DNA甲基化下调CLDN4表达对喉癌细胞增殖及侵袭能力的影响
发布时间：2018-07-15 14:29
【摘要】：细胞间连接是多细胞有机体中相邻细胞之间通过细胞质膜相互联系以及发挥协同作用的重要基础,在人类和动物体内主要存在四种形式,紧密连接是其中较为重要的一种。紧密连接是细胞/细胞间连接复合物的最顶端的组成成分,是液体和溶质通过细胞间隙的主要屏障,并在调节细胞增殖、分化和凋亡之间的平衡方面发挥关键作用,它主要由claudins(CLDNs)、闭合蛋白(occludin)、连接粘附分子(JAMs)等膜蛋白及紧密黏连蛋白(ZO-1、ZO-2和Z0-3)等胞浆蛋白组成,其中CLDNs是紧密连接最主要的成分,主要决定紧密连接的结构和功能。在肿瘤中CLDNs的表达经常发生改变,例如,CLDN1在食管癌中表达下调,CLDN 7在胰腺导管癌中表达下调,CLDN4在膀胱癌中表达和定位均发生改变。因此,推测CLDN s表达或定位的改变导致紧密连接结构和功能受到破坏,从而促进肿瘤的发生和转移。CLDN 4是CLDNs家族27个成员之一,同时也是产气荚膜梭菌肠毒素(CPE)C末端片段的受体。CLDN 4通过细胞外环能够与相邻细胞表面的CLDN 4相互作用,并为细胞旁扩散提供良好的屏障。有研究表明,CLDN 4在食管癌和胰腺癌中表达上调,在结肠癌和胃癌中表达下调。我们的前期工作中发现,与癌旁组织相比,喉癌组织中CLDN 4表达明显下调,甲基化特异性蛋白Me CP2表达明显上调,且两者的表达存在一定相关性;给予喉癌hep-2细胞去甲基化制剂5-aza-d C处理之后CLDN4表达上调,并且CLDN4表达上调可以抑制hep-2细胞的迁移及侵袭能力,那么5-aza-d C上调CLDN4表达是否是通过直接改变其DNA甲基化状态实现的,以及CLDN4是否具有调节喉癌hep-2细胞的迁移和侵袭等恶性表型的作用是本研究丞待解决的关键问题。目的:探讨喉癌hep-2细胞中CLDN4表达下调与DNA甲基化的关系以及CLDN4表达下调对喉癌hep-2细胞恶性表型的影响。方法:采用甲基化特异性PCR(methylation specific PCR,MSP)法检测5-aza-d C作用hep-2细胞对CLDN4基因DNA甲基化状态的影响;采用CCK8实验、划痕实验及Transwell实验分别检测RNAi技术沉默5-aza-d C作用组CLDN4基因对hep-2细胞增殖、迁移和侵袭能力的影响。结果:1、低表达CLDN4的hep-2细胞中检测到CLDN4基因的DNA甲基化,而5-aza-d C作用组hep-2细胞未检测到CLDN4基因的DNA甲基化;2、沉默5-aza-d C作用组的CLDN4基因后,hep-2细胞的增殖、迁移和侵袭能力均明显增强。结论:喉癌hep-2细胞中CLDN4表达下调与DNA甲基化有关;CLDN4表达下调可以促进喉癌hep-2细胞的恶性表型。
[Abstract]:Intercellular junction is an important basis for the interrelation and synergism of adjacent cells in multicellular organisms through the cytoplasmic membrane. There are four main forms in human and animal bodies, among which the close connection is one of the most important. Tight junctions are the top component of cellular / intercellular junction complexes, are the main barrier of liquid and solute passing through the intercellular space, and play a key role in regulating the balance between cell proliferation, differentiation and apoptosis. It is mainly composed of membrane proteins such as claudins (CLDNs), (occludin), binding adhesion molecules (jams) and cytosolic proteins such as tight adhesion proteins (ZO-1, ZO-2 and Z0-3), among which CLDNs are the most important components, which mainly determine the structure and function of tight junctions. The expression of CLDNs was frequently changed in tumors, such as the down-regulated expression of CLDN7 in esophageal carcinoma and the change of the expression and localization of CLDN4 in pancreatic ductal carcinoma. Therefore, it is assumed that changes in the expression or localization of CLDN s lead to the destruction of the tight junction structure and function, thus promoting the occurrence and metastasis of tumors. CLDN4 is one of the 27 members of the CLDNs family. At the same time, CLDN4, the receptor of the C-terminal fragment of Clostridium perfringens enterotoxin (CPE), can interact with CLDN4 on the surface of adjacent cells through the outer loop of the cell, and provide a good barrier for cell diffusion. Studies have shown that CLDN 4 is up-regulated in esophageal and pancreatic cancer, and down-regulated in colon and gastric cancer. In our previous work, we found that the expression of CLDN4 was down-regulated and the expression of methylation-specific protein me CP2 was significantly up-regulated in laryngeal carcinoma tissues compared with paracancerous tissues, and there was a certain correlation between the expression of CLDN4 and Me CP2. The expression of CLDN4 was upregulated after hep-2 cells were treated with 5-aza-d C, and the up-regulation of CLDN4 could inhibit the migration and invasion of hep-2 cells. So, whether the up-regulation of CLDN4 expression by 5-aza-d C was achieved by directly changing the methylation state of hep-2 cells? Whether CLDN4 can regulate the migration and invasion of hep-2 cells in laryngeal carcinoma is the key problem to be solved in this study. Aim: to investigate the relationship between the down-regulation of CLDN4 expression and methylation in laryngeal carcinoma hep-2 cells and the effect of the down-regulation of CLDN4 expression on the malignant phenotype of hep-2 cells. Methods: the effect of 5-aza-d C on the methylation status of hep-2 cells was detected by methylation specific (methylation specific PCR, and the proliferation of hep-2 cells was detected by CCK8 test, scratch test and Transwell test respectively. The effect of migration and invasiveness. Results DNA methylation of CLDN4 gene was detected in hep-2 cells with low expression of CLDN4, but no methylation of CLDN4 gene was detected in hep-2 cells treated with 5-aza-d C. Migration and invasion were significantly enhanced. Conclusion: the down-regulation of CLDN4 expression in laryngeal carcinoma hep-2 cells may promote the malignant phenotype of hep-2 cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65
本文编号：2124383
[Abstract]:Intercellular junction is an important basis for the interrelation and synergism of adjacent cells in multicellular organisms through the cytoplasmic membrane. There are four main forms in human and animal bodies, among which the close connection is one of the most important. Tight junctions are the top component of cellular / intercellular junction complexes, are the main barrier of liquid and solute passing through the intercellular space, and play a key role in regulating the balance between cell proliferation, differentiation and apoptosis. It is mainly composed of membrane proteins such as claudins (CLDNs), (occludin), binding adhesion molecules (jams) and cytosolic proteins such as tight adhesion proteins (ZO-1, ZO-2 and Z0-3), among which CLDNs are the most important components, which mainly determine the structure and function of tight junctions. The expression of CLDNs was frequently changed in tumors, such as the down-regulated expression of CLDN7 in esophageal carcinoma and the change of the expression and localization of CLDN4 in pancreatic ductal carcinoma. Therefore, it is assumed that changes in the expression or localization of CLDN s lead to the destruction of the tight junction structure and function, thus promoting the occurrence and metastasis of tumors. CLDN4 is one of the 27 members of the CLDNs family. At the same time, CLDN4, the receptor of the C-terminal fragment of Clostridium perfringens enterotoxin (CPE), can interact with CLDN4 on the surface of adjacent cells through the outer loop of the cell, and provide a good barrier for cell diffusion. Studies have shown that CLDN 4 is up-regulated in esophageal and pancreatic cancer, and down-regulated in colon and gastric cancer. In our previous work, we found that the expression of CLDN4 was down-regulated and the expression of methylation-specific protein me CP2 was significantly up-regulated in laryngeal carcinoma tissues compared with paracancerous tissues, and there was a certain correlation between the expression of CLDN4 and Me CP2. The expression of CLDN4 was upregulated after hep-2 cells were treated with 5-aza-d C, and the up-regulation of CLDN4 could inhibit the migration and invasion of hep-2 cells. So, whether the up-regulation of CLDN4 expression by 5-aza-d C was achieved by directly changing the methylation state of hep-2 cells? Whether CLDN4 can regulate the migration and invasion of hep-2 cells in laryngeal carcinoma is the key problem to be solved in this study. Aim: to investigate the relationship between the down-regulation of CLDN4 expression and methylation in laryngeal carcinoma hep-2 cells and the effect of the down-regulation of CLDN4 expression on the malignant phenotype of hep-2 cells. Methods: the effect of 5-aza-d C on the methylation status of hep-2 cells was detected by methylation specific (methylation specific PCR, and the proliferation of hep-2 cells was detected by CCK8 test, scratch test and Transwell test respectively. The effect of migration and invasiveness. Results DNA methylation of CLDN4 gene was detected in hep-2 cells with low expression of CLDN4, but no methylation of CLDN4 gene was detected in hep-2 cells treated with 5-aza-d C. Migration and invasion were significantly enhanced. Conclusion: the down-regulation of CLDN4 expression in laryngeal carcinoma hep-2 cells may promote the malignant phenotype of hep-2 cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65
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相关硕士学位论文 前2条
1 汪晓飞;CLDN4在PRRSV感染Marc-145细胞中的作用[D];山东农业大学;2015年
2 付渴心;DNA甲基化下调CLDN4表达对喉癌细胞增殖及侵袭能力的影响[D];吉林大学;2017年
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