發(fā)布時(shí)間：2018-06-09 13:38

  本文選題：LSD1 + Wnt3a　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：組蛋白特異性賴(lài)氨酸去甲基化酶1(Histone Lysine Specific Demethylase 1,簡(jiǎn)稱(chēng)LSD1)是由施洋教授課題組于2004年發(fā)現(xiàn)的首個(gè)具有黃素腺嘌呤二核苷酸(Flavin Adenine Dinucleotide,簡(jiǎn)稱(chēng)FAD)依賴(lài)的去甲基化酶。Wnt3a是一種分泌性蛋白,是由細(xì)胞分泌后可與細(xì)胞膜上的Wnt配體結(jié)合并激活Wnt信號(hào)通路的重要細(xì)胞因子。本論文通過(guò)分析胃癌組織病理切片中LSD1與Wnt3a的相關(guān)性,研究LSD1對(duì)Wnt經(jīng)典信號(hào)通路的影響。1)胃癌組織病理切片中LSD1與Wnt3a表達(dá)水平的相關(guān)性研究已有文獻(xiàn)報(bào)道,在乳腺癌、肺癌、前列腺癌、神經(jīng)母細(xì)胞瘤等多種惡性腫瘤細(xì)胞中LSD1呈高表達(dá)狀態(tài)。本課題組在前期的研究中發(fā)現(xiàn),LSD1在胃癌細(xì)胞HGC-27和MGC-803中的表達(dá)量遠(yuǎn)遠(yuǎn)高于正常胃粘膜上皮細(xì)胞GES-1。另外,前期探索Wnt經(jīng)典通路時(shí)發(fā)現(xiàn),在MGC-803細(xì)胞中,改變LSD1的表達(dá)量能夠通過(guò)調(diào)控Wnt通路關(guān)鍵蛋白Wnt3a的胞外分泌量而影響Wnt經(jīng)典通路。因此,本課題進(jìn)一步探索在胃癌病人癌組織中LSD1與Wnt3a表達(dá)量的相關(guān)性。首先選取收集到的94例胃癌病人的癌變組織和癌旁組織,并將其制作成病理組織切片,然后分別利用LSD1和Wnt3a的特異性抗體進(jìn)行LSD1和Wnt3a的免疫組化染色,檢測(cè)LSD1和Wnt3a在癌變組織和癌旁組織中的表達(dá)量,最后通過(guò)Spearman檢驗(yàn)對(duì)二者進(jìn)行相關(guān)性分析。結(jié)果顯示,在胃癌病人組織中,LSD1表達(dá)量與Wnt3a表達(dá)量呈顯著正相關(guān)。2)胃癌細(xì)胞中LSD1影響Wnt3a分泌的相關(guān)機(jī)制研究前面章節(jié)的研究發(fā)現(xiàn)在胃癌病人組織中LSD1和Wnt3a的表達(dá)呈現(xiàn)顯著正相關(guān),在此基礎(chǔ)上,本章節(jié)將進(jìn)一步在胃癌細(xì)胞和胃粘膜上皮細(xì)胞中研究LSD1與Wnt3a之間的相互調(diào)控機(jī)制。首先利用轉(zhuǎn)染試劑脂質(zhì)體3000將構(gòu)建好的低表達(dá)質(zhì)粒pLVX-shRNA-LSD1轉(zhuǎn)染入胃癌細(xì)胞MGC-803、HGC-27、SGC-7901和胃粘膜上皮細(xì)胞GES-1中,然后利用Elisa實(shí)驗(yàn)檢測(cè)細(xì)胞培養(yǎng)液中Wnt3a的蛋白表達(dá)量,結(jié)果顯示,在胃癌細(xì)胞HGC-27、MGC-803和SGC-7901中,下調(diào)LSD1的表達(dá)量后Wnt3a的胞外分泌量均下調(diào),而在胃粘膜上皮細(xì)胞GES-1中LSD1的表達(dá)下調(diào)后Wnt3a的胞外分泌量不發(fā)生明顯變化。與此同時(shí),提取細(xì)胞的RNA進(jìn)行反轉(zhuǎn)錄后,利用qPCR實(shí)驗(yàn)檢測(cè)PORCN蛋白的mRNA表達(dá)水平,結(jié)果顯示,在胃癌細(xì)胞HGC-27,MGC-803和SGC-7901中,下調(diào)LSD1的表達(dá)量后PORCN蛋白mRNA的表達(dá)受到抑制,而在胃粘膜上皮細(xì)胞GES-1中下調(diào)LSD1的表達(dá)后,PORCN蛋白mRNA的表達(dá)不發(fā)生明顯變化。說(shuō)明在胃癌細(xì)胞中LSD1正向調(diào)控PORCN的轉(zhuǎn)錄從而影響Wnt3a的分泌。3)LSD1通過(guò)Wnt通路影響胃癌細(xì)胞的侵襲和轉(zhuǎn)移本課題組的前期研究發(fā)現(xiàn),在胃癌細(xì)胞中,LSD1表達(dá)量上調(diào)后能夠促進(jìn)細(xì)胞的侵襲和遷移,同時(shí)能夠促進(jìn)細(xì)胞的EMT過(guò)程.。文獻(xiàn)調(diào)研發(fā)現(xiàn),Wnt信號(hào)通路能夠促進(jìn)腫瘤細(xì)胞的增殖、遷移和分化。本章節(jié)進(jìn)一步研究LSD1對(duì)胃癌細(xì)胞侵襲、遷移能力和EMT過(guò)程的調(diào)節(jié)是否是部分依賴(lài)于Wnt信號(hào)通路。利用慢病毒包裝的LSD1過(guò)表達(dá)載體感染MGC-803和SGC-7901細(xì)胞系,使該細(xì)胞系中LSD1的表達(dá)量升高,在確認(rèn)LSD1的過(guò)表達(dá)效果后,在過(guò)表達(dá)細(xì)胞中加入PORCN抑制劑WntC59,利用劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲和遷移能力的變化,結(jié)果發(fā)現(xiàn):在兩株細(xì)胞中,LSD1表達(dá)量的上調(diào)能夠促進(jìn)該細(xì)胞的遷移和侵襲能力,且在上調(diào)LSD1表達(dá)量之后加入WntC59,這種促進(jìn)作用將受到一定程度的抑制;通過(guò)Western Blot實(shí)驗(yàn)檢測(cè)EMT標(biāo)志蛋白E-Cadherin和N-Cadherin的表達(dá)量,結(jié)果顯示在MGC-803細(xì)胞和SGC-7901細(xì)胞中,LSD1的表達(dá)量上調(diào)后,E-cadherin的表達(dá)顯著降低,N-cadherin的表達(dá)明顯升高,而當(dāng)上調(diào)LSD1表達(dá)量的同時(shí)加入Wnt C59,E-cadherin的表達(dá)量降低的并不明顯,N-cadherin的表達(dá)量升高的也不明顯。因此我們認(rèn)為L(zhǎng)SD1對(duì)胃癌細(xì)胞的遷移,侵襲和EMT過(guò)程的影響很可能是通過(guò)Wnt信號(hào)通路實(shí)現(xiàn)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.2

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