抑癌基因SPARCL1的表达调控机制及其与互作基因在卵巢癌铂类耐药细胞中的功能研究

Ovarian cancer is the most lethal cancer of the female reproductive system, causes lots of death all over the world every year. Early-stage malignancy is frequently asymptomatic and difficult to detect, and thus diagnosis usually occurs after the disease has disseminated beyond the ovaries. Therefore, approximately 70% of ovarian cancers are diagnosed at advanced stage and only 40% of women with such cancers can expect to survive 5 years. Although cisplatin-centered chemotherapy, which is the currently preferred treatment modality in human ovarian cancer, can strongly cut down the mortality and lengthen the survival time of patients, the main obstacle to a successful treatment for ovarian cancer is the development of drug resistance to chemotherapy. Drug resistance results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumors. However, regardless of mechanisms, abnormal expression of tumor suppressor genes (TSGs) clearly contributes to drug resistance in ovarian and other cancers. SPARCL1, a TSG, was down regulated and contributed to cancer development in ovarian and many other cancers, while the study association with drug resistance is rare. Besides, SPARCL1 is not a classical TSG, which regulated by epigenetic modifications instead of a deletion or mutation. Previous studies mainly focused on DNA hypermethylation, and the regulation of SPARCL1 by microRNA or by co-regulation of microRNA with DNA methylation was never reported. Based on the RT-qPCR results, we found that the expression of SPARCL1 in platin-resistant ovarian cancer cells was apparently down-regulated compared with the platin-sensitive counterparts, and the positive expression rate of SPARCL1 in drug-resistant ovarian cancers was notably lower than the rate in controls. Besides, comprehensive bioinformatics analysis revealed that SPARCL1 which co-expressed with 8 genes might associate with drug resistance in ovarian cancer. Therefore, this program aims to illustrate the function of SPARCL1 with its co-expressed genes and its regulation in platin-resistant ovarian cancer cells. This program will set the stage for molecular-targeted therapies, drug resistance reversion and clinical applications in ovarian cancer.

卵巢癌是女性生殖器常见恶性肿瘤且死亡率高，严重威胁妇女健康和生命。对以铂类为主的化疗药物的耐药性是卵巢癌化疗失败的主要原因，而抑癌基因沉默表达则是多药耐药发生的重要根源之一。抑癌基因SPARCL1在很多癌中下调表达并调节肿瘤的发生发展，但其参与耐药调控的研究还非常罕见。表观遗传调控是SPARCL1沉默表达的主要修饰方式，然而现有研究仅集中在DNA甲基化上，而由microRNA及由DNA甲基化和microRNA协同调控SPARCL1表达的研究还未见报道。我们发现SPARCL1在卵巢癌铂类耐药细胞和多药耐药组织中都显著下调，且生物信息学分析表明该基因与多个基因共表达，并与卵巢癌多药耐药显著相关。因此，本研究旨在深入探讨SPARCL1在卵巢癌耐药细胞中下调表达的表观遗传机制，及SPARCL1与其共表达基因对卵巢癌铂类耐药的调节作用，为卵巢癌分子靶向治疗、耐药逆转及临床应用提供理论依据。

卵巢癌是女性生殖系统三大常见恶性肿瘤之一，死亡率高，严重威胁着妇女的健康和生命。以铂类为主的化疗药物的耐药性是卵巢癌治疗失败的主要原因，而抑癌基因沉默表达是卵巢癌耐药调节的重要机制。抑癌基因SPARCL1 在很多肿瘤中下调表达，通过调节细胞分化和侵润转移等方式调控肿瘤的发生发展。然而该基因与卵巢癌或肿瘤耐药相关的研究还较少，与卵巢癌耐药调控相关的研究更是罕见。本项目从细胞功能、分子机制、临床相关性及基因表达调控机制四个方面，系统阐述了SPARCL1与卵巢癌耐药调控的关系。和27例卵巢癌化疗敏感组织相比，抑癌基因SPARCL1在24例卵巢癌耐药组织中的mRNA和蛋白表达水平均显著下调；在卵巢癌A2780和SKOV3顺铂及卡铂耐药细胞中，该基因的表达较之对应亲本也显著下调。体外构建SPARCL1过表达和ShRNA干扰基因表达卵巢癌顺铂耐药细胞株，发现SPARCL1过表达能显著提高耐药细胞对顺铂的敏感性，并能抑制细胞增殖、克隆形成和细胞迁移，阻滞细胞周期；ShRNA干扰基因表达细胞实验结果则正好相反。基于转录组测序分析，发现卵巢癌耐药细胞中SPARCL1过表达与15条信号通路显著相关，其中包括Cell adhesion molecules及p53 signaling pathway等经典卵巢癌耐药调控通路；另外，SPARCL1还可能通过调节TRPC1而响应卵巢癌耐药调控。临床相关性分析显示，在51例卵巢癌组织中，SPARCL1低表达与不良无疾病生存期和总生存期显著相关，是潜在的卵巢癌预后标记物。在SPARCL1基因表达调控机制研究上，我们发现miR-448与SPARCL1在表达上显著负相关且miR-448能靶向调节该基因；但基于47例卵巢癌敏感组织和48例耐药组织的甲基化检测表明DNA甲基化不是SPARCL1在耐药组织中下调表达的调节因素。本研究首次明确了SPARCL1表达与卵巢癌耐药调控的相关性，探讨了其调控耐药的潜在分子机制，明确了其与临床因素特别是临床预后的相关性，为其应用于卵巢癌分子靶向治疗和耐药逆转提供了理论依据，并可作为潜在预后标记物应用于卵巢癌预后，具有重要的理论和应用价值。