NONO乙酰化修饰作用调控肿瘤化疗耐药的机制及临床意义

NONO is reported to be upregulated in variety of malignant tumors including colorectal cancer and this upregulation is positively correlated with the cancer progression and the resistance of cancer cells to chemotherapy. However, the mechanisms of NONO being upregulated in these cancers remain unknown. Our preliminary results indicated that NONO was stabilized by p300 mediated acetylation. Besides, DNA damage agents induced the acetylation of NONO and promoted the stabilization of NONO in the colorectal cells. Furthermore, silence of NONO increased the sensitivity of colorectal cells to chemotherapy agents. Based on these observations, we hypothesize that the dyregulation of the acetylation on NONO contributes the upregulation of NONO in the malignant tumors including colorectal cancer. Besides, the upregulated NONO expression promotes the progression and resistance of colorectal cancer cells to chemotherapy. To validate our hypothesis we design a variety of experiments. Firstly, we will identify the specific acetylation sites in NONO using TAP-MS and directed site mutationassay. To validate whether the specific acetylation site influence the progression and chemoresistance of cancer cells, we will construct the acetylation site mutated (K/R, K/Q) colorectal cells using CRISP-CAS9 system and examine their functional changes including the cell proliferation, apoptosis and the sensitivity to chemicals in vitro and in vivo. To reveal the clinical significance of NONO acetylation by p300, we will collect 200 colorectal cancer samples and explore the correlation of acetylated NONO with p300 expression. Besides, we will also analyzethe correlation of NONO acetylation level with the clinical characteristic, overall survival and the relapse rate of colorectal cancer patients. Overall, This study would strive to elucidate the mechanisms and clinical significance of NONO acetylaion on the colorectal cancer progression and chemoresistance.

NONO在结直肠癌等多种肿瘤中表达上调，并与肿瘤的恶性进展及化疗耐药相关。NONO表达调控机制尚未见报道。项目的前期研究发现NONO蛋白受p300介导的乙酰化修饰，并且乙酰化修饰调控NONO蛋白的稳定性；在结直肠癌模型中，化疗药物处理增强NONO的乙酰化修饰及蛋白水平，敲低NONO提高结直肠癌细胞对化疗药物的敏感性。因此我们推测乙酰化修饰失调是NONO蛋白在结直肠癌等肿瘤中表达上调的重要原因；NONO表达上调促进肿瘤的恶性进展及化疗耐药。本项目接下来将通过点突变鉴定乙酰化的位点；使用CRISP-CAS9系统建立NONO乙酰化位点K/R、K/Q突变的结直肠癌细胞株，并在细胞与动物水平验证乙酰化修饰对肿瘤恶性进展及化疗耐药的影响；在临床标本中分析NONO乙酰化水平与p300表达的相关性，以及与结直肠癌病人生存、预后及化疗后复发率的相关性，全面揭示乙酰化修饰调控NONO稳定性的机制及临床意义。

化疗耐药是晚期结直肠癌治疗失败的重要原因，阐明化疗耐药是当前结直肠癌研究的重要课题。阐明结直肠癌细胞生长、凋亡和化疗耐药的分子机制，有望发现结直肠癌化疗增敏的新的靶点，提高结直肠癌患者的治疗效率。 . 结直肠癌化疗耐药机制研究方面，我们通过构建5-FU耐药的SW480和HCT-116细胞株，发现SW480/5-FU和HCT-116/5-FU耐药细胞中PITX2表达显著高于亲本细胞。敲降PITX2后，结直肠癌5-FU耐药细胞活性减少，增殖能力减弱，S期期细胞减少。生物信息学分析、RIP实验及双荧光素酶实验表明，PITX2与Wnt3a启动子具有结合位点，PITX2能转录激活Wnt3a表达进而调控Wnt/β-catenin信号通路。此外，抑制Wnt /β-catenin通路减弱结直肠癌细胞对5-FU的耐药性，而激活Wnt /β-catenin通路后能逆转PITX2基因敲除对结直肠癌细胞5-FU耐药性的作用。整体动物实验进一步证实敲降PITX2能减弱结直肠癌细胞HCT-116对5-FU的耐药性。我们的研究结果证实，PITX2通过转录激活Wnt 3a进而激活Wnt /β-catenin通路，促进结直肠癌对5-FU耐药，揭示结直肠癌化疗耐药中的可能潜在分子机制。. 肿瘤细胞的生长、凋亡与化疗耐药密切相关，我们进一步研究探讨结直肠癌细胞增殖的分子机制。在大肠癌组织及细胞系中发现TTN-AS1分子高表达，TTN-AS1基因敲除可抑制结直肠癌细胞的增殖和侵袭，促进细胞凋亡。机制上，TTN-AS1可能通过调控miR-376a-3p发挥作用，转染miR-376a-3p减轻TTN-AS1敲除引起的变化，TTN-AS1通过海绵状miR-376a-3p正向调节KLF15，同时，整体动物水平也证实了TTN-AS1通过miR-376a-3p/KLF15轴促进大肠癌的增殖和侵袭。.我们的研究结果有利于阐明化疗耐药和生长增殖的分子机制，为结直肠癌化疗耐药提供新的标志物，为肿瘤的个体化治疗提供一定的理论基础。