PBRM1在非小细胞肺癌侵袭转移中功能及表达调控的研究

Epigenetic regulation disorders caused gene abnormal activating and/or losing plays a critical role in tumor development. Our preliminary study found that one of the members PBAF chromatin remodeling complexes PBRM1 (BAF180) expressed low or absent in some non-small cell lung cancer (NSCLC) cell lines, and which was correlated with tumor metastasis and staging. By analyzed Next Generation sequencing results, SNP and methylation microarray data, we found the relation between PBRM1 loss and some epigenetic regulation mechanisms that include gene mutation, allelic loss and CpG island methylation status to some extent. On the base of preliminary data, our group plan to further investigate epigenetic regulation mechanisms of PBRM1 loss with 3C technology and ChIP. We also plan to use Immunohistochemistry and single cell RT-PCR to compare PBRM1 expression level in primary and metastatic NSCLC tissue specimens. And we intend to apply some functional experiments in vitro, such as migration assay, invasion assay, and transwell Chamber to detect its biological behavior relating with NSCLC cells metastasis, and use gene chip and signal pathway molecular activators/inhibitors to investigate the mechanism of metastasis. In order to learn the effect of PBRM1 gene on metastatic tumor, we will establish orthotopic transplantation of human non-small cell lung cancer metastasis model in nude mice. We expected to provide a theoretical basis for exploring the molecular markers of lung cancer metastasis or therapeutic targets.

表观遗传调控紊乱所致的基因异常开启和/或关闭在肿瘤发生发展中起着重要的作用。我们前期工作发现PBAF染色质重塑复合物的成员之一PBRM1（BAF180）在部分非小细胞肺癌（NSCLC）细胞株中低表达或缺失，且与肿瘤转移和分期有关。通过对二代测序、SNP及甲基化芯片数据分析，在一定程度上明确了基因突变、等位基因缺失和启动子区甲基化等表观遗传调控情况。基于此，本课题组拟利用3C技术和ChIP深入探讨PBRM1缺失的表观遗传调控机制。采用免疫组化、单细胞RT-PCR比较PBRM1在原发和转移NSCLC组织标本中表达情况。通过细胞粘附、迁移等体外功能学实验方法和基因芯片、信号通路分子激活/抑制剂，检测PBRM1对NSCLC细胞粘附侵袭转移相关生物学行为的影响和机制。并通过原位移植建立人非小细胞肺癌裸鼠转移模型，研究PBRM1基因对转移瘤的影响。期望为探索肺癌转移的分子标志物或治疗靶标提供理论依据。

我们前期工作发现PBAF染色质重塑复合物的成员之一PBRM1（BAF180）在部分非小细胞肺癌（NSCLC）细胞株中低表达或缺失，且与肿瘤转移和分期有关。通过对二代测序、SNP及甲基化芯片数据分析，在一定程度上明确了基因突变、等位基因缺失和启动子区甲基化等表观遗传调控情况。基于此，本研究采用免疫组化检测BAF180在NSCLC原发灶癌组织、癌旁正常肺组织和肺部良性病变手术组织中的蛋白表达水平。用统计学方法分析BAF180蛋白表达水平与组织来源、临床分期的关系。密切随访明确BAF180蛋白表达水平与临床病情进展变化和预后的相关性。通过细胞粘附、迁移等体外功能学实验方法和基因芯片、信号通路分子激活/抑制剂，检测PBRM1缺失对NSCLC细胞粘附侵袭转移相关生物学行为的影响和机制。合成PBRM1基因的调控序列，构建luciferase报告基因载体。然后分别转染PBRM1野生型和低表达的细胞系，用Dual-Glo® Luciferase Assay System进行荧光素酶报告基因检测，明确PBRM1缺失表达的机制。期望为探索肺癌转移的分子标志物或治疗靶标提供理论依据。