LMCD1通过调节同源重组修复介导高基质硬度引起的结肠癌5-FU耐药的机制研究

耐药是5-FU化疗失败主因，触发机制有待完善。基质硬度在肿瘤恶性进程中的作用渐受重视，然而，尚无研究涉及其对结肠癌细胞5-FU敏感性的影响。预试验表明：高基质硬度可增强结肠癌细胞同源重组（HR）修复并抑制5-FU细胞毒性。运用文库筛选，我们挖掘出LMCD1这一功能未知的LIM家族蛋白，其表达被高基质硬度上调，沉默LMCD1可抑制HR修复并增强5-FU敏感性。分子机制上，沉默LMCD1可抑制NF-κB激活，而5-FU可触发LMCD1 与NEMO互作，后者的泛素化及出核是DNA损伤激活NF-κB的必要条件之一。综上，我们推测：LMCD1是连接基质硬度机械信号与结肠癌细胞5-FU耐药的桥梁分子，其表达丰度上调及与NEMO互作是高基质硬度下结肠癌细胞HR修复能力增强的机制之一。本项目将通过临床样本分析及体内外实验验证上述假设，以期从机械力学视角为结肠癌化疗增敏提供分子生物学依据与潜在的治疗靶点

Drug resistance accounts for most 5-FU chemotherapy failures, while its modulatory mechanism still remains unclear. The extracellular matrix stiffness has gained much focus for its role in tumor progression. However, the effects of matrix stiffness on chemosensitivity towards 5-FU in colon cancer cells has not yet been reported. Preliminary experiments indicated that high matrix stiffness enhanced the repair of homologous recombination (HR) and inhibited the cytotoxicity of 5-FU. Using CRISPR activation pooled library screening, we found that LMCD1, a LIM family protein with unknown function, was up-regulated by high matrix stiffness. Silencing of LMCD1 inhibited HR repair and enhanced 5-FU sensitivity. Molecularly, silencing of LMCD1 inhibited the activation of NF-κB transcription factor signaling, while 5-FU triggered the interaction between LMCD1 and NEMO. The ubiquitination and nuclear export of NEMO protein have been demonstrated as a key step for linking DNA damage to NF-κB activation. Collectively, we speculated that LMCD1 was one of the mediators for 5-FU resistance of colon cancer cells triggered by mechanical signals from high matrix stiffness. The upregulation of LMCD1 expression abundance and the interaction between LMCD1 and NEMO might both be key mechanisms for enhanced HR repair efficiency of colon cancer cells raised on substrates with higher stiffness. This project will verify the above hypothesis through clinical sample analysis, in vivo and in vitro experiments, in order to provide molecular biological evidence and potential therapeutic targets for the chemosensitization of colon cancer from the perspective of tumor mechanical environment.