基于高通量筛选及测序技术的三阴性乳腺癌吉西他滨耐药基因谱的筛选及其功能研究

Gemcitabine is considered as an essential first-line chemotherapy drug for patients with advanced triple-negative breast cancer (TNBC), and resistance to gemcitabine will result in tumor progression. The applicant has constructed human cell lines of gemcitabine-resistance in TNBC, and found that miR-484-CDA axis played a part in regulating the chemo-sensitivity of breast cancer cells to gemcitabine. In addition, using the Gene Ontology analysis, it was revealed that differential expression genes were related to drug metabolism processes, also may modulate the sensitivity to gemcitabine by multiple pathways. Based on the previous studies, the project was designed to establish the Barcode library, to obtain candidate resistance gene profiles of differential expression by next-generation sequencing technology. And by widely conducting the research of microRNA and interactive proteins in cells, animal models and tissue specimens, the mechanism of gemcitabine resistance in breast cancer is systematically analyzed. Meanwhile, the relationship between resistance genes and malignant biological phenotype of breast cancer is investigated, and its potential value to predict therapy response and prognosis of breast cancer is explored. Our study sought to expound on the mechanism of gemcitabine resistance comprehensively, to establish the predictive markers for curative effect, and to identify the population benefited from chemotherapy to the greatest extent, thereby, to provide theoretical support for the precise individualized treatment and improve the survival rate of TNBC.

吉西他滨是晚期三阴性乳腺癌的重要一线化疗药物，肿瘤对其耐药可导致肿瘤进展。申请人前期构建了人三阴性乳腺癌耐吉西他滨的细胞株，发现miR-484-CDA调控轴参与调控乳腺癌细胞对吉西他滨的敏感性；通过基因本体分析发现差异表达基因不仅与药物代谢相关，还可能通过多种途径调节吉西他滨的药物敏感性。本项目立足于前期研究基础，构建条形码文库，利用二代测序技术平台，遴选出差异表达的候选耐药基因谱，通过微小RNA及互作蛋白在细胞学、动物模型及乳腺癌组织标本中充分展开研究，系统性地剖析乳腺癌中吉西他滨的耐药机理；同时，研究耐药基因与乳腺癌恶性生物学表型之间的关系，探讨其在乳腺癌疗效预测及预后方面的潜在价值。本研究将在全局层面阐明吉西他滨的耐药机制，确立疗效预测标记物，最大化甄别化疗获益人群，为精准的个体化治疗提供理论支持，提高三阴性乳腺癌的生存率。

化疗是乳腺癌患者疾病控制和治愈的重要手段之一，然而化疗耐药问题往往是治疗失败的主要原因。寻找肿瘤化疗耐药的内在机制和逆转耐药的策略是当下科学研究的热点之一。本项目基于乳腺癌吉西他滨耐药问题展开系列研究。我们通过对耐吉西他滨的MDA-MB-231Gem细胞及其亲本细胞进行RNA-seq检测获取耐药相关基因，并利用文库筛选结合高通量二代测序方法进一步筛选出潜在的吉西他滨耐药候选基因。通过细胞毒检测、细胞活性和细胞凋亡等实验研究KCNN4等耐药候选基因对乳腺癌细胞生物学行为的影响。并从细胞分子水平和小鼠体内实验进一步探索吉西他滨耐药的分子机制和逆转耐药的策略。本研究建立了一个包含145个基因的吉西他滨耐药基因标签（GRGB）文库,并从中确定了KCNN4和BCL2A1作为主要研究对象。研究发现KCNN4高表达能够显著降低乳腺癌细胞对吉西他滨的敏感性，同时能够促进细胞的增殖，抑制细胞凋亡。进一步发现KCNN4的表达也会影响乳腺癌细胞对阿糖胞苷的敏感性，但是对紫衫类化疗药物的敏感性无明显影响。BCL2A1的表达对乳腺癌细胞也有相似的作用。在机制上，我们发现KCNN4通过激活RAS-MAPK/PI3K-AKT信号通路影响下游靶基因BCL2A1的表达，从而发挥促增长、抑凋亡，促耐药的功能。TRAM-34是KCNN4的特异性抑制剂，体内外实验均证实它能够逆转KCNN4高表达所致的吉西他滨耐药。此外，我们利用公共数据库进一步证实KCNN4和BCL2A1高表达的患者其生存预后越差。这些发现丰富了乳腺癌化疗耐药的分子机制，为将来药物靶点的研究提供了理论基础，也为乳腺癌患者个体化治疗提供参考，具有广阔的应用前景和重要的临床意义。