kank1基因在鼻咽癌中表达下调及其机制和功能研究

发布时间：2018-04-19 13:22

本文选题：鼻咽癌 + kank1　；参考：《中南大学》2012年博士论文

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)高发于我国南方及东南亚地区,而在北美和其他西方国家的白人中少见。流行病学及病因学研究表明,鼻咽癌的发病与遗传因素、EB病毒感染和环境因素有关。鼻咽癌显著的种族易感性和家族聚集现象表明,遗传因素特别是瘤基因的激活和抑瘤基因的失活在鼻咽癌发病中具有十分重要的作用,因此寻找鼻咽癌遗传易感基因是防治鼻咽癌的关键。 kank1基因是kank家族的一个重要成员,位于9p24.3区域。研究显示,Kank1在脑、肺、肾肿瘤组织及相应的细胞系中存在杂合性缺失(loss of heterozygosity, LOH)和启动子区甲基化,导致该基因表达下调/缺失,提示kankl基因可能是一个抑瘤基因。鼻咽癌组织比较基因组杂交(comparative genomic hybridization, CGH)的结果显示,染色体9p存在高频率LOH,随后将高频缺失定位在更精细的9p21.3-p24.3区域。这些研究结果高度提示,在9p21.3-p24.3可能存在与鼻咽癌相关的抑瘤基因。目前已发现该区的抑瘤基因有p16、NGX6和kank1等,而kank1基因与鼻咽癌的关系尚未见报的。因此,本研究开展kank1基因在鼻咽癌中的表达情况、失活机制、功能及其临床病理学意义研究。 1、kank1基因在鼻咽癌中的表达研究采用RT-PCR、Real-time RT-PCR方法检测kank1mRNA在正常鼻咽上皮细胞系NP69、4株鼻咽癌细胞系(CNE1、CNE2、5-8F、6-10B)、11例非癌组织和77例鼻咽癌组织中的表达。结果显示,kank1在正常鼻咽细胞系NP69和几乎全部非癌组织均有表达,而在4株鼻咽癌细胞系和大于50%的鼻咽癌组织中表达下调或缺失。Western blotting结果显示,kank1蛋白在60%(12/20)的鼻咽癌组织中表达下调。结果表明,鼻咽癌中kank1基因存在高频率的表达下调/缺失。 2、kank1基因在鼻咽癌中表达下调的机制研究由于kank1位于鼻咽癌的高频缺失区域,而基因内部及其附近微卫星位点的丢失可以间接反映该基因的缺失情况。为此,我们采用PCR-变性聚丙烯酰胺凝胶电泳-银染方法,分析kank1基因上下游的D9S1858和WI-12779二个微卫星位点在鼻咽癌组织中的杂合性缺失情况。启动子区DNA异常甲基化是抑瘤基因表观遗传学改变最为常见的方式,生物信息学分析发现kank1启动子区存在CpG岛。我们采用甲基化特异性PCR分析鼻咽癌组织kank1启动子甲基化情况。结果显示,鼻咽癌组织D9S1858和WI-12779发生LOH的频率分别为31.4%(11/35)和34.3%(12/35),kank1基因LOH的频率为51.4%(18/35)；鼻咽癌细胞株和鼻咽癌组织中kank1基因全部存在甲基化,而正常组织中甲基化频率为60%(6/10)。提示kank1等位基因LOH和启动子高甲基化可能是导致其在鼻咽癌中表达下调/缺失的重要原因。 3、鼻咽癌细胞kank1的功能研究为了探讨kank1在鼻咽癌发病中的作用,我们采用脂质体转染技术将kank1表达质粒和空白质粒转染高成瘤高转移5-8F鼻咽癌细胞,建立kank1高表达的5-8F鼻咽癌细胞系(5-8Fkank1)和对照细胞系(5-8Fvector)。随后观察kank1表达上调对5-8F细胞生物学特征的影响。MTT分析结果显示,5-8Fkankl细胞的增殖速度明显低于5-8Fvector细胞和未转染细胞(p0.05)；软琼脂集落形成实验的结果显示,5-8Fkank1细胞的克隆形成率明显低于5-8Fvector细胞和未转染细胞(p0.05)；流式细胞术分析的结果显示,与5-8Fvector细胞和未转染细胞相比,5-8Fkank1细胞Go/G1期细胞比例明显增加(p0.05),S期和G2/M期细胞比例明显减少(P0.05)；流式细胞术和Hoechst33258染色结果显示,5-8Fkankl细胞的凋亡明显高于5-8Fvector细胞和未转染细胞(p0.05)；细胞体外迁移和侵袭实验显示,与5-8Fvector细胞和未转染细胞相比,5-8Fkankl细胞的运动能力和侵袭能力均明显降低(p0.05)。研究结果提示,kank1具有抑制鼻咽癌细胞增殖、促进鼻咽癌细胞凋亡以及抑制鼻咽癌细胞体外迁移和侵袭的作用。 4、kank1的临床病理意义分析为探讨鼻咽癌kank1表达下调/缺失的临床病理意义,我们采用免疫组化(IHC)染色检测kank1在14例非癌组织和69例鼻咽癌组织中的表达。结果显示,kank1在72.5%(50/69)的鼻咽癌组织中表达下调,而在14例正常鼻咽粘膜上皮中只有1例表达下调。kank1表达下调/缺失与鼻咽癌淋巴结转移和临床分期负相关,kank1表达下调/缺失的鼻咽癌更易发生淋巴结转移、临床分期更晚,而与肿瘤组织学类型、原发灶大小、患者的性别和年龄无关。本文研究结果表明,kank1基因在非癌组织中稳定表达,在鼻咽癌组织和细胞中表达明显下调或缺失,LOH和启动子区甲基化可能是导致kank1基因表达下调的机制之一,通过在鼻咽癌细胞系中过表达kank1的表达发现,kank1基因能够抑制细胞的增殖、促进细胞的凋亡、抑制细胞侵袭和迁移,kank1基因表达下调在鼻咽癌发病中具有重要作用。结果提示,kank1是候选的鼻咽癌抑瘤基因。
[Abstract]:Nasopharyngeal carcinoma (NPC) is highly prevalent in southern and Southeast Asia and is rare among white people in North America and other western countries. Epidemiological and etiological studies have shown that the incidence of nasopharyngeal carcinoma is related to genetic factors, EB virus infection and environmental factors. The genetic factors especially the activation of the tumor gene and the inactivation of the tumor suppressor genes play an important role in the pathogenesis of nasopharyngeal carcinoma. Therefore, it is the key to find the genetic susceptibility gene of nasopharyngeal carcinoma (NPC) to prevent and control nasopharyngeal carcinoma.
The Kank1 gene is an important member of the kank family, located in the 9p24.3 region. The study shows that Kank1 has the absence of heterozygosity (loss of heterozygosity, LOH) and promoter region methylation in the brain, lung, kidney tumor and corresponding cell lines, which leads to the downregulation / loss of the gene expression, suggesting that the kankl gene may be a tumor suppressor gene. Nasopharyngeal carcinoma is a gene for nasopharyngeal carcinoma. The results of comparative genomic hybridization (CGH) show that there is a high frequency LOH in chromosome 9p, and then high frequency deletion is located in a more fine 9p21.3-p24.3 region. These results suggest that the tumor suppressor genes associated with nasopharyngeal carcinoma may exist in 9p21.3-p24.3. The tumor suppressor of the region has been found at present. Because of p16, NGX6 and Kank1, the relationship between the Kank1 gene and nasopharyngeal carcinoma has not yet been reported. Therefore, this study carried out the study of the expression of Kank1 gene in nasopharyngeal carcinoma, the mechanism of inactivation, function and its clinicopathological significance.
Study on the expression of 1, Kank1 gene in nasopharyngeal carcinoma
The expression of kank1mRNA in normal nasopharyngeal epithelial cell line NP69,4 strain of nasopharyngeal carcinoma cell line (CNE1, CNE2,5-8F, 6-10B), 11 non cancerous tissues and 77 nasopharyngeal carcinoma tissues was detected by RT-PCR and Real-time RT-PCR. The results showed that Kank1 was expressed in normal nasopharyngeal cell line NP69 and almost all noncancerous tissues, but in 4 nasopharyngeal carcinoma cell lines. The expression of down-regulation or deletion of.Western blotting in nasopharyngeal carcinoma tissues greater than 50% showed that the expression of Kank1 protein was down regulated in 60% (12/20) nasopharyngeal carcinoma tissues. The results showed that the expression of Kank1 gene in nasopharyngeal carcinoma had a high frequency of downregulation / deletion.
2, the mechanism of down regulation of Kank1 gene expression in nasopharyngeal carcinoma.
Because Kank1 is located in the high frequency region of nasopharyngeal carcinoma, the loss of the microsatellite loci within and near the gene can be indirectly reflected. Therefore, we use PCR- denatured polyacrylamide gel electrophoresis and silver staining method to analyze two microsatellite loci in the upstream of Kank1 gene and WI-12779 in nasopharyngeal carcinoma tissue DNA abnormal methylation in the promoter region is the most common way of epigenetic changes in tumor suppressor genes. Bioinformatics analysis found that there is a CpG island in the Kank1 promoter region. Methylation specific PCR was used to analyze the Kank1 promoter methylation status of nasopharyngeal carcinoma tissue. The results showed that the nasopharyngeal carcinoma tissues were D9S1858 and WI-1. 2779 the frequency of LOH was 31.4% (11/35) and 34.3% (12/35), and the frequency of Kank1 gene LOH was 51.4% (18/35). The Kank1 gene in nasopharyngeal carcinoma cell lines and nasopharyngeal carcinoma tissues was all methylation, while the normal methylation frequency was 60% (6/10). It suggests that Kank1 allele LOH and promoter hypermethylation may lead to nasopharyngeal carcinoma. The important cause of downregulation / deletion in the expression.
3, functional study of nasopharyngeal carcinoma cell Kank1
In order to explore the role of Kank1 in the pathogenesis of nasopharyngeal carcinoma, we transfected Kank1 expression plasmids and blank plasmids into high metastasis 5-8F nasopharyngeal carcinoma cells by liposome transfection, and established a 5-8F nasopharyngeal carcinoma cell line (5-8Fkank1) and a control cell line (5-8Fvector) with high expression of Kank1. Then we observed that the expression of Kank1 was up-regulated to 5-8F cell biology. The results of.MTT analysis showed that the proliferation rate of 5-8Fkankl cells was significantly lower than that of 5-8Fvector cells and untransfected cells (P0.05). The result of the formation of soft agar colony formation showed that the clone formation rate of 5-8Fkank1 cells was significantly lower than that of 5-8Fvector cells and untransfected cells (P0.05); the results of flow cytometry analysis showed that the 5-8Fkank1 cells were and 5-8. Compared with untransfected cells, the proportion of Go/G1 phase cells in 5-8Fkank1 cells increased significantly (P0.05), and the proportion of cells in S and G2/M stages decreased significantly (P0.05). The results of flow cytometry and Hoechst33258 staining showed that the apoptosis of 5-8Fkankl cells was significantly higher than that of 5-8Fvector cells and untransfected cells (P0.05), and the migration and invasion of cells in vitro. Compared with 5-8Fvector cells and untransfected cells, the exercise ability and invasion ability of 5-8Fkankl cells were significantly decreased (P0.05). The results suggest that Kank1 can inhibit the proliferation of nasopharyngeal carcinoma cells, promote the apoptosis of nasopharyngeal carcinoma cells and inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro.
The clinicopathological significance of 4, Kank1
To investigate the clinicopathological significance of down-regulation / deletion of Kank1 expression in nasopharyngeal carcinoma (NPC), immunohistochemical (IHC) staining was used to detect the expression of Kank1 in 14 non cancerous tissues and 69 nasopharyngeal carcinoma tissues. The results showed that Kank1 was down regulated in 72.5% (50/69) nasopharyngeal carcinoma tissues, while only 1 cases in 14 normal nasopharyngeal mucosa epithelium were expressed as down regulated.Kank1. Down regulation / deletion was negatively correlated with lymph node metastasis and clinical staging of nasopharyngeal carcinoma. Nasopharyngeal carcinoma with down-regulation and deletion of Kank1 was more likely to occur lymph node metastasis. The clinical stage was later, but it was not related to the histological type, the size of the primary focus, and the sex and age of the patients.
The results of this study show that the expression of Kank1 gene in non cancer tissues is stable, and the expression in the tissues and cells of the nasopharyngeal carcinoma is obviously downregulated or deleted. The methylation of LOH and promoter region may be one of the mechanisms that lead to the downregulation of Kank1 gene expression. By overexpression of Kank1 expression in the nasopharyngeal carcinoma cell lines, the Kank1 gene can inhibit the cell. Proliferation, cell apoptosis, inhibition of cell invasion and migration, the down regulation of Kank1 gene expression plays an important role in the pathogenesis of nasopharyngeal carcinoma. The results suggest that Kank1 is a candidate tumor suppressor gene for nasopharyngeal carcinoma.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

【参考文献】