活性维生素D通过DNMT3B诱导DKK1去甲基化抑制结直肠癌细胞增殖的机制研究

Colorectal cancer is a malignant tumor that threatens human health seriously. Its pathogenesis and progression are closely related to the abnormal activation of Wnt/β-catenin signaling pathway. Active vitamin D could promote the expression of DKK1, an antagonist of Wnt/β-catenin signaling pathway, and inhibit the proliferation of colorectal cancer cells, however, the underlying mechanism by which active vitamin D increased DKK1 expression was unknown. The applicant's preliminary experimental results showed that active vitamin D could inhibit the expression of DNMT3B, which is a member of DNA methyltransferases, what’s more, abnormal hypermethylation of promoter region was an important reason for the down-regulation of DKK1 expression in colorectal cancer. Therefore, applicant hypothesized that active vitamin D could inhibit the proliferation of colorectal cancer cells by inhibiting the expression of DNMT3B, thus inducing DKK1 demethylation to promote its expression. To confirm the above hypothesis，this project intends to detect the demethylation effect of active vitamin D on DKK1 in vivo and in vitro experiments, and to reveal the mechanism of DKK1 demethylation induced by active vitamin D through the construction of DNMT3B overexpressed colorectal cell model and nude mouse xenograft model. This study will enrich the function of active vitamin D in regulating gene methylation, and provide theoretical support for the application of active vitamin D in the prevention and treatment of colorectal cancer.

结直肠癌是严重威胁人类健康的恶性肿瘤。Wnt/β-catenin信号通路异常激活与结直肠癌发病及进展密切相关，活性维生素D可促进该信号通路拮抗分子DKK1的表达，发挥抑制结直肠癌细胞增殖的作用，然而其促进DKK1表达的机制尚不明确。申请人前期实验结果表明，活性维生素D可抑制DNA甲基转移酶DNMT3B的表达，而启动子区异常高甲基化是DKK1在结直肠癌中表达下调的重要原因，因此申请人提出活性维生素D通过抑制DNMT3B的表达，诱导DKK1去甲基化促进其表达，进而抑制结直肠癌细胞增殖的科学假说。为证实上述假说，本项目拟在体内及体外实验中检测活性维生素D对DKK1的去甲基化作用，并通过构建DNMT3B过表达结直肠癌细胞模型及裸鼠移植瘤模型，探讨活性维生素D诱导DKK1去甲基化的机制。研究结果将丰富活性维生素D在调控基因甲基化方面的功能，为其应用于结直肠癌的预防及治疗提供理论支持。

维生素D是人体必需的一种脂溶性维生素，维生素D缺乏与结直肠癌发病率及病死率升高密切相关，Wnt/β-catenin信号通路的激活在结直肠癌的发病及进展中发挥重要的作用。Dickkopf-1（DKK1）是 Wnt/β-catenin信号通路的拮抗分子，在结直肠癌中扮演抑癌基因的角色。前期研究结果表明活性维生素D可促进DKK1的表达，发挥抑制结直肠癌细胞增殖的作用，然而其促进DKK1表达的机制尚不明确。本项目主要进行了四方面的研究，首先，检测了1,25(OH)2D3对结直肠癌细胞增殖的影响并对相关机制进行了探讨；其次，在肠癌细胞和患者标本中检测了DKK1表达及甲基化情况，并通过焦磷酸测序探讨了1,25(OH)2D3对结直肠癌细胞DKK1启动子区及5’UTR区甲基化的调控作用；再次，构建了DNMT3B干涉肠癌细胞系，对1,25(OH)2D3通过DNMT3B诱导DKK1去甲基化的机制进行了探讨。最后，在结直肠癌患者及健康对照者血清中检测了25(OH)D水平，并分析了其与患者临床病理学指标的相关性。研究结果表明，1,25(OH)2D3可通过抑制Wnt/β-catenin 信号通路，发挥抑制结直肠癌细胞增殖的作用。结直肠癌患者血清DKK1水平显著下降，DNA异常甲基化是抑制DKK1表达的重要原因，1,25(OH)2D3可使结直肠癌细胞DKK1 5’UTR区发生去甲基化进而影响其表达。DNMT3B可通过影响DNA甲基化修饰在DKK1表达调控中发挥作用。结直肠癌患者血清25(OH)D缺乏率高达86.25%，显著高于健康对照者（P<0.05），女性患者维生素D缺乏率显著高于男性（P<0.05）。项目的研究从表观遗传学的角度阐明了活性维生素D抗结直肠癌的一种新机制，为结直肠癌的防治提供了新的思路。

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