羟基脲去除恶性实体肿瘤中双微体的分子机制及外源性双微体转入细胞的作用研究

Double minute chromosomes (DMs) are the extrachromosome cytogenetic structures in tumor cells, which carry many oncogenes and drug resistant genes amplification. In the previous study, low dose treatment of hydroxyurea (HU) was found could induce DMs reduction from cells. While the molecular mechanism of HU-induced DMs reduction remains to be clarified. In this study, we will focus on three direct points, including HU-induced DNA synthesis pressure, loss of checkpoint and DMs binding proteins, to investigate the molecular mechanisms of DMs formation and reduction in tumors. The study will be conducted in DMs containing human ovarian cancer and colon cancer cell lines. Chemical inhibitors treatment, RNA interference performed to knockdown and co-knockdown, and overexpression techniques, as well as immunofluorescence (IF) and fluorescence in-situ hybridization (FISH) in combination analyses, will be performed to elucidate the major factors involved in DMs formation, and to identify the specific proteins binding to DMs. After that, we prepare to separate micronuclei by density gradient centrifugation, and extract the DMs binding proteins for further protein mass spectrometry (MS) analysis for acquiring the proteomics information of DMs recruit. That will help us to recognize the specificity of the proteins that DMs recruit, to identify the DNA repair pathway that DMs process, and then to investigate the molecular mechanisms of HU-induced DMs reduction. The new discovery of this study will provide a theoretical basis for clinical multi-target combination therapy selection for reducing DMs from malignant solid tumors. On the other hand, we are going to obtain much ringed DMs DNA from tumor cells with pulsed-field gel electrophoresis (PFGE) method, and then positively transfect the exogenous DMs into normal cells by electronic transfection. We plan to detect if DMs could process extrachromosome duplication in the recipient cells, and to check the amplification and the expression levels of the amplified genes that DMs carried. Later, we will examine the effect of the exogenous DMs on cell phenotypes. This will be greatly helpful for exploring targeted therapy by DMs carrying useful genes transfected into tumor cells in the future.

双微体（DMs）是肿瘤细胞中基因扩增的染色体外遗传结构，携带癌基因和耐药基因的扩增。双微体形成与减少的分子机制尚不明确，我们拟将此作为主要研究内容。从羟基脲可诱导双微体减少着手，从羟基脲引起的DNA合成压力、细胞周期检验点缺失和双微体结合蛋白这3个最直接切入点进行研究。选择含有双微体的人卵巢癌、结肠癌等实体瘤细胞株为研究材料，通过化学抑制剂、RNA干扰和过表达等技术，并联合应用免疫荧光和FISH技术进行特异蛋白的双微体定位，分离微核、提取微核中双微体结合蛋白，结合蛋白质谱分析，确定羟基脲去除双微体的分子机制，确定募集到双微体上的蛋白的特殊性。这对于临床去除恶性实体肿瘤中双微体的多靶点联合治疗的选择提供理论依据。脉冲场凝胶电泳技术分离肿瘤细胞中环状双微体DNA转入正常细胞，确认双微体进行染色体外复制和对细胞表型的影响，为今后开发双微体作为载体携带有益基因导入细胞进行靶向治疗进行有价值的探索。

双微体（DMs）是肿瘤细胞中基因扩增的染色体外遗传结构，携带癌基因和耐药基因的扩增，双微体形成与减少的分子机制尚不明确。我们研究从羟基脲可诱导双微体减少着手，研究发现DNA合成压力与肿瘤双微体的存在密切相关。选用含有双微体的人卵巢癌细胞为研究材料，应用针对DNA合成压力的抑制剂核糖核苷酸还原酶的抑制剂羟基脲和吉西他滨作用会去除细胞中的双微体数目和减少携带癌基因扩增，从而有效降低含有双微体的肿瘤细胞的恶性程度。吉西他滨的有效作用浓度低于羟基脲7500倍，在纳摩级就可有效去除肿瘤细胞中的双微体。抑制DNA合成压力关键点核糖核苷酸还原酶的表达，同样会去除细胞中的双微体数目和减少携带癌基因扩增，从而有效降低含有双微体的肿瘤细胞的恶性程度。研究发现这一过程的分子机制是DNA合成压力的抑制剂核糖核苷酸还原酶的抑制剂可以引起肿瘤细胞中双微体DNA双链损伤，双微体随微核排出引起的。研究中发现肿瘤细胞中出现的姐妹染色单体交换的频次与肿瘤细胞含有双微体有关，包括人卵巢癌、结肠癌、胃癌、神经系统肿瘤等，同种类型肿瘤中含有双微体的肿瘤细胞中更易出现姐妹染色单体交换，基因组不稳定。本研究提出针对含有双微体的恶性肿瘤治疗的靶向策略，大大降低化疗药物剂量，对于肿瘤基础研究和临床治疗改进提出了新的思路和建议。. 经过4年的努力，课题组完成了研究计划，发表期刊论文7篇，包括SCI收录文章3篇，国家核心期刊4篇。同时，通过研究培养博士研究生1名，硕士研究生3名已经毕业。