LSD1介导Wnt通路促进胃癌细胞侵袭、迁移和EMT的机制研究

发布时间：2018-06-09 13:38

本文选题：LSD1 + Wnt3a　；参考：《郑州大学》2017年硕士论文

【摘要】：组蛋白特异性赖氨酸去甲基化酶1(Histone Lysine Specific Demethylase 1,简称LSD1)是由施洋教授课题组于2004年发现的首个具有黄素腺嘌呤二核苷酸(Flavin Adenine Dinucleotide,简称FAD)依赖的去甲基化酶。Wnt3a是一种分泌性蛋白,是由细胞分泌后可与细胞膜上的Wnt配体结合并激活Wnt信号通路的重要细胞因子。本论文通过分析胃癌组织病理切片中LSD1与Wnt3a的相关性,研究LSD1对Wnt经典信号通路的影响。1)胃癌组织病理切片中LSD1与Wnt3a表达水平的相关性研究已有文献报道,在乳腺癌、肺癌、前列腺癌、神经母细胞瘤等多种恶性肿瘤细胞中LSD1呈高表达状态。本课题组在前期的研究中发现,LSD1在胃癌细胞HGC-27和MGC-803中的表达量远远高于正常胃粘膜上皮细胞GES-1。另外,前期探索Wnt经典通路时发现,在MGC-803细胞中,改变LSD1的表达量能够通过调控Wnt通路关键蛋白Wnt3a的胞外分泌量而影响Wnt经典通路。因此,本课题进一步探索在胃癌病人癌组织中LSD1与Wnt3a表达量的相关性。首先选取收集到的94例胃癌病人的癌变组织和癌旁组织,并将其制作成病理组织切片,然后分别利用LSD1和Wnt3a的特异性抗体进行LSD1和Wnt3a的免疫组化染色,检测LSD1和Wnt3a在癌变组织和癌旁组织中的表达量,最后通过Spearman检验对二者进行相关性分析。结果显示,在胃癌病人组织中,LSD1表达量与Wnt3a表达量呈显著正相关。2)胃癌细胞中LSD1影响Wnt3a分泌的相关机制研究前面章节的研究发现在胃癌病人组织中LSD1和Wnt3a的表达呈现显著正相关,在此基础上,本章节将进一步在胃癌细胞和胃粘膜上皮细胞中研究LSD1与Wnt3a之间的相互调控机制。首先利用转染试剂脂质体3000将构建好的低表达质粒pLVX-shRNA-LSD1转染入胃癌细胞MGC-803、HGC-27、SGC-7901和胃粘膜上皮细胞GES-1中,然后利用Elisa实验检测细胞培养液中Wnt3a的蛋白表达量,结果显示,在胃癌细胞HGC-27、MGC-803和SGC-7901中,下调LSD1的表达量后Wnt3a的胞外分泌量均下调,而在胃粘膜上皮细胞GES-1中LSD1的表达下调后Wnt3a的胞外分泌量不发生明显变化。与此同时,提取细胞的RNA进行反转录后,利用qPCR实验检测PORCN蛋白的mRNA表达水平,结果显示,在胃癌细胞HGC-27,MGC-803和SGC-7901中,下调LSD1的表达量后PORCN蛋白mRNA的表达受到抑制,而在胃粘膜上皮细胞GES-1中下调LSD1的表达后,PORCN蛋白mRNA的表达不发生明显变化。说明在胃癌细胞中LSD1正向调控PORCN的转录从而影响Wnt3a的分泌。3)LSD1通过Wnt通路影响胃癌细胞的侵袭和转移本课题组的前期研究发现,在胃癌细胞中,LSD1表达量上调后能够促进细胞的侵袭和迁移,同时能够促进细胞的EMT过程.。文献调研发现,Wnt信号通路能够促进肿瘤细胞的增殖、迁移和分化。本章节进一步研究LSD1对胃癌细胞侵袭、迁移能力和EMT过程的调节是否是部分依赖于Wnt信号通路。利用慢病毒包装的LSD1过表达载体感染MGC-803和SGC-7901细胞系,使该细胞系中LSD1的表达量升高,在确认LSD1的过表达效果后,在过表达细胞中加入PORCN抑制剂WntC59,利用划痕实验和Transwell实验检测细胞侵袭和迁移能力的变化,结果发现:在两株细胞中,LSD1表达量的上调能够促进该细胞的迁移和侵袭能力,且在上调LSD1表达量之后加入WntC59,这种促进作用将受到一定程度的抑制;通过Western Blot实验检测EMT标志蛋白E-Cadherin和N-Cadherin的表达量,结果显示在MGC-803细胞和SGC-7901细胞中,LSD1的表达量上调后,E-cadherin的表达显著降低,N-cadherin的表达明显升高,而当上调LSD1表达量的同时加入Wnt C59,E-cadherin的表达量降低的并不明显,N-cadherin的表达量升高的也不明显。因此我们认为LSD1对胃癌细胞的迁移,侵袭和EMT过程的影响很可能是通过Wnt信号通路实现的。
[Abstract]:Histone specific lysine demethylation enzyme 1 (Histone Lysine Specific Demethylase 1, abbreviated as LSD1) is the first secretory protein that is found by Professor Shiyang in 2004 with the dependence of Flavin Adenine Dinucleotide (Flavin Adenine Dinucleotide, abbreviated FAD) as a secretory protein, which is secreted by cells and may be secreted by cells. Wnt ligands on the cell membrane bind and activate important cytokines in the Wnt signaling pathway. In this paper, the correlation between LSD1 and Wnt3a in the pathological sections of gastric carcinoma was analyzed, and the influence of LSD1 on the classical signaling pathway of Wnt.1) the correlation of LSD1 to Wnt3a expression in the pathological sections of gastric cancer was reported in the literature, in breast cancer and lung The expression of LSD1 in cancer, prostate cancer, neuroblastoma and other malignant tumor cells is highly expressed. In our previous study, we found that the expression of LSD1 in HGC-27 and MGC-803 of gastric cancer cells was much higher than that of GES-1. in normal gastric mucosa epithelial cells. In the early exploration of the classical Wnt pathway, the expression of LSD1 was found in MGC-803 cells, and LSD1 was changed in MGC-803 cells. The expression can affect the Wnt classical pathway by regulating the exocytosis of the key protein Wnt3a of the Wnt pathway. Therefore, we further explore the correlation between the expression of LSD1 and Wnt3a in cancer tissues of patients with gastric cancer. First, select the cancerous tissues and para cancerous tissues of 94 cases of gastric cancer, and make them into pathological sections. The expression of LSD1 and Wnt3a in cancerous tissues and para cancerous tissues was detected by using specific antibodies of LSD1 and Wnt3a to detect the expression of LSD1 and Wnt3a in cancerous tissues and adjacent tissues. Finally, the correlation analysis was carried out by Spearman test on the two groups. The results showed that the expression of LSD1 and Wnt3a expressed a significant positive correlation in the tissues of the patients with gastric cancer. The results showed that the expression of LSD1 and Wnt3a expressed a positive correlation in the tissues of the patients with gastric cancer. Research on the effect of LSD1 on the secretion of Wnt3a in gastric cancer cells research in the previous chapter found that the expression of LSD1 and Wnt3a in gastric cancer tissues has a significant positive correlation. On this basis, this chapter will further study the mutual regulation mechanism between LSD1 and Wnt3a in gastric cancer cells and gastric epithelial cells. First, the transfection test will be used. The agent liposome 3000 transfected the constructed low expression plasmid pLVX-shRNA-LSD1 into gastric cancer cells MGC-803, HGC-27, SGC-7901 and gastric mucosal epithelial cells GES-1. Then the Elisa test was used to detect the protein expression of Wnt3a in the cell culture fluid. The results showed that the Wnt3a was in the gastric cancer cell HGC-27, MGC-803 and SGC-7901, down LSD1 expression. The exocrine volume was down, and the exocrine quantity of Wnt3a was not significantly changed after the expression of LSD1 in the gastric epithelial cell GES-1 was down. Meanwhile, after the RNA of the extracted cells was reverse transcribed, the mRNA expression of PORCN protein was detected by qPCR test. The results showed that the decrease of LSD1 in HGC-27, MGC-803 and SGC-7901 in gastric cancer cells was reduced. The expression of PORCN protein mRNA was inhibited, while the expression of LSD1 was down regulated in GES-1 of gastric mucosa epithelial cells, and the expression of PORCN protein mRNA did not change obviously. It indicated that LSD1 was regulating the transcription of PORCN in gastric cancer cells and thus affecting Wnt3a secreting.3) LSD1 through Wnt pathway to influence the invasion and metastasis of gastric cancer cells. The earlier study of the group found that in gastric cancer cells, the up-regulated expression of LSD1 can promote cell invasion and migration and promote cell EMT process. The literature survey found that Wnt signaling pathway can promote the proliferation, migration and differentiation of tumor cells. This chapter studies the invasion, migration ability and EMT of LSD1 in gastric cancer cells. Whether the regulation of the process is partly dependent on the Wnt signaling pathway. Using the LSD1 overexpressed vector packed with lentivirus to infect MGC-803 and SGC-7901 cell lines, the expression of LSD1 in the cell line is increased. After the overexpression of LSD1 is confirmed, the PORCN inhibitor WntC59 is added to the overexpressed cells, and the scratch test and Transwell test are used. The changes in cell invasion and migration ability showed that up regulation of LSD1 expression in two cells promoted the migration and invasion of the cell, and added WntC59 after the up regulation of LSD1 expression, which would be inhibited to a certain extent, and EMT marker protein E-Cadherin and N-Cadherin were detected by Western Blot test. The results showed that in MGC-803 and SGC-7901 cells, after the expression of LSD1 was up-regulated, the expression of E-cadherin decreased significantly, and the expression of N-cadherin increased obviously, while Wnt C59 was added to the LSD1 expression while the expression of E-cadherin decreased not obviously, and the expression of N-cadherin was not obvious. Therefore, the expression of N-cadherin was not obvious. It is believed that the effects of LSD1 on migration, invasion and EMT process of gastric cancer cells may be achieved through Wnt signaling pathway.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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