SMYD3/RIZ信号通路调控食管癌发病的表观遗传学机制研究

In our previous work, several techniques, such as gene transfection, gene silence, MSP, Western Blotting and et al, were applied. We have found that, SMYD3 expressed in a high level in esophageal carcinoma, while RIZ1 was in a low level and its tumor-suppressor activity has diminished. We believe that these phenomena are relevant with methylation, and we have successfully realized the increase of expression level of RIZ1 and the recovery of its tumor-suppressor activity through silencing SMYD3, reversing methylation, transfecting RIZ1 and etc. In the meantime, the expression at C-myc, VEGF and some other important target points which can trigger the tumor has been well lowered down. Accordingly, we plan to study the fashion and mechanism that the SMYD3 mediated histone methylation modification affects the RIZ1 in the SMYD3/RIZ pathway and its relationship with DNA promoter methylation by means of histone H3K4 qualitative methylation detecting, MSP analysis, DZNep, LSD1 and 5-aza-CdR methylation reversion. In addition, the influence of the RIZ balance, which is regulated by SMYD3 on the biological behavior of esophageal carcinoma cells is also within our research extent through flow cytrometry, SRB, MTT, transwell and et al, for we wish to find the molecular mechanism of the signal pathways relevant with SMYD3/RIZ during the occurrence and development of esophageal carcinoma. It is of great significance and application expectation to create a theoretical basis and necessary experimental proofs for the molecular therapy of esophageal carcinoma.

前期工作应用基因转染、基因沉默、MSP、免疫印迹等技术发现：SMYD3在食管癌中高表达；RIZ1低表达抑癌活性减低，与DNA启动子甲基化相关；通过沉默SMYD3、逆转甲基化、转染RIZ1的多种手段均使得RIZ1表达上调且抑癌活性恢复，同时伴随有C-myc及VEGF等促肿瘤发病重要靶点的表达水平下降。在此基础上拟通过组蛋白H3K4甲基化定量检测、MSP分析，DZNep、LSD1、5-aza-CdR逆转甲基化等方法研究SMYD3/RIZ通路中SMYD3介导组蛋白甲基化修饰作用于RIZ1的方式与机制及其与DNA启动子甲基化的关系，并通过流式细胞术、SRB、MTT、transwell等研究SMYD3调控RIZ平衡对食管癌细胞生物学行为的影响，为SMYD3/RIZ相关信号通路在食管癌发生发展中的分子机理、为食管癌分子治疗提供理论基础和实验依据，具有重要意义及应用前景。

食管癌是威胁人类健康重要疾病，我国食管癌发病率和死亡率均居世界首位，并且表现为食管鳞癌为主。SMYD3作为一种组蛋白甲基化酶在多种肿瘤中起重要作用，课题组前期已发现其在食管鳞癌组织高表达，在细胞系中通过基因沉默下调后可以导致RIZ1的升高，并起到抑癌作用，因此认为SMYD3是RIZ1的上游调控基因。食管癌中RIZ1表达上调后，其抑癌活性得以恢复前期已被课题组证实。本研究试图明确SMYD3调控RIZ1的具体机制。通过研究我们得出以下结论：1. RIZ基因有两种表达产物RIZ1和RIZ2，两者区别在于PR-domain，RIZ1由于具备这一结构而具有抑癌作用，而RIZ1和RIZ2的表达之间是一种阴阳平衡，课题组体外实验转染RIZ2后观察到RIZ1 mRNA表达减少，另外当把PR-domain转染后得到了与RIZ1相同的抑癌效果，因此证实了SMYD3在食管癌中调控RIZ表达平衡。2. 课题组研究中发现SMYD3过表达并不能影响到RIZ1 mRNA和蛋白水平的表达，因此推测SMYD3对RIZ1的调控仅发生在一定条件下，而SMYD3作为一种H3K4组蛋白甲基化酶，其可通过调控H3K4 me2和me3甲基化程度而调控RIZ1。3. 组蛋白甲基化与DNA甲基化有诸多联系，SMYD3下调后可部分逆转RIZ1 DNA启动子甲基化，进而调控RIZ1表达。另外，课题组对H3K4me1、me2、me3甲基化的水平在癌和癌旁组织有无区别进行定量检测，发现三种甲基化在食管癌中均明显高于癌旁，说明组蛋白甲基化对癌症发生有重大意义。当应用5-aza-CdR逆转食管癌DNA启动子甲基化时，H3K4甲基化水平和甲基化酶活性发生相应改变，证明“组蛋白甲基化与DNA甲基化”、“组蛋白甲基化酶和去甲基化酶”有密切关联。后期通过对大样本人临床标本进行检测，进一步观察到SMYD3、RIZ1在食管癌表达方式，并发现其等与临床疾病特征（TNM分期、组织分化、淋巴结转移）关联，并对预后、放疗效果等产生重要影响，影响生存。相关工作不仅为SMYD3/RIZ 信号通路在食管癌发生发展中的分子机理、为食管癌分子治疗提供理论基础和实验依据，另外联合两者可作为食管癌临床评测预后和疾病进展十分有意义的指标。