ARTEMIN在喉鳞状细胞癌进展中的作用及其分子机制

发布时间：2018-05-07 23:14

本文选题：喉鳞状细胞癌 + Artemin　；参考：《安徽医科大学》2014年博士论文

【摘要】：背景:Artemin(ARTN)是一种星形胶质细胞系起源的神经营养因子,参与了人类多种生理和病理进程。新近研究发现ARTN对恶性肿瘤的发生发展也发挥了促进作用。另一方面,研究发现micro RNAs(mi RNAs)通过抑制靶m RNA的翻译或降解靶m RNA负性调节基因的表达,从而参与机体生命过程中一系列的重要进程,包括恶性肿瘤中多种细胞因子表达的调控。然而,ARTN在人喉鳞状细胞癌中的角色和作用机制尚未见报道。本课题首先观察ARTN及其受体在人喉鳞状细胞癌中表达及临床意义,并且进一步分析ARTN对人喉癌细胞侵袭、迁移和增殖的影响;其次,观察mi RNA合成重要因子Dicer在喉癌中表达及其意义,进一步筛选并验证调控ARTN表达的特异性mi RNA,探讨mi RNA对ARTN调控的分子机制。方法:(1)运用免疫组化的方法检测76例石蜡包埋喉鳞状细胞癌及26例喉息肉样本中ARTN及其受体GFRα1的表达情况,分析ARTN和GFRα1表达对喉鳞状细胞癌患者的临床病理意义及预后意义;通过Real-time PCR方法检测16例喉鳞状细胞癌及14例喉息肉新鲜样本中ARTN及其受体GFRα1的表达。(2)用脂质体介导的转染方法将ARTN小干扰RNA(si RNA)转染人喉鳞状细胞癌细胞株Hep-2,设立阴性对照;通过MTS试剂盒和Transwell侵袭实验检测细胞增殖能力和侵袭力;采用Transwell迁移实验检测细胞迁移能力。(3)运用免疫组化方法检测76例石蜡包埋喉鳞状细胞癌及26例喉息肉样本中Dicer的表达水平,喉鳞状细胞癌Dicer表达水平与患者临床病理特征及预后的相关性。(4)利用生物信息学方法预测靶向ARTN的微小RNA,并进行实验验证:①对Hep-2细胞分别转染mi RNA模拟物(mimics)、抑制物(ASO)和相应对照(negative control)后,通过real-time PCR检测转染后ARTN表达的改变。②克隆ARTN基因3′UTR,将其与质粒psi CHECK-2(含荧光素酶报告基因)连接后,与该mi RNA共转染Hep-2,做荧光素酶报告实验(Luciferase Reporter Assay)验证ARTN是该mi RNA的直接靶基因。结果:(1)喉鳞状细胞癌石蜡组织中ARTN和GFRα1的表达显著高于喉息肉组织(P0.05);ARTN和GFRα1的表达与喉鳞状细胞癌的p TNM分期呈显著正相关(P0.05),与患者预后呈显著负相关(P0.05);相关性分析表明ARTN表达与GFRα1的表达呈显著正相关(P0.05);喉鳞状细胞癌新鲜样本ARTN和GFRα1的表达也显著高于喉息肉组织(P0.05)。(2)使用特异性si RNA下调Hep-2细胞内源性ARTN表达后,细胞增殖、侵袭和迁移能力均显著降低(P0.01)。(3)Dicer在喉鳞状细胞癌中的表达显著高于其在喉息肉中的表达水平(P0.05);Dicer的表达水平与肿瘤的p TNM分期及淋巴结转移密切相关(P0.05);KM生存曲线提示Dicer表达水平与喉鳞状细胞癌患者的生存时间密切相关(P0.05)。(4)生物信息学软件Targetscans预测mi R-223可能靶向ARTN基因;外源性转染Hep-2细胞mi R-223 mimics可以显著下调ARTN表达,外源性转染Hep-2细胞mi R-223 ASO可以显著上调ARTN表达;荧光素酶报告实验证实mi R-223与ARTN的3′UTR直接相互作用。结论:(1)ARTN及其受体在喉鳞状细胞癌中表达失调,对喉鳞状细胞癌的发生和发展可能发挥了重要作用。(2)mi RNA可能参与了喉鳞状细胞癌ARTN表达的调控,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.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.65

【参考文献】