赖氨酸特异性去甲基化酶1对结肠癌侵袭转移能力的影响及其机制的研究

Metastasis is the major cause of death in patients with colon cancer. Our previous studies have shown that Lysine-specific demethylase 1 (LSD1) correlated closely with metastasis and poor prognosis of colon cancer.Silencing of LSD1 gene impaired proliferation and invasiveness, and induced apoptosis of colon cancer cells in vitro. In this project, the downstream target genes of LSD1 in colon cancer cell will be detected by Microarray Gene Expression Profiling and ChIP-DSL Promoter Array, the metastasis-related target genes will be identified by bioinformatics technology, and the transcriptional regulatory sites of target gene promoter will be detected by Luciferase Assay. Then the metastasis-related target gene in colon cancer will be confirmed by gene silencing and overexpression experiments. And the methylation levels of histone H3 lysine 4 (H3K4) and histone H3 lysine 9 (H3K9) at LSD1 targeted gene promoter will be detected by ChIP to investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. Finally, the influence of LSD1 and its targeted gene on growth and metastasis of colon cancer xenografts will be detected by whole-body fluorescence imaging in vivo, in order to assess the feasibility of using exogenous LSD1 inhibitors to inhibit invasion and metastasis of colon cancer cells, which will provide a new idea for the treatment of colon cancer.

转移是导致结肠癌患者死亡的主要原因。我们在前期研究中发现赖氨酸特异性去甲基化酶LSD1与结肠癌的转移密切相关，靶向沉默LSD1基因能有效抑制结肠癌细胞的增殖、侵袭和转移能力，诱导细胞凋亡。本研究拟利用基因表达谱芯片和ChIP-DSL启动子芯片技术探测结肠癌细胞中LSD1调控的下游靶基因，生物信息学技术确定与转移相关的靶基因；进而利用Luciferase实验验证LSD1与靶基因启动子区域的特异性结合。通过沉默和过表达上述靶基因，证实其对结肠癌侵袭性的影响。然后利用ChIP技术检测LSD1下游靶基因启动子区域H3K4、H3K9的甲基化水平，探讨LSD1影响结肠癌侵袭转移的分子机制。最后通过动物实验检测LSD1及其靶基因在活体内对结肠癌转移的影响，并评估应用外源性LSD1抑制剂抑制结肠癌转移的可行性，为结肠癌的治疗提供新的思路。

转移是导致结肠癌患者死亡的主要原因，开展结肠癌转移机制的研究非常必要。我们在本研究中发现：LSD1在结肠癌组织中的阳性率高达66.7%，明显高于正常结肠粘膜上皮组织。LSD1在较晚TNM分期、远处转移的结肠癌组织中的表达率明显增高，且阳性表达LSD1的结肠癌患者预示着更差的预后。LSD1的表达水平与结肠癌细胞株的侵袭性呈正相关。体外抑制LSD1的表达能抑制结肠癌细胞的增殖、侵袭转移能力，诱导细胞凋亡，并能上调E钙黏蛋白的表达，下调N钙黏蛋白的表达，三者可能在结肠癌的侵袭转移过程中发挥了重要作用。通过基因表达谱芯片检查发现：在LSD1敲除的SW620细胞中，3633个基因表达明显上调，4642个基因表达明显下调。而在LSD1过表达的HT29细胞中，4047个和4240个基因表达分别明显上调和下调。进一步通过分子生物学技术确定备选的LSD1下游与转移相关的靶基因共四个：CABYR, FOXF2, TLE4和CDH1。通过基因沉默和过表达实验，我们发现沉默LSD1基因能明显上调SW620细胞中CABYR和CDH1的表达，而过表达LSD1基因则能明显抑制HT-29中CABYR和CDH1的表达。我们利用IP和ChIP实验检测LSD1对四个候选靶基因的靶向调控作用，结果发现LSD1能与CABYR和CDH1的启动子区域结合，并通过下调CABYR和CDH1基因启动子区域组蛋白的H3K4me2和H3K4me水平，下调CABYR和CDH1的表达。同时我们发现，沉默CABYR和CDH1基因能促进结肠癌的侵袭性，从而证实LSD1能通过调控CABYR和CDH1基因的表达影响结肠癌的侵袭转移。因此，LSD1能通过组蛋白去甲基化修饰促进结肠癌的转移，LSD1有望成为结肠癌的新的治疗靶点。