以人转录组为靶点的shRNA-mir文库筛查肺癌相关的新肿瘤抑制基因及候选基因INPP4B在肺癌发生中的机制

肿瘤抑制基因的功能缺陷或缺失在肺上皮细胞恶性转化的过程中起关键作用，在全基因组范围内筛查肺细胞恶变过程中起关键作用的新型肿瘤抑制基因是肺癌研究中的一个重要任务。我们应用针对人类细胞转录组为靶点的逆转录病毒小发卡RNA干扰文库shRNA-mir，先以肺支气管上皮细胞系体外转化试验为筛选模型，筛查shRNA-mir转染所引起的锚定不依赖性转化细胞克隆，PCR扩增各个转化细胞克隆中的shRNA片段，经DNA 测序推断靶点基因。同时分组收集所有转化细胞克隆，在裸鼠成瘤模型上进行体内致瘤筛选。用基因芯片技术鉴定瘤体所富集的shRNA。通过靶点比对，明确候选基因目录。再以初筛候选基因磷酸肌醇4-磷酸酶B（INPP4B）为侧重，检测INPP4B在肺癌组织中的基因突变和表达水平，以定量磷酸化蛋白组学筛查肺上皮细胞内INPP4B 潜在的蛋白底物群，为肺癌的发生和治疗提供新型分子机制和个体化药物作用靶点。

Lung cancer is emerging rapidly as the leading cause of cancer death in China. The loss-of-function mutations, deletions, and/or epigenetic silencing of a variety of tumor suppressors play a crucial role in lung tumorigenesis. Thus, to identify novel tumor suppressors in a genome-wide and unbiased manner is one of the central tasks for lung cancer research. To achieve that goal, we are employing an shRNA-mir library-based approach targeting human transcriptome to interrogate potential tumor suppressor candidates systematically in immortalized BEAS-2B human bronchial epithelial cells. .Starting with soft agar colony formation assays for screening BEAS-2B cells transformed by the retroviral shRNA-mir library with the feature of anchorage-independent growth, large (>0.1mm in diameter), well-separated colonies are isolated for further proliferation. PCR is performed to amplify the integrated shRNA fragments from individual genomic DNA extracted from each colony, and each PCR product is submitted for DNA sequencing to reveal the identification of the integrated shRNA and its target gene. Meanwhile, the rest transformed cells on each 10cm soft agar plate will be pooled together by centrifugation, and cultured in regular medium, each cell group will be subjected to tumor formation screens in nude mice. Extraction of genomic DNA from the tumor tissues formed in nude mice and amplification of shRNAs by PCR will be carried out again . The resulting PCR products will be pooled together and submitted for the half-hairpin-assisted microarray analysis to discover all shRNAs enriched in the nude mouse model. By comparing the list of candidate target genes from the single colony-picking assays with the list from the colony-pooling, nude mouse tumor-forming screens , a priority list of tumor suppressor candidates will be established. Moreover, a couple of promising hits will be validated by using shRNAs targeting their 3' UTRs combined with plasmids expressing their ORFs to exclude off-target effects of shRNAs in both the cultured BEAS-2B cells and the nude mouse model..Furthermore, we are focusing on the inositol polyphosphate 4-phosphatase type II (INPP4B) gene，a top candidate identified from our pilot screen, to reveal its mechanism underlying suppression of malignant transformation in BEAS-2B cells. The mutations, expression levels of INPP4B in various lung cancer cell lines and clinical tissues will be examined by PCR-SSCP, RT-PCR , Western Blotting and Immunohistochemistry, and the potential de-phosphorylation substrates of INPP4B will be profiled by using a quantitative phosphoproteomics approach in BEAS-2B cells upon oxidative stress. The ultimate goal of our research is to shed new light on the mechanism by which INPP4B suppresses lung tumorigenesis and provide novel drug targets for therapeutics development of lung cancer.