增殖抑制基因（HSG）协同顺铂促进人肺腺癌细胞线粒体途径凋亡的机制研究

Platinum-based chemotherapy is the first-line treatment in advanced lung adenocarcinoma. Unfortunately, the research on profiling of genes expression in predicting the efficacy of this treatment has not got exciting results. Our previous study suggested that hyperplasia suppressor gene (HSG) induced apoptosis of mitochondrial pathway via MAPK signal transduction pathway, which had the similar pathway on tumor cell apoptosis induced by cisplatin. So we supposed that it might be in synergistic effects in apoptosis mechanisms with HSG and cisplatin. The current study would be performed in vitro and in vivo. Three human lung adenocarcinoma cell lines, A549, Calu-6 and NCI-H1299, were chosen according to two factors, the expression of p53, an important regulator in mitochondrial pathway of apoptosis, and drug resistance to another regulator, ABT-737. Adenoviral-mediated gene transfer would be performed to make human HSG overexpression in the cell lines. In vitro, the apoptotic rate of the cell lines and the expression of the apoptosis-related genes and proteins would be analyzed in relation to the degrees of differential HSG expression and the effects of cisplatin and ABT-737. In vivo, tumor models in nude mice would be generated. Tumor formation and progression in these mice and the expression of the apoptosis-related genes and proteins would also be analyzed on the effect of the expression of HSG and with/without the treatment of cisplatin and/or ABT-737. The research aims to study on the apoptosis mechanisms via mitochondrial pathway induced by synergistic effects of HSG and cisplatin on human lung adenocarcinoma cells, and the role of p53 and ABT-737 in the molecular regulation of it.

晚期肺腺癌主要以铂类为基础的联合化疗方案治疗，但缺乏疗效预测基因。本课题组前期研究发现，增殖抑制基因（HSG）可通过MAPK信号传导通路诱导细胞线粒体途径凋亡，与顺铂诱导肿瘤细胞凋亡有相同的信号传导通路，两者可能有协同性。本课题拟通过细胞水平和整体动物水平研究，根据线粒体途径凋亡调控基因p53表达情况及调控药物ABT-737耐药情况不同，选取A549、Calu-6、NCI-H1299三种人肺腺癌细胞株，运用腺病毒转染技术分别建立HSG高表达细胞株。在细胞水平层面，在顺铂、ABT-737干预与否的体系中，分析HSG表达程度不同细胞株凋亡率及凋亡基因和蛋白表达的差异；在整体动物层面，建立裸鼠瘤荷模型，比较HSG表达程度不同细胞株在顺铂、ABT-737干预与否时的成瘤情况、凋亡基因和蛋白表达的差异，探讨HSG基因协同顺铂促进人肺腺癌细胞线粒体途径凋亡机制及p53基因、ABT-737对其调控作用。

晚期肺腺癌主要以铂类为基础的联合化疗方案治疗，但缺乏疗效预测基因。本课题组前期研究发现，增殖抑制基因（HSG）可通过MAPK信号传导通路诱导细胞线粒体途径凋亡，与顺铂诱导肿瘤细胞凋亡有相同的信号传导通路，两者可能有协同性。本研究中，我们首先利用30对人肺腺癌组织标本及癌旁正常组织样本，采用免疫组化实验比较，证实HSG基因在人肺腺癌组织中表达高于癌旁组织。进一步通过RNA干扰技术在人肺腺癌细胞株A549中敲除HSG基因，细胞学实验提示细胞增殖、细胞周期及细胞侵袭行为在HSG基因敲减后均受到抑制。细胞学实验提示HSG基因沉默与药物顺铂、ABT-737在促进人肺腺癌细胞株凋亡上均具有协同作用。裸鼠成瘤动物模型提示HSG基因敲减后的A549细胞株成瘤能力下降，肿瘤生长较HSG基因未敲减的A549细胞株生长缓慢。基因芯片分析也证实了HSG基因敲减后细胞周期相关通路受到抑制。芯片分析显示，诸如RAP1A、RALB及ITGA2等不同癌基因在HSG基因下调后出现反向调节，该现象表明，HSG基因在肿瘤发生发展中的机制具有双向调节性。进一步基因芯片分析表明，MLF1IP基因是HSG基因的下游靶基因。MLF1IP与PLK1相互作用是细胞周期和细胞分裂的关键步骤；MLF1IP在肺腺癌样本中异常高表达，且MLF1IP基因沉默同样抑制A549细胞的增殖力。据此我们认为肺腺癌中可能存在MFN2/MLF1IP/PLK1信号轴调控肿瘤发生发展。在我们未来的研究中，将在大样本上检测MFN2/MLF1IP/PLK1的表达相关性；利用慢病毒介导的RNAi和过表达技术调节HSG，MLF1IP和/或PLK1基因的表达，探讨HSG调控MLF1IP表达的分子机制，在细胞和整体动物水平进一步研究MLF1IP和PLK1相互作用对肺腺癌发生发展的影响，为肺腺癌诊断和治疗提供新的靶标。