臭参调控Acacb巴豆酰化在肠癌中的分子机制

Intestinal cancer is one of the five diseases that report the highest incidence and mortality in China. It is caused by chronic constipation. In the preliminary study, the applicant noted that Choushen could efficaciously relieve constipation, prevent intestinal cancer and directly suppress the proliferation of intestinal cancer cells; Acacb mediated the inhibition of the molecular mechanism of intestinal cancer with Choushen as the core regulatory factor. In turn, Choushen played a regulatory role in the proliferation and apoptosis of intestinal cancer cells through Acacb’s control over crotonylation. On this basis, the applicant made a scientific assumption: Choushen could be used to regulate the molecular mechanism of the Acacb-Hdac3-crotonylation signal axis of intestinal cancer. This presents a study that involves the following tests and experiments: (1) testing target proteins with crotonylation modified quantitative proteomics using tandem mass tags (TMTs) and analyzing the modification function of the target proteins by means of point mutation; (2) verifying the Acacb-Hdac3→crotonylation regulatory signal axis with co-immunoprecipitation (CO-IP) and mass spectrographic analysis; (3) lentivirus knock-down experiment: using Acacb for transfection of intestinal cancer cells after deactivation of the HIV-based lentivirus vector construction, then carrying out a phenotypic analysis on mutant cells by means of medication with Choushen and antibody detection with QPCR and WB crotonylation and subsequently, investigating the phenotypic change mechanisms of micromolecules and biomacromolecule interaction through post-translational modification by proteins and the corresponding significance in the physiological and pathological process of intestinal cancer.

肠癌我国发生率和死亡率均排前五，慢性便秘是诱因。前期研究中，申请人发现，林药臭参可缓解便秘预防肠癌且具有直接抑制肠癌细胞增殖的作用；Acacb是介导臭参抑制肠癌分子机制的核心调控因子，臭参经由Acacb调控巴豆酰化而调节肠癌细胞增殖、凋亡。由此，提出本项目科学假设：臭参调控Acacb-Hdac3→巴豆酰化这条信号轴在肠癌中的分子机制。拟研究：①全蛋白巴豆酰化修饰TMT标记定量蛋白质组学检测靶蛋白，并用点突变实验方法研究靶蛋白修饰功能；②Acacb-Hdac3→巴豆酰化调控信号轴的验证：CO-IP结合质谱分析对调控信号轴进行验证研究；③慢病毒敲减实验，Acacb慢病毒载体构建后转染肠癌细胞，再用臭参药物处理、结合QPCR、WB巴豆酰化泛抗体检测等方法对突变细胞做表型分析，结合Rescue基因回补实验，研究小分子与生物大分子互作经过蛋白质翻译后修饰的表型变化机制和其在生理病理过程中的意义。

结直肠癌是最常见的恶性肿瘤之一，在世界范围内的癌症相关死亡原因中位列第二。我们前期研究发现臭参提取物能够抑制结直肠癌肿瘤细胞的增殖并促进其凋亡，利用DMH诱导的结直肠癌小鼠模型进一步在体内证实了臭参提取物都结直肠癌的抑制效果。结肠肿瘤组织中赖氨酸位点的巴豆酰化水平相较正常对照小鼠肠组织显著降低，臭参提取物治疗后赖氨酸位点的巴豆酰化水平明显恢复。利用全蛋白巴豆酰化修饰TMT标记定量蛋白质组学检测并通过相关生物信息学分析我们发现，Acacb可能是臭参提取物发挥结直肠癌治疗作用的关键作用因子，其经由调控HDAC2表达影响下游靶蛋白赖氨酸的巴豆酰化修饰水平发挥治疗作用。 我们进一步在结肠癌肿瘤细胞HCT116中通过RNA干扰以及高表达质粒转染调控Acacb表达，观察其对HDAC2、HDAC3表达水平的影响以及赖氨酸巴豆酰化水平的调控。我们发现，干扰Acacb表达后HDAC2表达水平显著降低，HCT116细胞中赖氨酸巴豆酰化水平亦明显降低；而高表达Acacb的HCT116细胞中HDAC2转录水平较对照质粒转染细胞明显升高，赖氨酸巴豆酰化水平亦明显升高，验证了Acacb调控HDAC2表达影响下游靶蛋白赖氨酸巴豆酰化水平的研究假说。我们进一步利用免疫荧光染色在结肠癌模型小鼠肠组织中验证Acacb及HDAC2表达与赖氨酸巴豆酰化水平的相关性，以及臭参治疗后结肠组织中Acacb及HDAC2表达均明显上调，赖氨酸巴豆酰化水平显著升高。本研究在体内外探索了臭参提取物通过调控Acacb-HDAC2信号轴影响赖氨酸巴豆酰化水平治疗结直肠癌的作用机制，为将臭参提取物应用于结直肠癌肿瘤治疗提供依据。