非小细胞肺癌中黄连素干预TF/FVIIa通路抑制转移的作用研究

Lung cancer metastasis is the major cause of death in patients with non-small cell lung cancer (NSCLC), tissue factor (TF) is the key factor for tumor dissemination and metastasis. So far there is no domestic and international research about using traditional Chinese medicine with low toxicity and high efficiency to affect lung cancer metastasis. In previous work, we have demonstrated that Berberine (BBR) can inhibit the invasion and migration of NSCLC cells, and BBR can inhibit the expression of TF, then in the further research, we also found that miRNA19a and miRNA20a could target TF through luciferase reporter assay and BBR could up-regulate the expression of TF-relevant miRNAs. Therefore, we speculated that BBR could regulate the TF/FVIIa pathway through up-regulating miRNA to inhibit the metastasis of NSCLC. So in this project we plan to 1) determine the TF and relevant miRNAs expression in NSCLC tissues of advanced stage combined with thrombus; 2) determine TF and relevant miRNAs expression under different concentrations of BBR in NSCLC cells, and determine the expression of relevant proteins during TF/FVIIa pathway, the downstream metastatic proteins and angiogenesis genes through gain or loss of function of TF with BBR intervention; 3) establish orthophoric transplanted NSCLC nude mice and conditional TF knock out mice, to further develop the BBR role in regulating TF/FVIIa pathway contributing to mechanism of NSCLC by up-regulating miRNA.

肺癌转移是非小细胞肺癌(NSCLC)主要死因，组织因子(TF)是肿瘤播散与转移的关键因素。利用低毒高效中药调控TF影响肺癌转移机制国内外尚无报道。我们前期研究证实黄连素（BBR）能够抑制NSCLC细胞侵袭与迁移，并发现其能抑制TF表达，通过荧光素酶报告实验发现miRNA19a/20a能靶向TF，且BBR能够上调TF相关miRNA表达。因此我们推测BBR通过上调相关miRNA调控TF/FVIIa通路抑制NSCLC的转移。本课题拟进一步1）在晚期合并血栓NSCLC组织中验证TF及相关miRNA表达；2）细胞水平验证不同浓度BBR干预下TF及相关miRNA表达情况，并通过TF功能性获得或缺失验证BBR干预下TF/FVIIa通路及下游转移蛋白和血管生成相关基因表达情况；3）构建正位肺癌移植裸鼠及TF条件性基因敲除小鼠，进一步探索BBR上调miRNA调控TF/FVIIa通路在肺癌中的转移机制。

肺癌转移是非小细胞肺癌(NSCLC)主要死因，组织因子(TF)是肿瘤播散与转移的关键因素。利用低毒高效中药调控TF影响肺癌转移机制国内外尚无报道。我们前期研究证实黄连素（BBR）能够抑制NSCLC细胞侵袭与迁移，并发现其能抑制TF表达，通过荧光素酶报告实验发现miRNA-19a能靶向TF，且BBR能够上调TF相关miRNA表达。本课题组前期已经证明将hsa-miR-19a-mimic或hsa-miR-19a-inhibitor 转染PC9 细胞后发现,hsa-miR-19a-mimic 可抑制细胞增殖，阻滞细胞于G0/G1 期， 下调TF 的表达；hsa-miR-19ainhibitor则可促进细胞增殖，促进细胞周期进程,上调TF 的表达,并进一步检测NSCLC患者血清中TF的表达水平，TF的表达越高预后越差。本研究旨在通过鉴定黄连素作用于miR-19a来完善我们之前的研究，并评估其在肺癌中的生物学功能。我们进一步通过QRT-PCR测定不同浓度黄连素处理肿瘤细胞后miR-19a 、TF的表达情况。cck8实验测定BBR对转染hsa-miR-19ainhibitor或 TF-overexpression肺癌细胞的增殖能力，流式方法检测BBR对转染hsa-miR-19ainhibitor或 TF-overexpression的肺癌细胞中凋亡比例， Western blotting方法检测BBR对转染hsa-miR-19ainhibitor或 TF-overexpression的肺癌细胞中凋亡相关蛋白表达的影响，动物实验检测BBR对A549、PC9肺癌细胞异体移植瘤的作用。结果发现，黄连素通过上调miR-19a的表达抑制细胞增殖和诱导细胞凋亡来抑制NSCLC生长并可能通过miR-19a/ TF激活MAPK通路起到肿瘤抑制剂的作用。