抑制A-NHEJ途径去除双微体/均质染色区逆转肿瘤耐药的研究

Double minutes (DMs) and homogeneously staining regions (HSRs) play a very important role in the process of drug-resistance in cancer cells. Previously, our group found the association between alternative non-homologous end joining (A-NHEJ) pathway and DMs/HSRs formation. To further explore the molecular mechanism of DMs/HSRs formation and drug-resistance involving A-NHEJ in cancer cells, stable clones will be acquired by transfecting lentivirus shRNAs of A-NHEJ into HSRs- and DMs-containing cells, respectively. Next, in HSRs- and DMs-containing cells, (1) the effects of DSBs repairment, gene amplification and amplified forms will be detected by inhibiting A-NHEJ, (2) the effects of micronucleus formation rate, efflux rate of amplified DHFR gene and the colocalization of it and DSBs will be examined by inhibiting A-NHEJ in short- and long-term, respectively, (3) the effects of proliferation capability, drug sensitivity and DHFR expression by inhibiting A-NHEJ will also be examined. Finally, xenograft model will be constructed by injecting HSRs- and DMs-containing cells into nude mice subcutaneously, and the effects of survival conditions, tumor sizes, and the DMs/HSRs condition in primary culture cells by inhibiting A-NHEJ will be observed. From an evolutionary point of view, our study will explore the molecular mechanism of DMs/HSRs formation in different stages of cancer drug-resistance (HSRs and DMs), which can provide promising direction for reversing drug-resistance in cancer cells.

双微体（DMs）和均质染色区（HSRs）在肿瘤耐药中发挥重要作用，我们前期发现选择性非同源末端连接（A-NHEJ）途径与DMs/HSRs形成相关。为了揭示A-NHEJ参与DMs/HSRs形成并影响肿瘤耐药的分子机制，我们拟构建A-NHEJ慢病毒shRNAs并分别转染入含HSRs和含DMs细胞获得稳定克隆。之后，检测干涉A-NHEJ途径对DNA双链断裂（DSBs）修复、基因扩增及扩增基因存在形式的影响。分别观察抑制剂短期抑制、稳定克隆长期干涉A-NHEJ对微核形成、扩增DHFR基因外排及其与DSBs共定位情况。观察干涉A-NHEJ对细胞增殖能力、药物敏感性和DHFR表达的影响。最后，裸鼠皮下成瘤，观察A-NHEJ抑制剂对裸鼠生存、瘤大小及肿瘤原代培养细胞中DMs/HSRs的影响。本课题从进化角度针对肿瘤耐药不同阶段（HSRs、DMs）揭示DMs/HSRs形成的分子机制，为逆转耐药提供新思路。

双微体（DMs）和均质染色区（HSRs）是基因扩增的主要形式，在肿瘤进展和耐药过程中发挥重要作用。基因扩增多源于DNA双链断裂（DSBs）及断裂后的异常修复。选择性非同源末端连接（A-NHEJ）途径是一种新发现的DSBs修复方式，易产生基因组的大片段错误。因此，我们猜想A-NHEJ可能与基因扩增相关。我们发现含DMs和含HSRs的HT-29 MTX耐药细胞中A-NHEJ途径关键蛋白的表达明显高于HT-29亲代细胞。将shPARP1（A-NHEJ途径核心蛋白）慢病毒表达载体分别转染入含DMs和含HSRs的细胞中获得稳定克隆。在含DMs的细胞中：干扰组细胞中DMs数目明显减少、DHFR基因及位于同一DMs上的其他基因的拷贝数均明显减少、DHFR表达降低、H2AX表达减少但是HR和NHEJ途径关键蛋白表达增高、微核/核出芽形成率及外排率没有变化、生长减慢、凋亡增多、IC50值降低。而在含HSRs细胞中，干扰组细胞除HR途径关键蛋白表达增高外，其他各项指标均无明显变化。PARP1抑制剂ABT-888短期作用于含DMs的细胞导致DMs数目明显下降；作用于SCID鼠皮下肿瘤导致肿瘤细胞的耐药性明显下降。因此，我们发现A-NHEJ途径可能通过影响DMs（而不是HSRs）形成影响肿瘤耐药性，这个发现也为肿瘤耐药的个体化治疗提供理论依据。另外，在含有DMs的细胞中，抑制A-NHEJ途径可以导致其他修复途径代偿性增强，提示联合抑制A-NHEJ和其他修复通路可能会更好地治疗DMs相关的肿瘤。