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

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.

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。

我们应用针对全转录组为靶点的小发卡RNA病毒文库转染BEAS-2B细胞，以转化细胞软琼脂克隆形成实验为指标，筛查肺癌相关的新型肿瘤抑制基因。并利用体内外细胞实验，小鼠成瘤模型及临床样本对新肿瘤抑制基因进行功能验证。同时以定量蛋白组学筛查能够指导肺癌个体化治疗的分子靶点。还研发了一个新型阳离子抗瘤肽，为肿瘤的治疗提供了优良的候选药物。本课题完成了下述研究内容。..（一）本课题发现三个肺癌相关的新肿瘤抑制基因：1，首次揭示Sesn2是新型肺癌抑制基因，是肿瘤抑制因子p53的转录靶点。Sesn2通过激活AMPK、活化TSC2，从而抑制mTOR通路活性，降低细胞增殖能力；2，发现新肿瘤抑制因子INPP4B与PTEN具有协同作用，参与DNA同源重组修复过程，调控肿瘤细胞的老化；3，TIAR是本课题筛选出来的又一个新型肿瘤抑制因子，是一个RNA结合蛋白，主要位于细胞核内。在各种DNA损伤刺激下，TIAR由细胞核向细胞质聚集，通过结合长链非编码RNA Gas5，参与细胞周期的控制和基因组稳定性的维护。..（二）本课题应用定量蛋白组学揭示两个能够指导肿瘤个体化治疗的分子靶点：PDCD4及TCTP1。1，PDCD4是一种常见的肿瘤抑制因子，PDCD4表达水平的高低可以影响常用一线抗癌药物紫杉醇的敏感性和药效。我们证实PDCD4通过结合UBE2S的mRNA，抑制细胞有丝分裂期的进展，从而影响紫杉醇的药物敏感性；2，翻译控制肿瘤蛋白TCTP在多种肿瘤细胞中高表达，它调控了Rad51介导的DNA同源重组修复过程。用小分子药物盐酸曲妥霉素可抑制TCTP的表达，并强化传统化疗药物的治疗效果。..（三）本课题研发了一种新型、高效的小分子抗瘤肽：AIK。AIK是本课题组在筛选具有抗瘤活性的人工合成多肽过程中发现的一种能高效杀伤肿瘤细胞的多肽。体内外实验表明AIK对白血病细胞，肺癌细胞，肝腹水细胞等具有明显的抑制作用，且毒副作用比常用化疗药物低。我们又建立了重组AIK多肽的基因工程表达和纯化方法。AIK具有分子量小，活性高，不受经典耐药机制的影响，是优良的抗肿瘤候选药物。..本项目共培养博士研究生3名，硕士研究生7名，已发表本课题直接相关的研究论文9篇，SCI收录2篇，全国肿瘤学术会议文摘1篇。申请国家发明专利2项，获授权专利1项。本课题负责人培养的首届博士毕业生中标国家自然基金青年基金1项。