非小细胞肺癌新融合基因的鉴定及其功能研究

Fusion genes are important molecular alterations in lung cancers, however, we know little about the whole fusion genes profile except recently identified fusion genes like EML4-ALK, CD74- or SLC34A2-ROS1 and KIF5B-RET,etc. On the basis of substantial study on the ALK epidemic and molecular characteristics of ALK rearrangements in Chinese lung cancers, in this grant, (1) we will profile the fusion genes in a large-scale manner in pulmonary adenocarcinoma (Ad) as well as in squamous cell carcinoma (SCC) by whole-transcriptome sequencing (WTS) technology. Briefly, through isolation of high-quality RNA from 10 cases of adenocarcinoma and 10 cases of squamous cell carcinoma, high-throughput data of WTS will be generated on the platform of Illumina HiSeq2000 or GA IIx with more than average 50× coverage. According to the human reference genome assembly (NCBI build 36.3, hg18), large-scale of alignment will be performed to identify potential gene rearrangements, which will be further validated by specific RT-PCR and capillary sequencing. Tumor samples harboring potentially oncogenic fusion genes will also be validated using whole genome sequencing to test the exact information on the structural chromosomal rearrangement. (2) To testify the clinical implications of fusion genes of interest (fGOI), we will further test the recurrence of target fusions in a cohort of 200 lung cancers with 100 in either histology of Ad and SCC by specific RT-PCR and/or immunohistochemistry (IHC). (3) To clone the whole-length cDNA of target fusion genes from freshly frozen tumor samples, then do ecotopic expression of fGOI with or without co-transfected fGOI-targeted siRNA vectors in model cells NIH 3T3 and 293, then the phenotypes of colony-forming capacity in soft-agar medium, tumorogenicity capacity in inoculated nude mice will be assessed. In this study we seek to obtain the primary whole profile of fusion genes in Chinese pulmonary adenocarcinoma as well as in squamous cell carcinoma, and to clarify the detailed information of 1 or 2 fusion genes with functional study on their potential oncogenic ability. These results will be very helpful to the molecular classification of lung cancers and the establishment of personalized medicine by providing vital preclinical data and materials.

融合基因是肺癌中重要的分子变异，但目前了解甚少。本课题组对ALK融合基因在我国肺癌中的分子特征进行了系统研究。本研究拟采用转录组深度测序技术分析肺癌中的融合基因序列信息。挑选10例腺癌和10例鳞癌新鲜标本，采用Illumina HiSeq200或GA IIx测序仪分析获得50×以上的深度测序结果。参照人类基因组参照序列（build36.3，hg18）比对分析潜在的融合序列。以全基因组深度测序结果验证基因重排信息。对目标融合基因采用RT-PCR或/和免疫组化技术扩大在200例肺癌标本中进行验证。从新鲜组织克隆融合基因的全长序列并进行模式细胞异位表达或表达联合siRNA靶向干扰，采用体外克隆形成实验、动物接种致瘤实验鉴定其驱动细胞恶性转化的功能。最终获得相对全面的肺癌融合基因谱信息、明确1～2个致癌性融合基因及其分子机制，为建立新的肺癌分子亚型及其个体化靶向诊治模式奠定重要的临床前研究数据。

全转录组测序技术能够在分析转录本的结构和表达水平的同时，发现融合基因和未知的转录本，精确地识别可变剪切位点以及SNV，提供最全面详细的转录组信息。鉴于潜在具有恶性转化能力的驱动基因与肺癌关系密切，但目前了解甚少，缺乏对肺癌的融合基因谱的全面分析。对10例未经治疗的IIIA期肺鳞癌患者手术标本进行全转录组测序，数据质量高，Q30均高于85％。研究发现肺鳞癌存在大量SNV，平均12.15/Mb，单碱基变异在非吸烟标本与吸烟标本中未见明显差异，最常见的类型是C:G-T:A和A:T-G:C，与肺腺癌不同。Defuse软件进行融合基因的分析，平均每个标本鉴定出97个融合基因，其中有50个融合基因分别在4个或4个以上的标本中同时出现。 TopHat软件在10例标本中一共筛选出132个融合基因，70.5％为同一染色体内的基因融合，根据其提供的3类证据的高低，在26各融合基因中共验证出20个融合基因，验证率达到76.9%。在验证出的20个融合基因中，对存在5个框内融合基因 （ECE2-PSMD2、ARHGAP42-TMEM123、WWTR1-AGTR1、TNFSF10-FXR1、FAM38A-APRT）肿瘤组织及相应的正常组织进行RT-PCR及直接测序法测序，这些框内融合基因在肿瘤组织中都有表达水平的上调。同时在GLCI_6号患者的正常肺组织中也存在ECE2-PSMD2基因融合，未在相应的正常组织中检测到其余4个融合基因。进一步我们挑选ARHGAP42-TMEM123和WWTR1-AGTR1两个融合基因进行验证。构建目的融合基因的慢病毒进行克隆形成实验、细胞增殖实验MTT和划痕实验。结果提示上述融合基因对细胞增殖及侵袭能力无明显影响。本课题主要阐述了转录组测序对筛选肺癌融合基因的潜在优势，我们的研究将为后续探讨肺癌融合基因临床及转化研究奠定了基础。