【作者】 张海利；

【导师】 王斌全；

【作者基本信息】 山西医科大学 ， 耳鼻咽喉头颈外科学， 2012， 博士

【摘要】 背景与目的：、侵袭性与转移性是恶性肿瘤的两个主要特征,也是影响恶性肿瘤患者预后的关键因素,在肿瘤侵袭转移的复杂过程中受到众多基因的调控。肿瘤转移相关基因1(MTA1)是新近发现的与恶性肿瘤转移相关的基因,该基因在肿瘤转移过程中表达明显上调。MTA1具有组蛋白脱乙酰基酶活性,调控组蛋白脱乙酰基,通过影响染色质的状态来调整复制,从而发挥其生物学作用。基质降解、细胞间的粘附及肿瘤的血管生成在肿瘤侵袭转移的过程中均发挥着重要作用。本研究通过检测喉鳞癌组织、喉部非典型增生组织及正常黏膜组织中MTA1、MMP-9、β-catenin及EGFR的表达,以了解这些参与肿瘤侵袭转移的基因表达在喉鳞癌发生发展的作用及其相关性。利用RNA干扰技术及质粒转染技术观察MTA1对HEP-2细胞生物学特性变化的影响,进一步认识MTA1对喉癌细胞侵袭转移的调控作用。方法：采用免疫组织化学法检测MTA1蛋白在40例正常喉黏膜上皮、37例喉不典型增生组织及40例喉鳞癌组织中的表达水平,分析MTA1在喉鳞癌组织中的作用与临床分期、分型、病理学分级及颈淋巴结转移的关系；采用免疫组织化学法检测MMP-9、EGFR及β-catenin蛋白在40例喉鳞癌组织与喉正常黏膜组织中的表达,分析MMP-9、EGFR及β-catenin蛋白在喉鳞癌组织中的作用,并分析MTA1与MMP-9、EGFR及β-catenin表达的相关性。在喉癌细胞株HEP-2细胞中,采用RNA干扰技术转染针对MTA1的siRNA (MTA1-siRNA), RT-PCR法及western-blot法分别从转录水平与翻译水平鉴定其基因沉默效果,并采用MTT法绘制细胞生长曲线、细胞侵袭及迁移实验、平板克隆形成实验、细胞黏附实验及细胞划痕愈合实验等观察细胞生物学特性的改变；稳定转染MTA1的表达质粒及干扰质粒,挑选阳性克隆,进行MTT法绘制细胞生长曲线、细胞侵袭迁移实验、细胞黏附实验、平板克隆形成实验及划痕愈合实验,检测MTA1基因表达水平的变化,对肿瘤细胞生物学特性的影响。结果：MTA1蛋白表达于细胞核中。在喉正常黏膜组织中,MTA1蛋白呈低水平表达,阳性表达率为5.0%,而在喉鳞癌组织中,MTA1蛋白阳性表达细胞数量及染色强度均明显增高,阳性率达到67.5%,在不典型增生组织中,MTA1表达率为37.8%,介于正常组织与喉鳞癌组织之间。MTA1核表达水平在不典型增生组织与癌组织中的表达水平均高于喉正常鳞状上皮组织(p<0.01)；癌组织中MTA1表达水平高于不典型增生组织,且统计学意义显著(p<0.01)；MTA1蛋白在从喉正常黏膜组织、不典型增生组织到喉鳞癌组织中的表达呈进行性增加。分析MTA1在喉鳞癌组织中的表达与临床病理学参数的关系中显示,在40例喉鳞癌组织标本中,MTA1蛋白在低、中及高分化组织中阳性表达率分别为90.0%、72.2%及41.7%,表现为随着分化程度的增高,其阳性表达率降低,有统计学差异(χ2=6.141,p<0.05)；在喉鳞癌原发灶中MTA1蛋白在伴有颈淋巴结转移的组织中阳性表达率为78.6%,而在不伴有颈淋巴结转移组织中的表达率为41.7%,统计学分析结果显示,MTA1蛋白的表达水平与喉鳞癌患者的颈淋巴结转移相关,MTA1蛋白的高表达能导致颈淋巴结转移的发生(χ2=5.215,p<0.05)；在与喉鳞癌临床分期及分型的关系中,可见MTA1在Ⅰ、Ⅱ期的表达率较Ⅲ、Ⅳ期低,分别为53.8%、92.9%,但二者之间没有差异(χ2=0.623,p>0.05)；MTA1与喉鳞癌临床分型也无关(χ2=1.138,p>0.05)。MMP-9、EGFR及β-catenin在喉鳞癌组织中的表达率分别为82.5%、87.5%及87.5%,而在喉正常组织中的阳性表达率分别为35.0%、37.5%及35.0%,有统计学差异(p<0.05)；经过相关分析显示,在喉鳞癌中,MTA1与MMP-9、EGFR及β-catenin的表达呈正相关(p<0.05)。在HEP-2细胞株,转染靶向MTA1-siRNA后,MTA1基因蛋白及mRNA的表达水平明显下降,与空白对照组及无义对照MTA1-NiRNA组相比,MTA1基因的表达被显著抑制(p<0.05)。在检测随后的细胞生物学表型中发现,转染MTA1-siRNA后,细胞的生长速度减慢、体外侵袭迁移能力下降、细胞的体外黏附及克隆形成能力均下降；稳定转染MTA1表达质粒及干扰质粒后,MTT法检测细胞的生长速度,发现细胞的生长速度在表达质粒组最快,而在于扰质粒组最慢,有统计学差异；在细胞体外侵袭实验中,表达质粒组、对照组及干扰质粒组穿膜细胞数量分别为423.6±14.15,301.2±25.4和115.4±15.52；在迁移实验中的穿膜细胞数分别为549.2±21.51,352±25.03和120.8±17.28。在细胞迁移及侵袭实验中,穿膜细胞数量最多的是表达质粒转染组,明显高于对照组及干扰质粒组,干扰质粒组中细胞的穿膜能力最弱,细胞数量最少；平板克隆形成实验中,表达质粒组、对照组及干扰质粒组中克隆形成数量分别为168.67±9.5、132.57±5.5和57.33±4.51,可见MTA1的高表达会促进肿瘤细胞的克隆形成能力,而降低其表达,则会导致克隆形成能力的下降；在细胞划痕愈合实验及细胞黏附实验中,MTA1表达质粒转染组细胞的划痕愈合时间缩短、黏附能力增强,干扰质粒组细胞划痕愈合时间最长、黏附能力下降。结论：喉鳞癌组织中,MTA1的高表达在喉鳞癌的恶性进展、侵袭转移及颈淋巴结的转移中起着重要的作用,或许可以作为判断喉鳞癌恶性程度的一个分子生物学标志；MMP-9、EGFR及(3-catenin蛋白在喉鳞癌的发生发展中起着重要的作用,MTA1的过度表达与MMP-9、EGFR及β-catenin成正相关,MTA1过度表达导致肿瘤的浸润转移是与MMP-9、EGFR及β-catenin的异常表达共同作用的结果。在人喉癌细胞株HEP-2中,改变MTA1基因的表达水平,导致喉癌癌细胞侵袭转移能力及其它恶性行为的变化。上调MTA1基因的表达,会增强肿瘤细胞的恶性行为,导致肿瘤的侵袭转移；而抑制其表达,则可以降低肿瘤细胞的恶性表型。为研究MTA1基因在喉鳞癌中的作用及进行以MTA1为靶点的基因治疗研究打下了一定的基础。更多还原

【Abstract】 Background and Objective:Invasion and metastasis are the most important characteristics of malignant cancer and life-threatening aspects of cancer which is a most complex process involving multi-steps and mult-ifactors.Metastasis-associated gene1(MTA1) is positively correlated with cancer metastasis in many cancer types and is a subunit of the NURD (nucleosome remodelling and deacetylase) complex which is involved in chromatin remodelling and histone deacetylation to regulate gene expression. To explore significance of MTA1in the invasion and migration processes in laryngeal squamous cell carcinoma(LSCC) we detected the expression of MTA1in the different stages of LSCC including normal epithelial cell, dysplasia and invasive cancer. We explored the expression of MMP-9,EGFR ang β-catenin in the using immunohistochemistry and analyzed the important role in LSCC and the correlation of MTA1with MMP-9,EGFR and β-catenin.We performed RNA interference against MTA1to study the effect of interference and the biological function of MTA1in LSCC cell line HEP-2. HEP-2cells transfected with cDNA3-MTA1and RNA interference plasmid and analyzed malignant phenotype in HEP-2and the function in LSCC.Method:Immunohistochemistry staining was adopted to detect the expression of MTA1in the different stages of LSCC including40cases with normal epithelial cell,37cases with dysplasia and40cases with invasive cancer and analyzed the function of MTA1in LSCC and the correlation between the expression of MTA1and clinicopathological parameters of LSCC.We detected the expression of MMP-9,EGFR and β-catenin in the tissues derived from40patients by immunohistochemistry and observed their role in LSCC and correlation between the expression of MTA1and MMP-9,EGFR, β-catenin.The siRNA against MTAl was chemically synthesized and transfected into HEP-2cells. The MTA1protein and mRNA levels in the transfected cells were assayed by RT-PCR and western blot respectively and cellular biological characteristics were observed by wound healing assay, Colony formation, invasion assay and adhesion assay. HEP-2Cells were tranfected with MTA1high expression plasmid and RNA interference plasmid against MTA1and observed the cellular biological characteristics by wound healing assay, Colony formation, invasion assay and adhesion assay. Result:The MTA1locates in the nucleus with low level of expression in the normal laryngeal squamous epithelial cells and the positive expression rate of MTA1was5.0%.But the positive expression rate of MTA1was67.5%in LSCC which was higher significantly than that in normal and dysphasia tissues(37.8%)(P<0.05).The MTA1expression level in normal laryngeal squamous cells was lower than in dysplasia cells and carcinoma cells (P<0.05). In the studied specimens, from the normal squamous epithelial cells to invasive carcinoma cells, the nuclear expression of MTA1in the different stages of carcinogenesis of LSCC increased. In well, moderately and poorly differentiated tissues of LSCC, the frequency of MTA1protein positivity was90.0%,72.2%and41.7%respectively. The expression of MTA1decreased gradually from poorly, moderately and well differentiated tissues and had significant difference(p<0.05). The positive staining rate was41.7%in patients without cervical lymph node metastasis, and78.6%in those with cervical lymph node metastasis and the expression of MTA1was correlated with lymphatic metastasis in LSCC(p<0.05). The relationship of MTA1protein with clinical stages and clinical classification were not found. The expression rate of MMP-9,EGFR and β-catenin was82.5%,87.5%and87.5%respectively in cancer tissues and35.0%,37.5%and35.0%in normal tissues, which had significant difference(p<0.05).MTA1expression levels in LSCC were positively correlated with MMP-9,EGFR and β-catenin protein(p<0.05).The expression levels of MTA1in the HEP-2cells transfected with siRNA against MTA1were significantly decreased than that in ceiil transfected with NiRNA.The siRNA against MTA1could efficiently inhibit MTA1gene expression and cell adhesion, invasion and movement as well as colony formation and cell growth were decreased.RNA interference against MTA1in HEP-2cell played its role to decrease cancer cell invasion,movement,adhesion,colony formation and cell growth.HEP-2Cells were tranfected with MTA1high expression plasmid and RNA interference plasmid against MTA1and observed the cellular biological characteristics. The healing ability of MTAl-siRNA transfected cells significantly lagged behind the pcDNA3-MTA1and control siRNA transfected cells by wound healing assay. In the migration assay, responding to pcDNA3-MTA1,control-siRNA and MTA1-siRNA, the number of cells migrated to the bottom side of the membrane were549.2±21.51,352±25.03and120.8±17.28respectively. The number of cells migrated to the bottom side of the membrane in the invasion assay were423.6±14.15,301.2±25.4and115.4±15.52respectively. Compared to the control-siRNA transfected cells,MTA1-siRNA significantly decreased the migration and invasion ability, while pcDNA3-MTA1moderately enhanced the migration and invasion ability. The colony number of pcDNA3-MTAl, control-siRNA and MTA1-siRNA group were168.67±9.5,132.57±5.5and57.33±4.51in colony formation assay, we can observed that those cells tranfected with MTA1high expression plasmid promoted colony formation. The healing ability of MTA1-siRNA transfected cells significantly lagged behind the pcDNA3-MTA1and control siRNA transfected cells in wound healing assay.Conclusion:Studies have indicated that MTA1played a role in the process of malignant progression and metastasis of carcinoma. The overexpression of MTA1causing the occurrence and development of LSCC may be realize by the abnormal expression of MMP-9,EGFR and β-catenin protein. In biological studies with human laryngeal squamous cell carcinoma cell line, MTA1plays its important roles to promote cancer cell adhesion,invasion and movement. RNA interference against MTA1decreased the malignant phenotypes. This study started an effective beginning to explore cancer gene therapy strategy targeting MTA1.更多还原