抗bFGF介导肺癌化疗耐药；同时发现益气养肺方联合bFGF单抗，抑制肺癌细胞增殖并诱导凋亡，降低Akt/mTOR磷酸化。因此，本课题组提出益气养肺方可能在干预及调控肺癌bFGF/FGFR1通路，抑制肺癌侵袭转移中发挥重要作用。基于此，本研究遵循中医辨证施治、临证经验，以体内外肺癌转移为研究对象，建立肺癌转移动物模型，运用迁移侵袭检测、生物芯片、流式细胞术、定量PCR和Western blot 手段，探索益气养肺方对肺癌bFGF/FGFR1通路影响及其调控的精确分子机制，确立益气养肺方抗肺癌转移关键干预靶点，为阐明益气养肺方抑制肺癌侵袭转移机制提供新科学依据。

本项目根据任务书计划，顺利完成益气养肺方干预bFGF/FGFR1通路抑制肺癌侵袭转移的作用机制研究，主要研究工作结果如下： .1.明确益气养肺方及组分与作用靶点研究，建立复方益气养肺方的质量标准，采用薄层色谱法对复方进行定性定量鉴别，通过高效液相色谱法法测定复方益气养肺方中补骨脂素含量。益气养肺方有效活性成分槲皮素抑制肺癌增殖，促进凋亡，增加顺铂对肺癌杀伤作用，在体内外均能抑制SPP1的表达，调控PI3K/AKT /NF-kB通路。.2.通过bFGF/FGFR1通路在临床肺癌侵袭转移作用研究，显示bFGF，FGFR1在肺癌组织中表达均上调，bFGF与FGFR表达有明显相关性，bFGF与FGFR在肺癌的表达与病理分级、淋巴结转移、临床分期有关，肺癌患者血清中成纤维细胞激活蛋白、血小板源生长因子水平均高于健康对照组。.3.开展益气养肺中药有效成分及bFGF单抗抑制肺癌新靶点的作用研究，显示汉防己甲素抑制高侵袭肺癌细胞相关的特异miRNAs，汉防己甲素通过LINC00467/miR-335 -3p/PTK2轴，抑制PI3K/Akt 通路抑制肺癌侵袭转移能力。bFGF抗体联合伊立替康在抑制肺癌生长及转移中有协同作用，具有抑制肺癌增殖、迁移、侵袭作用，bFGF抗体联合伊立替康通过抑制p-AKT蛋白和ERK蛋白的水平，抑制肿瘤细胞的增殖和转移。.4.参与肺癌侵袭转移及其干预作用的新靶点研究，bFGF和FGFR1可激活PI3K/Akt/mTOR信号通路，并且促进肺癌细胞增殖、粘附、侵袭和迁移、抑制细胞凋亡；bFGFmAb和AP24534可以抑制PI3K/Akt/mTOR信号通路，并且抑制肺癌细胞增殖、侵袭和迁移、促进细胞凋亡。在RNA m6A甲基化影响肺癌侵袭转移研究中，表明RNA m6A阅读子YTHDF2通过上调AXIN1/Wnt/β-catenin信号通路参与肺腺癌的发展，bFGF信号可通过丝裂原活化蛋白激酶(MAPK)/ERK磷酸化LRP6来放大经典Wnt /β-catenin信号通路作用。.5.本项目形成的主要成果有①发表学术论文SCI和核心期刊论文4篇，并在中国肿瘤大会作大会报告，培养博士后、博士、硕士共7名。本研究为针对性筛选抑制肺癌侵袭转移的有效中药，提供研究依据和实验探索。