CHD5基因调控喉癌细胞EMT过程的机制研究

Laryngeal squamous cell carcinoma, as one of the most common malignancies of HNSCC, constitutes almost 2% to 3% of all malignant tumors. Disappointingly, survival has not markedly improved in recent decades because patients still frequently develop loco regional recurrence, distal metastases and second primary tumors. Cancer cell metastasis to distal organs is the major cause of death in almost all forms of cancer. .In the early stages of metastasis, certain epithelial tumor cells undergo epithelial-mesenchymal transition (EMT), which is characterized by the loss of epithelial polarity and differentiation markers such as E-cadherin, β-catenin and the gain of mesenchymal markers usually expressed by cells with a mesodermal origin. It was widely accepted that aberrant Snail or slug expression results in loss of expression epithelial architecture and the cell adhesion molecule E-cadherin. Interestingly, loss of E-cadherin is necessary and sufficient to cause EMT. The Mi-2/NuRD complex represents a major macro associated with changes in epithelial architecture and molecular form of histone deacetylase in vertebrates' invasive growth. The cancer cells with an EMT phenotype are capable of moving away from their parent tumor, interacting with stromal cells, invading their adjacent tissues and the circulation systems. Since certain tumor cells, such as some laryngeal tumor cells, often disseminate even before their parent tumor can be clinically diagnosed, it is important to explore the currently unclear molecular networks governing the above multi-step invasion- metastasis cascade..Chromodomain helicase DNA-binding protein 5 (CHD5) was identified as a member of the CHD family and as a candidate TSG located at 1p36. CHD family members belong to the group of ATP-dependent chromatin-remodeling enzymes and have previously been implicated in regulating the expression of their corresponding genes by remodeling chromatin structure. Our previous study identified and reported that CHD5 is silenced in LSCC by aberrant hypermethylation. Downregulated CHD5 mRNA levels are significantly associated with advanced TNM stages, which implies that CHD5 might inhibit the metastasis of LSCC. Our study indicates that reduced CHD5 expression that results in decreased apoptosis, increased proliferation and invasiveness could contribute to the invasion and aggression of LSCC. Recent study showed that CHD5 is an integral component of the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex. The Mi2/NuRD complexes function primarily in gene repression involved in many biological processes, including cancer initiation and progression. .Here,we focus our investigation on the hypothesis that CHD5/NuRD complexes might regulate the metastatic potential of laryngeal cancer cells and suppress laryngeal cancer metastasis. These results will establish a mechanism that was independently isolated in several different links between CHD5 status and invasive growth of laryngeal cancer.

喉癌是头颈部最常见的恶性肿瘤之一，占全身恶性肿瘤的2-3%。喉癌患者最常见的死因为癌细胞颈部淋巴结转移或远隔器官转移。肿瘤细胞的早期转移过程表现为上皮细胞发生间质细胞转化（EMT），E-cadherin表达缺失是EMT的标志性事件。Mi-2/NuRD 复合物能够抑制Snail基因转录并上调E-cadherin表达，抑制上皮细胞EMT过程。CHD5基因是新近发现的候选抑癌基因，其所在的CHD家族蛋白质具有染色质重塑的作用。前期试验中，我们发现并报道了CHD5在喉癌组织中发生表观遗传学沉默现象并与喉肿瘤侵袭和转移能力密切相关。最新研究表明，CHD5基因是Mi-2/NuRD 复合物的亚单位之一，参与NuRD复合物的转录调控过程。根据以上研究成果，我们假定CHD5通过NuRD复合物亚单位的形式参与调控喉癌细胞EMT过程。本课题组将就该假设进行研究，以阐明CHD5基因在喉癌侵袭转移过程中的作用机制。

喉鳞状细胞癌是头颈部最常见的恶性肿瘤之一。染色质域解旋酶DNA结合蛋白5（chromodomain helicase DNA-binding protein 5,CHD5）是染色质重塑酶CHD家族的成员之一，具有控制细胞增殖、凋亡和衰老的功能。国外研究发现，CHD5蛋白是Mi2/NuRD复合物的一个亚单位，在上皮源性恶性肿瘤细胞发生上皮-间质化过程中，NuRD复合物能够抑制上皮钙黏蛋白(E-cadhein，E-cad)上游重要的负性调控者Snail基因转录，促进E-cadherin基因的表达，从而在癌细胞转移和侵袭过程中发挥重要作用。本研究通过检测101例原发喉癌组织、癌旁正常组织中CHD5与Snail、Slug、E-cadherin，β-catenin基因mRNA和蛋白质水平的表达情况及其相互关系。CHD5过表达或沉默后在体内和体外环境下，我们检测了喉癌细胞侵袭转移能力的变化，以及裸鼠成瘤组织细胞内CHD5、Snail、Slug、E-cadherin、β-catenin基因的表达情况。实验结果表明CHD5、β-catenin和E-cad基因mRNA及蛋白水平的表达在喉癌组织中显著低于癌旁正常组织（P < 0.05）。喉癌组织中，CHD5蛋白与β-catenin、E-cad蛋白呈显著正相关（P < 0.05）；CHD5蛋白与Snail、Slug蛋白表达呈显著负相关（P < 0.05）。同时萤光素酶方法分别检测结果显示随着剂量CHD5表达水平增加的刺激下Snail启动子活性的减低（P < 0.05）。体外实验显示CHD5能够上调β-catenin、E-cad的表达，抑制Snail和Slug基因蛋白表达水平（P < 0.05）。CHD5沉默后Hep-2细胞的增殖能力显著增强（P < 0.05）。裸鼠成瘤组织中CHD5蛋白水平的表达与Snail、Slug基因呈负相关，而与E-cadherin，β-catenin基因表达水平呈正相关（P < 0.05）。本实验进一步证实了CHD5基因可能通过调控E-cadherin，β-catenin，Snail、Slug基因的表达参与喉癌细胞EMT的过程，从而为喉癌除手术外的综合治疗提供进一步的研究依据。