肺癌干细胞标志基因microRNA结合位点SNP的筛选与功能研究

In china, the most malignant tumor is lung carcinoma, with non-small cell lung cancer (NSCLC) accounting for 80% of the cases. Chemotherapy is one of the main strategies for the treatment of NSCLC. However, with the wide application of anticancer drugs, drug resistance has become one of the most common and most difficult problems in the clinic. Cancer is a genetic disease. The American Cancer Genome Project found averagely a solid tumor has obtained about 100 genetic and epigenetic changes in the genome. Moreover, studies from Pharmacogenetics / Pharmacogenomics demonstrated that the effects of chemotherapeutic drugs on tumor cell killing depend on the expression and / or polymorphism of a specific (group) gene, suggesting the importance of individualized cancer treatment. Very interestingly, genome-wide association studies (GWAS) showed that the single nucleotide polymorphism (SNP) plays an important role in the tumorigenesis and cancer drug resistance, and that SNP alters the regulation of gene expression by microRNA (miRNA). Increasing data demonstrated that cancer stem cells (CSC) are the drivers for carcinogensis and play key roles in cancer drug resistance. Thus, elimination of CSC would be the ultmate goal for cancer therapy. Whether CSC marker gene SNP changes the binding of miRNA and hence the effect of miRNA on CSC to resist to cancer drugs is totally unknown. The recent advances in the studies of lung CSC, CSC markers, lung cancer individualized chemotherapy, SNP and miRNA, triggered us to hypothesize that NSCLC stem cell marker gene SNP of miRNA binding sites may alter the regulation of gene expression by miRNA and affect the sensitivity of NSCLC stem cell to chemotherapeutic drugs. To this end, we will explore the GWAS database of NSCLC from MD Anderson Cancer Center to screen the lung CSC marker gene SNP that are related to the prognosis and outcome of NSCLC patients with chemotherapy; to mine lung CSC marker gene SNP with potential miRNA binding sites; to investigate the transcriptional changes of lung CSC marker genes by the SNP with potential miRNA binding sites; to screen the miRNAs that regulate the expression of lung CSC marker genes at the post-transcriptional levels; finally to valiate the effects of miRNA on the sensitivity of NSCLC cells to cisplatin in vitro to probe the possibility of targeting SNP for cancer therapy. With the completion of this proposal, we will systematically demonstrate the mechanisms by which the SNP of miRNA binding sites alters the gene expression of NSCLC stem cell marker genes at both transcriptional and post-transcriptional levels. Our study will provide data to show whether SNP of miRNA binding sites plays a role in cancer drug resistance and the maintenance of NSCLC stem cells. Most importantly, our study will provide evidence if SNP of miRNA binding sites can be used as biomarkers for patient stratification, and the basis to explore new targets for the reversal of resistance to chemotherapy.

耐药性是化疗失败最常见、难克服的问题。而耐药性与肿瘤干细胞密切相关，GWAS证明SNP在肿瘤耐药性起重要作用。由于SNP会改变miRNA对靶基因的调控，肿瘤干细胞标志基因SNP是否影响miRNA的结合而导致耐药是癌症领域的重大课题。我们认为：NSCLC干细胞标志基因miRNA结合位点SNP改变miRNA对靶基因的调控，影响化疗药物的敏感性。为此将利用安德森癌症中心GWAS数据筛选预测含铂方案化疗生存的NSCLC干细胞标志基因miRNA结合位点SNP；研究miRNA结合位点SNP对这类基因表达的影响；筛选调节干细胞标志基因表达的miRNA；观察体外调节靶miRNA对顺铂耐药性的影响。本研究将阐明miRNA结合位点SNP与NSCLC干细胞标志基因表达的转录和转录后调控机制，说明靶miRNA与NSCLC干细胞的联系，为miRNA结合位点SNP作为筛选化疗优势人群、逆转耐药的新靶点提供理论基础。

具有自我更新和无限增殖能力的肿瘤干细胞是恶性肿瘤发生的根源；恶性肿瘤的局部复发、远处转移转移、化疗耐药、抗辐射等恶性表型特征都与肿瘤干细胞有关。GWAS证明SNP在肿瘤耐药性起重要作用。我们的假说是：NSCLC干细胞标志基因miRNA结合位点SNP改变miRNA对靶基因的调控，影响化疗药物的敏感性。为此利用安德森癌症中心GWAS数据筛选预测含铂方案化疗生存的NSCLC干细胞标志基因miRNA结合位点SNP；研究16种干细胞相关基因mRNA表达与NSCLC患者生存期之间的关系；验证胆固醇与NSCLC患者含铂化疗疗效之间的关系及调节机制。本研究发现：1.肿瘤干细胞标志基因CD133的功能位点 rs2240688的基因型与无局部复发生存时间、无远处转移生存时间以及总生存时间显著相关；2.CD24 mRNA低表达NSCLC的患者生存期更长；3.胆固醇通过上调ABCG2降低了肺腺癌对铂类化疗药的敏感性。提示肿瘤干细胞标志基因CD133的功能位点 rs2240688 可预测非小细胞肺癌患者的放化疗抗性，可能是一个可预测临床预后以及选择治疗策略的有希望的标志物。CD24表达可能预测NSCLC患者的预后。首次提出了肺腺癌患者血清胆固醇对化疗的影响，揭示了肿瘤干细胞因子ABCG2在高胆固醇引起的耐药性增加中的重要作用；支持了胆固醇先引起耐药性增加的观点。通过降脂达到化疗增敏的作用是一个可行的措施。