發(fā)布時(shí)間：2018-05-07 23:14

  本文選題：喉鱗狀細(xì)胞癌 + Artemin　； 參考：《安徽醫(yī)科大學(xué)》2014年博士論文

【摘要】：背景:Artemin(ARTN)是一種星形膠質(zhì)細(xì)胞系起源的神經(jīng)營(yíng)養(yǎng)因子,參與了人類多種生理和病理進(jìn)程。新近研究發(fā)現(xiàn)ARTN對(duì)惡性腫瘤的發(fā)生發(fā)展也發(fā)揮了促進(jìn)作用。另一方面,研究發(fā)現(xiàn)micro RNAs(mi RNAs)通過(guò)抑制靶m RNA的翻譯或降解靶m RNA負(fù)性調(diào)節(jié)基因的表達(dá),從而參與機(jī)體生命過(guò)程中一系列的重要進(jìn)程,包括惡性腫瘤中多種細(xì)胞因子表達(dá)的調(diào)控。然而,ARTN在人喉鱗狀細(xì)胞癌中的角色和作用機(jī)制尚未見(jiàn)報(bào)道。本課題首先觀察ARTN及其受體在人喉鱗狀細(xì)胞癌中表達(dá)及臨床意義,并且進(jìn)一步分析ARTN對(duì)人喉癌細(xì)胞侵襲、遷移和增殖的影響;其次,觀察mi RNA合成重要因子Dicer在喉癌中表達(dá)及其意義,進(jìn)一步篩選并驗(yàn)證調(diào)控ARTN表達(dá)的特異性mi RNA,探討mi RNA對(duì)ARTN調(diào)控的分子機(jī)制。方法:(1)運(yùn)用免疫組化的方法檢測(cè)76例石蠟包埋喉鱗狀細(xì)胞癌及26例喉息肉樣本中ARTN及其受體GFRα1的表達(dá)情況,分析ARTN和GFRα1表達(dá)對(duì)喉鱗狀細(xì)胞癌患者的臨床病理意義及預(yù)后意義;通過(guò)Real-time PCR方法檢測(cè)16例喉鱗狀細(xì)胞癌及14例喉息肉新鮮樣本中ARTN及其受體GFRα1的表達(dá)。(2)用脂質(zhì)體介導(dǎo)的轉(zhuǎn)染方法將ARTN小干擾RNA(si RNA)轉(zhuǎn)染人喉鱗狀細(xì)胞癌細(xì)胞株Hep-2,設(shè)立陰性對(duì)照;通過(guò)MTS試劑盒和Transwell侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力和侵襲力;采用Transwell遷移實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力。(3)運(yùn)用免疫組化方法檢測(cè)76例石蠟包埋喉鱗狀細(xì)胞癌及26例喉息肉樣本中Dicer的表達(dá)水平,喉鱗狀細(xì)胞癌Dicer表達(dá)水平與患者臨床病理特征及預(yù)后的相關(guān)性。(4)利用生物信息學(xué)方法預(yù)測(cè)靶向ARTN的微小RNA,并進(jìn)行實(shí)驗(yàn)驗(yàn)證:①對(duì)Hep-2細(xì)胞分別轉(zhuǎn)染mi RNA模擬物(mimics)、抑制物(ASO)和相應(yīng)對(duì)照(negative control)后,通過(guò)real-time PCR檢測(cè)轉(zhuǎn)染后ARTN表達(dá)的改變。②克隆ARTN基因3′UTR,將其與質(zhì)粒psi CHECK-2(含熒光素酶報(bào)告基因)連接后,與該mi RNA共轉(zhuǎn)染Hep-2,做熒光素酶報(bào)告實(shí)驗(yàn)(Luciferase Reporter Assay)驗(yàn)證ARTN是該mi RNA的直接靶基因。結(jié)果:(1)喉鱗狀細(xì)胞癌石蠟組織中ARTN和GFRα1的表達(dá)顯著高于喉息肉組織(P0.05);ARTN和GFRα1的表達(dá)與喉鱗狀細(xì)胞癌的p TNM分期呈顯著正相關(guān)(P0.05),與患者預(yù)后呈顯著負(fù)相關(guān)(P0.05);相關(guān)性分析表明ARTN表達(dá)與GFRα1的表達(dá)呈顯著正相關(guān)(P0.05);喉鱗狀細(xì)胞癌新鮮樣本ARTN和GFRα1的表達(dá)也顯著高于喉息肉組織(P0.05)。(2)使用特異性si RNA下調(diào)Hep-2細(xì)胞內(nèi)源性ARTN表達(dá)后,細(xì)胞增殖、侵襲和遷移能力均顯著降低(P0.01)。(3)Dicer在喉鱗狀細(xì)胞癌中的表達(dá)顯著高于其在喉息肉中的表達(dá)水平(P0.05);Dicer的表達(dá)水平與腫瘤的p TNM分期及淋巴結(jié)轉(zhuǎn)移密切相關(guān)(P0.05);KM生存曲線提示Dicer表達(dá)水平與喉鱗狀細(xì)胞癌患者的生存時(shí)間密切相關(guān)(P0.05)。(4)生物信息學(xué)軟件Targetscans預(yù)測(cè)mi R-223可能靶向ARTN基因;外源性轉(zhuǎn)染Hep-2細(xì)胞mi R-223 mimics可以顯著下調(diào)ARTN表達(dá),外源性轉(zhuǎn)染Hep-2細(xì)胞mi R-223 ASO可以顯著上調(diào)ARTN表達(dá);熒光素酶報(bào)告實(shí)驗(yàn)證實(shí)mi R-223與ARTN的3′UTR直接相互作用。結(jié)論:(1)ARTN及其受體在喉鱗狀細(xì)胞癌中表達(dá)失調(diào),對(duì)喉鱗狀細(xì)胞癌的發(fā)生和發(fā)展可能發(fā)揮了重要作用。(2)mi RNA可能參與了喉鱗狀細(xì)胞癌ARTN表達(dá)的調(diào)控,ARTN是mi R-223的直接靶基因。
[Abstract]:Background: Artemin (ARTN) is a neurotrophic factor derived from astrocyte lines and participates in a variety of physiological and pathological processes in human beings. Recent studies have found that ARTN has also played a role in the development of malignant tumors. On the other hand, the study found that micro RNAs (MI RNAs) inhibits the translation of target m RNA and degrades the RNA negative modulation of target m. The expression of the gene is involved in a series of important processes in the life process of the body, including the regulation of the expression of a variety of cytokine in the malignant tumor. However, the role and mechanism of ARTN in human laryngeal squamous cell carcinoma have not yet been reported. The first observation of the expression and clinical significance of ARTN and its receptor in human laryngeal squamous cell carcinoma, and the clinical significance, and the clinical significance of the study, are first observed and the clinical significance of the expression and clinical significance of the human laryngeal squamous cell carcinoma. And further analyze the effect of ARTN on the invasion, migration and proliferation of human Laryngocarcinoma Cells. Secondly, to observe the expression and significance of MI RNA synthesis important factor Dicer in larynx cancer, to further screen and verify the specific mi RNA regulating ARTN expression, and to explore the molecular mechanism of MI RNA to ARTN regulation. Method: (1) 76 cases of paraffin wax were detected by immunohistochemical method. The expression of ARTN and its receptor GFR alpha 1 in 26 cases of laryngeal squamous cell carcinoma and 26 cases of laryngeal polyp were expressed. The clinicopathological significance and prognostic significance of ARTN and GFR alpha 1 expression for laryngeal squamous cell carcinoma were analyzed. The expression of ARTN and its receptor GFR alpha 1 in 16 cases of laryngeal squamous cell carcinoma and 14 cases of laryngeal polyps were detected by Real-time PCR. (2) ARTN small interference RNA (Si RNA) was transfected into human laryngeal squamous cell carcinoma cell line Hep-2 with liposome mediated transfection, and negative control was set up. Cell proliferation ability and invasive ability were detected by MTS kit and Transwell invasion test. Cell migration ability was detected by Transwell migration test. (3) 76 cases of paraffin paraffin were detected by immunohistochemistry. The expression level of Dicer in the laryngeal squamous cell carcinoma and 26 cases of laryngeal polyps, the correlation between the expression of Dicer in laryngeal squamous cell carcinoma and the clinicopathological features and prognosis of the patients. (4) using bioinformatics methods to predict the tiny RNA of the target ARTN, and the experimental verification: (1) transfection of Hep-2 cells to MI RNA analogue (mimics) and inhibitor (ASO) After the corresponding control (negative control), the expression of ARTN expression after transfection was detected by real-time PCR. (2) the ARTN gene was cloned 3 'UTR, which was connected with the plasmid psi CHECK-2 (containing luciferase reporter gene) and co transfected Hep-2 with the MI RNA, and the luciferase report was proved to be the direct target. Results: (1) the expression of ARTN and GFR a 1 in the paraffin tissues of laryngeal squamous cell carcinoma was significantly higher than that of laryngeal polyps (P0.05); the expression of ARTN and GFR alpha 1 was positively correlated with the P TNM staging of laryngeal squamous cell carcinoma (P0.05), and had a significant negative correlation with the prognosis of the patients (P0.05), and the correlation analysis showed that the expression of ARTN was positively correlated with the expression of GFR alpha 1. P0.05): the expression of fresh samples of laryngeal squamous cell carcinoma ARTN and GFR alpha 1 was also significantly higher than that of laryngeal polyp (P0.05). (2) the proliferation, invasion and migration of Hep-2 cells decreased significantly (P0.01) using specific Si RNA down regulation of endogenous ARTN expression in Hep-2 cells (P0.01). (3) the expression of Dicer in laryngeal squamous cell carcinoma was significantly higher than that in laryngeal polyps. Level (P0.05); the expression level of Dicer was closely related to P TNM staging and lymph node metastasis (P0.05); KM survival curve suggested that the expression level of Dicer was closely related to the survival time of the patients with laryngeal squamous cell carcinoma (P0.05). (4) the bioinformatics software Targetscans predicted that MI R-223 might target ARTN genes. 3 mimics can significantly downregulate the expression of ARTN. Exogenous transfection of Hep-2 cells mi R-223 ASO can significantly up-regulate the expression of ARTN; the luciferase reporter experiment confirms the direct interaction of MI R-223 and ARTN's 3 'UTR. Conclusion: (1) the expression of ARTN and its receptors in laryngeal squamous cell carcinoma may be unbalanced and may play a heavy role in the occurrence and development of laryngeal squamous cell carcinoma. (2) mi RNA may be involved in the regulation of ARTN expression in laryngeal squamous cell carcinoma, and ARTN is a direct target gene of MI R-223.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 吳一波;沈志森;余星;劉洋;于雪林;趙曉彥;郭俊明;;喉癌相關(guān)miRNA的研究進(jìn)展[J];基礎(chǔ)醫(yī)學(xué)與臨床;2012年05期

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本文編號(hào)：1858886