【作者】 张婷；

【导师】 郑一超；

【作者基本信息】 郑州大学 ， 药物化学， 2019， 硕士

【摘要】 LSD1(Histone lysine specific demethylase 1)作为第一个被发现的组蛋白去甲基化酶可利用黄素腺嘌呤二核苷酸(FAD,Flavin adenine dinucleotide)为辅因子,特异性地去除组蛋白H3K4和H3K9的单、双甲基。Wnt通路可调控细胞增殖、转移和死亡,越来越多的证据表明,在人类癌症中遗传或者表观遗传缺陷常常引起Wnt/β-catenin通路的调节因子表达失调。目前已有多篇文章刊登了LSD1与Wnt/β-catenin通路中某些关键蛋白共同调节EMT相关蛋白表达,进而促进乳腺癌和结肠癌的增殖。课题组前期发现LSD1被抑制后可下调男性胃癌细胞MGC-803培养基中的Wnt3a,但是女性胃癌细胞的Wnt3a分泌不受影响。因此,本文旨在研究LSD1是否以性别特异性方式调节Wnt3a分泌进而调控胃癌细胞转移。1)Wnt3a和LSD1、EMT相关蛋白在胃癌组织中表达量的相关性分析本课题对269例胃癌组织进行免疫组化实验检测Wnt3a和LSD1以及EMT标志蛋白(包括Snail、N-Cadherin和Vimentin)的表达情况。分析结果显示在男性来源或者具有淋巴转移患者的胃癌组织中Wnt3a和LSD1蛋白表达具有较为明显的相关性;然而在女性来源患者或者未发生转移患者的胃癌组织中,Wnt3a和LSD1蛋白表达没有显著相关性。基于以上发现,我们推测在男性来源的胃癌细胞中Wnt3a和LSD1可相互调节。为了进一步探究性别对Wnt3a和其他蛋白之间相关性的影响,我们又将数据按性别分为两组,对Wnt3a、LSD1以及EMT标志蛋白进行相关线性分析。数据显示,在女性来源胃癌组织中Wnt3a分别与LSD1、Snail、N-Cadherin之间没有显著相关性,表明Wnt3a可能并不影响女性来源胃癌患者的EMT进程。然而,在男性来源胃癌组织中,LSD1、Snail、N-Cadherin和Vimentin的表达量都与Wnt3a为正相关。据报道在胃癌中LSD1过表达,所以我们接着研究在LSD1低表达和高表达的胃癌组织中Wnt3a与其他蛋白的相关性。Pearson相关性分析结果显示,男性来源且LSD1高表达胃癌组织中,Wnt3a与LSD1、Snail、N-Cadherin以及Vimentin有更高的相关系数。因此,我们认为在男性来源且LSD1高表达的胃癌细胞中,LSD1可能以性别特异性方式与Wnt3a相互作用进而诱导EMT进程。2)胃癌细胞中Wnt3a介导LSD1调控Wnt/β-catenin通路的机制研究本课题上述研究工作显示男性来源且LSD1高表达的胃癌组织中,Wnt3a与LSD1的表达量呈现显著正相关性,因此我们认为两者之间可能存在相互作用,在此基础上,本章节将进一步研究在胃癌细胞系和胃粘膜上皮细胞中LSD1对Wnt3a是否存在调控以及LSD1对Wnt/β-catenin通路的影响,以期探究这种调控是否只发生于男性来源且LSD1高表达的胃癌细胞。通过Western blot、Elisa和RT-qPCR实验,我们发现只有在MGC-803细胞中观察到LSD1敲低后可下调Wnt3a的分泌但不影响mRNA水平,但是在MKN45、SGC-7901以及GES-1细胞中没有得到相似的结果;同样地,只有在MGC-803细胞中LSD1过表达可上调Wnt3a分泌。随后对涉及Wnt/β-catenin通路激活的蛋白以及靶基因的检测结果表明,只有在MGC-803细胞中,LSD1敲低引起PORCN下调,造成Wnt3a分泌减少,下调Wnt/β-catenin通路中的关键蛋白β-catenin入核以及EMT的转录因子Snail入核,而在MKN45细胞中无此结果。RT-qPCR的检测结果表明,在MGC-803细胞中,LSD1敲低后Wnt/β-catenin通路靶基因c-Myc和CyclinD1的mRNA也降低,而MKN45细胞中c-Myc和CyclinD1的mRNA同样没有检测到显著变化。说明在MGC-803细胞中,LSD1敲低可失活Wnt/β-catenin通路并阻止Snail入核。体内MGC-803皮下移植瘤免疫组化进一步证实,MGC-803中LSD1的敲低下调Wnt3a和β-catenin表达,上调P-β-catenin表达,说明在体内环境下,MGC-803中LSD1的敲低也可以下调Wnt/β-catenin通路。以上所有结果说明了在男性来源胃癌细胞MGC-803中LSD1可能通过PORCN调控Wnt3a的分泌进而激活Wnt/β-catenin通路。因此,我们推测LSD1通过调节Wnt/β-catenin通路进一步影响EMT进程。3)LSD1对Wnt/β-catenin通路介导MGC-803侵袭和转移的调控能力研究目前已有多篇报道表明LSD1参与细胞转移和侵袭,而LSD1活性缺失可逆转EMT,进而抑制细胞转移和侵袭。同时,在多种癌症中Wnt3a可诱导EMT并参与疾病的发生。因此,本章节进一步探索Wnt3a是否介导LSD1促进胃癌细胞侵袭及转移。通过Elisa、Western blot、划痕实验和transwell实验可以发现在MGC-803中,LSD1过表达促进Wnt3a分泌、转移侵袭以及EMT。然而PORCN抑制剂LGK974通过抑制Wnt3a的棕榈酰化进而抑制Wnt3a分泌,同时也在一定程度上阻止LSD1过表达诱导的转移、侵袭以及EMT。因此,我们认为在MGC-803中,阻断Wnt3a信号通路可抑制LSD1介导的细胞转移、侵袭和EMT,Wnt3a在此过程中起到非常重要的角色。为了进一步验证这一结论,我们建立了MGC-803 Control和MGC-803 LSD1-KD细胞全身转移小鼠模型,经过7周观察我们发现LSD1敲低可以在一定程度上抑制MGC-803在小鼠体内的转移,并延长小鼠生存期。HE染色进一步验证了LSD1促进了MGC-803在小鼠体内的肺转移和肝转移。随后的免疫组化实验显示在体内LSD1低表达可下调Wnt3a、β-catenin以及间质细胞标志蛋白N-Cadherin、Vimentin和Snail的表达,从而证实LSD1通过调节Wnt/β-catenin通路促进男性胃癌细胞MGC-803的转移和EMT。综上所述,只有在男性胃癌细胞中LSD1能够通过调控PORCN进而促进Wnt3a分泌,激活Wnt/β-catenin通路上调Wnt通路靶基因c-Myc、CyclinD1和Snail表达,进而促进胃癌细胞转移和EMT。本课题探索了LSD1以性别特异性方式通过调节Wnt/β-catenin通路促进胃癌细胞转移的机制,为胃癌个性化治疗提供了新思路。更多还原

【Abstract】 As the first identified histone lysine demethylase,lysine specific demethylase 1(LSD1)utilizes the flavin adenine dinucleotide(FAD)as a cofactor to specifically demethylates di-and monomethylated histones H3K4 and H3K9.Wnt signaling pathway regulates many cell functions,including proliferation,migration and cell death.And an increasing body of evidence indicates that regulators controlling Wnt/β-catenin pathway are frequently dysregulated in human cancers owing to genetic and epigenetic defects.Until now,several articles have published the cooperation between LSD1 and some key proteins in Wnt/β-catenin pathway to promote EMT,cell proliferation in breast cancer,colon cancer.In the previous study,it was found that abrogation of LSD1 inhibited Wnt3 a secretion in male derived gastric cancer cell MGC-803,while it was not affected in female derived gastric cancer cells.Therefore,the aim of this study is to investigate whether Wnt3 a mediated LSD1 induced gastric cancer cell metastasis in a gender specific manner.1)The correlation analysis between Wnt3 a and LSD1,EMT related proteins in gastric cancer tissuesIn this study,the expression of Wnt3 a,LSD1 and EMT marker proteins(including Snail,N-Cadherin and Vimentin)were detected in 269 gastric cancer tissues by immunohistochemical staining.The correlation analysis results showed that the expression of Wnt3 a was strong significantly correlated with LSD1 expression in male and metastasis specimens while there was no significant correlation between the expression of LSD1 and Wnt3 a in female or in non-metastasis patients.Based on the above findings,we hypothesized that Wnt3 a and LSD1 may affect each other’s expression.To further explore the effect of gender,269 specimens were subject to immunohistochemical analysis to clarify the expression relationship among Wnt3 a,LSD1 and EMT markers in female and male derived gastric cancer tissue specimens.As indicated in results,in female derived gastric cancer tissue specimens,the correlationship between Wnt3 a and LSD1,Snail,N-Cadherin are not significant,suggesting that Wnt3 a may not impact on EMT in female derived gastric cancer cells.On the other hand,all these proteins were positively correlated with Wnt3 a in male derived gastric cancer tissue specimens.As LSD1 was reported to be overexpressed in gastric cancer,LSD1 lowexpressed and overexpressed gastric cancer tissues were selected and subjected to correlationship analysis between Wnt3 a and others.As shown in results,in male derived LSD1 overexpressed gastric cancer tissues,Wnt3 a performed stronger correlationship with LSD1,Snail,N-Cadherin and Vimentin.Therefore,we deem that LSD1 may interregulate with Wnt3 a and then contribute to EMT in male derived LSD1 overexpressed gastric cancer cells in a gender specific manner.2)Wnt3a-mediated LSD1 regulates Wnt/β-catenin pathway in gastric cancer cellsThe aforesaid research work of this subject showed that there was a significant positive correlation between the expression of Wnt3 a and LSD1 in male derived LSD1 overexpressed gastric cancer tissues.Therefore,we believe that there may be the impact of LSD1 on Wnt3 a.In this chapter,we will further investigate the role of LSD1 in the regulation of Wnt3 a and the effect of LSD1 on Wnt/β-catenin pathway in gastric cancer cell lines and gastric mucosal epithelial cells,in order to investigate whether this regulation occurs only in male derived LSD1 overexpressed gastric cancer cells.By Western blot,Elisa and RT-qPCR experiments,we found that when LSD1 was knocked down,Wnt3 a protein secretion in the cell culture medium were significantly decreased in MGC-803 cells,but not in MKN45,SGC-7901 and GES-1 cells while no significant change in Wnt3 a mRNA can be detected when LSD1 was knocked down in these cells.Similarly,amount of Wnt3 a was greatly increased when LSD1 was overexpressed in MGC-803 cells.Subsequent detection of proteins involved in Wnt/β-catenin pathway activation revealed that LSD1 knockdown decreased expression of PORCN and secretion of Wnt3 a as well as nucleus translocation of β-catenin and Snail in MGC-803 cells but not in MKN45 cells.The results of RT-qPCR showed that the mRNA level of Wnt/β-catenin pathway targets c-Myc and CyclinD1 were also decreased in MGC-803 cells,but not in MKN45 cells.These suggested that LSD1 knockdown inactivate the Wnt/β-catenin pathway and prevent nucleus translocation of Snail in MGC-803 cells.MGC-803 cells subcutaneous xenografts detected by immunohistochemical staining showed that deficiency of LSD1 led to low expression of Wnt3 a and β-catenin but up-regulated P-β-catenin in MGC-803 cells,suggesting that knockdown of LSD1 can also down-regulate the Wnt/β-catenin pathway in vivo.All of the above results indicated that LSD1 may regulate Wnt3 a secretion via PORCN and then activate Wnt/β-catenin pathway in male derived gastric cancer cell line MGC-803 cells.Therefore,we speculate that LSD1 may make further effect on EMT by Wnt/β-catenin pathway.3)The ability of LSD1 to mediate invasion and metastasis of MGC-803 by Wnt/β-catenin pathwayAt present,there are many reports that LSD1 is involved in cancer cells metastasis and invasion,the activity loss of LSD1 leads to the reversal of EMT process and then inhibits cells metastasis and invasion.Meanwhile,Wnt3 a is as an inducer of EMT to participate in the occurrence of diseases in a variety of cancers.Therefore,whether Wnt3a-mediated LSD1 promotes invasion and metastasis of gastric cancer cells was further explored in this chapter.By Elisa,Western blot,wound healing and transwell experiment,it was found that LSD1 overexpression promoted Wnt3 a secretion,metastasis and invasion as well as EMT in MGC-803.However,the PORCN inhibitor LGK974 disputes the secretion of Wnt3 a by preventing palmitoylation of Wnt3 a,and also prevents the accelerated invasion and EMT by LSD1 overexpression.Therefore,we believe that blockade of Wnt3 a signaling pathway can inhibit LSD1-induced cell metastasis and EMT in MGC-803,so Wnt3 a plays a very important role in this process.To further validate this conclusion,we established a mouse model of MGC-803 Control and MGC-803 LSD1-KD systemic transfer.After 7 weeks of observation,we found that LSD1 knockdown can inhibit the migration of MGC-803 in mice to some extent,meanwhile,prolong the survival time of the mouse.HE staining experiments further confirmed that LSD1 promoted lung metastasis and liver metastasis of MGC-803 in mice.Subsequent immunohistochemical staining experiments showed that LSD1 lowexpression reduced the expression of Wnt3 a,β-catenin,and the mesenchymal marker proteins,including N-Cadherin,Vimentin,and Snail.The above results suggested that LSD1 promotes metastasis and invasion of MGC-803 by the Wnt/β-catenin pathway.In summary,LSD1 may promote the secretion of Wnt3 a by regulating PORCN,and then activate Wnt/β-catenin pathway,subsequently upregulate Wnt pathway target genes c-Myc,CyclinD1 and Snail,thereby promoting the metastasis and EMT of male derived gastric cancer cell lines.This study explored the mechanism of Wnt3a-mediated LSD1 promoting gastric cancer cells metastasis by Wnt/β-catenin pathway in a gender specific manner,presented a new idea for personalized treatment of gastric cancer.更多还原