本文選題：鼻咽癌 + kank1��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)高發(fā)于我國(guó)南方及東南亞地區(qū),而在北美和其他西方國(guó)家的白人中少見(jiàn)。流行病學(xué)及病因?qū)W研究表明,鼻咽癌的發(fā)病與遺傳因素、EB病毒感染和環(huán)境因素有關(guān)。鼻咽癌顯著的種族易感性和家族聚集現(xiàn)象表明,遺傳因素特別是瘤基因的激活和抑瘤基因的失活在鼻咽癌發(fā)病中具有十分重要的作用,因此尋找鼻咽癌遺傳易感基因是防治鼻咽癌的關(guān)鍵。 kank1基因是kank家族的一個(gè)重要成員,位于9p24.3區(qū)域。研究顯示,Kank1在腦、肺、腎腫瘤組織及相應(yīng)的細(xì)胞系中存在雜合性缺失(loss of heterozygosity, LOH)和啟動(dòng)子區(qū)甲基化,導(dǎo)致該基因表達(dá)下調(diào)/缺失,提示kankl基因可能是一個(gè)抑瘤基因。鼻咽癌組織比較基因組雜交(comparative genomic hybridization, CGH)的結(jié)果顯示,染色體9p存在高頻率LOH,隨后將高頻缺失定位在更精細(xì)的9p21.3-p24.3區(qū)域。這些研究結(jié)果高度提示,在9p21.3-p24.3可能存在與鼻咽癌相關(guān)的抑瘤基因。目前已發(fā)現(xiàn)該區(qū)的抑瘤基因有p16、NGX6和kank1等,而kank1基因與鼻咽癌的關(guān)系尚未見(jiàn)報(bào)的。因此,本研究開(kāi)展kank1基因在鼻咽癌中的表達(dá)情況、失活機(jī)制、功能及其臨床病理學(xué)意義研究。 1、kank1基因在鼻咽癌中的表達(dá)研究 采用RT-PCR、Real-time RT-PCR方法檢測(cè)kank1mRNA在正常鼻咽上皮細(xì)胞系NP69、4株鼻咽癌細(xì)胞系(CNE1、CNE2、5-8F、6-10B)、11例非癌組織和77例鼻咽癌組織中的表達(dá)。結(jié)果顯示,kank1在正常鼻咽細(xì)胞系NP69和幾乎全部非癌組織均有表達(dá),而在4株鼻咽癌細(xì)胞系和大于50%的鼻咽癌組織中表達(dá)下調(diào)或缺失。Western blotting結(jié)果顯示,kank1蛋白在60%(12/20)的鼻咽癌組織中表達(dá)下調(diào)。結(jié)果表明,鼻咽癌中kank1基因存在高頻率的表達(dá)下調(diào)/缺失。 2、kank1基因在鼻咽癌中表達(dá)下調(diào)的機(jī)制研究 由于kank1位于鼻咽癌的高頻缺失區(qū)域,而基因內(nèi)部及其附近微衛(wèi)星位點(diǎn)的丟失可以間接反映該基因的缺失情況。為此,我們采用PCR-變性聚丙烯酰胺凝膠電泳-銀染方法,分析kank1基因上下游的D9S1858和WI-12779二個(gè)微衛(wèi)星位點(diǎn)在鼻咽癌組織中的雜合性缺失情況。啟動(dòng)子區(qū)DNA異常甲基化是抑瘤基因表觀遺傳學(xué)改變最為常見(jiàn)的方式,生物信息學(xué)分析發(fā)現(xiàn)kank1啟動(dòng)子區(qū)存在CpG島。我們采用甲基化特異性PCR分析鼻咽癌組織kank1啟動(dòng)子甲基化情況。結(jié)果顯示,鼻咽癌組織D9S1858和WI-12779發(fā)生LOH的頻率分別為31.4%(11/35)和34.3%(12/35),kank1基因LOH的頻率為51.4%(18/35)；鼻咽癌細(xì)胞株和鼻咽癌組織中kank1基因全部存在甲基化,而正常組織中甲基化頻率為60%(6/10)。提示kank1等位基因LOH和啟動(dòng)子高甲基化可能是導(dǎo)致其在鼻咽癌中表達(dá)下調(diào)/缺失的重要原因。 3、鼻咽癌細(xì)胞kank1的功能研究 為了探討kank1在鼻咽癌發(fā)病中的作用,我們采用脂質(zhì)體轉(zhuǎn)染技術(shù)將kank1表達(dá)質(zhì)粒和空白質(zhì)粒轉(zhuǎn)染高成瘤高轉(zhuǎn)移5-8F鼻咽癌細(xì)胞,建立kank1高表達(dá)的5-8F鼻咽癌細(xì)胞系(5-8Fkank1)和對(duì)照細(xì)胞系(5-8Fvector)。隨后觀察kank1表達(dá)上調(diào)對(duì)5-8F細(xì)胞生物學(xué)特征的影響。MTT分析結(jié)果顯示,5-8Fkankl細(xì)胞的增殖速度明顯低于5-8Fvector細(xì)胞和未轉(zhuǎn)染細(xì)胞(p0.05)；軟瓊脂集落形成實(shí)驗(yàn)的結(jié)果顯示,5-8Fkank1細(xì)胞的克隆形成率明顯低于5-8Fvector細(xì)胞和未轉(zhuǎn)染細(xì)胞(p0.05)；流式細(xì)胞術(shù)分析的結(jié)果顯示,與5-8Fvector細(xì)胞和未轉(zhuǎn)染細(xì)胞相比,5-8Fkank1細(xì)胞Go/G1期細(xì)胞比例明顯增加(p0.05),S期和G2/M期細(xì)胞比例明顯減少(P0.05)；流式細(xì)胞術(shù)和Hoechst33258染色結(jié)果顯示,5-8Fkankl細(xì)胞的凋亡明顯高于5-8Fvector細(xì)胞和未轉(zhuǎn)染細(xì)胞(p0.05)；細(xì)胞體外遷移和侵襲實(shí)驗(yàn)顯示,與5-8Fvector細(xì)胞和未轉(zhuǎn)染細(xì)胞相比,5-8Fkankl細(xì)胞的運(yùn)動(dòng)能力和侵襲能力均明顯降低(p0.05)。研究結(jié)果提示,kank1具有抑制鼻咽癌細(xì)胞增殖、促進(jìn)鼻咽癌細(xì)胞凋亡以及抑制鼻咽癌細(xì)胞體外遷移和侵襲的作用。 4、kank1的臨床病理意義分析 為探討鼻咽癌kank1表達(dá)下調(diào)/缺失的臨床病理意義,我們采用免疫組化(IHC)染色檢測(cè)kank1在14例非癌組織和69例鼻咽癌組織中的表達(dá)。結(jié)果顯示,kank1在72.5%(50/69)的鼻咽癌組織中表達(dá)下調(diào),而在14例正常鼻咽粘膜上皮中只有1例表達(dá)下調(diào)。kank1表達(dá)下調(diào)/缺失與鼻咽癌淋巴結(jié)轉(zhuǎn)移和臨床分期負(fù)相關(guān),kank1表達(dá)下調(diào)/缺失的鼻咽癌更易發(fā)生淋巴結(jié)轉(zhuǎn)移、臨床分期更晚,而與腫瘤組織學(xué)類(lèi)型、原發(fā)灶大小、患者的性別和年齡無(wú)關(guān)。 本文研究結(jié)果表明,kank1基因在非癌組織中穩(wěn)定表達(dá),在鼻咽癌組織和細(xì)胞中表達(dá)明顯下調(diào)或缺失,LOH和啟動(dòng)子區(qū)甲基化可能是導(dǎo)致kank1基因表達(dá)下調(diào)的機(jī)制之一,通過(guò)在鼻咽癌細(xì)胞系中過(guò)表達(dá)kank1的表達(dá)發(fā)現(xiàn),kank1基因能夠抑制細(xì)胞的增殖、促進(jìn)細(xì)胞的凋亡、抑制細(xì)胞侵襲和遷移,kank1基因表達(dá)下調(diào)在鼻咽癌發(fā)病中具有重要作用。結(jié)果提示,kank1是候選的鼻咽癌抑瘤基因。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R739.63

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