RNA编辑在肺腺癌中的临床意义及作用机制

发布时间：2018-03-24 14:33

本文选题：肺腺癌　切入点：RNA编辑　出处：《天津医科大学》2016年博士论文

【摘要】：目的:1.研究早期肺腺癌患者基因编码区的RNA编辑现象。2.研究腺苷脱氨酶1(Adenosine Deaminases that Act on RNA 1,ADAR1)、腺苷脱氨酶2(Adenosine Deaminases that Act on RNA 2,ADAR2)和鸟氨酸脱羧酶(Ornithine Decarboxylase,ODC)在肺腺癌中表达的临床意义及预后价值。3.研究抗酶蛋白抑制因子1(Antizyme inhibitor 1,AZIN1)的RNA编辑对A549细胞增殖与转移的影响。方法:1.应用微流控芯片多重聚合酶联反应与RNA测序(microfluidics-based multiplex PCR and RNA sequencing)检测早期肺腺癌患者基因编码区的RNA编辑现象。应用荧光定量PCR(RT-q PCR)检测肺腺癌肿瘤组织与癌旁正常组织中ADAR1、ADAR2和ODC的基因表达水平。2.应用免疫组织化学染色技术检测ADAR1、ADAR2和ODC蛋白在肺腺癌肿瘤组织与癌旁正常组织中的表达,分析ADAR1与ODC在肺腺癌中表达的相关性和预后价值。3.构建稳定表达编辑型AZIN1基因的A549细胞(A549-edt/AZIN1)和稳定表达野生型AZIN1基因的A549细胞(A549-wt/AZIN1)。应用XTT实验和Transwell细胞侵袭实验,研究AZIN1的RNA编辑对于A549增殖和侵袭的影响,应用免疫印迹法检测ODC蛋白的表达水平。结果:1.相对于癌旁正常组织,肺腺癌组织的RNA编辑水平升高。在肺腺癌肿瘤组织编辑水平最高的20个基因中,在基因编码区的共涉及14个基因位点,包括:COG3,SRP9,COPA,SRP9,CACNA1D,RICTOR,ZNF669,METTL6,FLNA,AZIN1,C20orf30,PODXL,FLNB,PDZD7。2.ADAR1基因和ODC基因在肺腺癌肿瘤组织中的表达显著高于癌旁正常组织,P(27)0.05。ADAR2基因在肺腺癌肿瘤组织中的表达与癌旁正常组织无统计学差异,其中P(29)0.05。3.ADAR1蛋白定位于细胞核,呈棕黄色或棕褐色颗粒。60.4%的肺腺癌组织ADAR1高表达,而癌旁正常组织仅20.8%为高表达,两组差异有统计学意义(P(27)0.05)。ADAR1蛋白的表达与肺腺癌患者的TNM分期和淋巴结转移情况明显相关(P(27)0.05);而与性别、年龄、吸烟史、胸膜侵犯情况、肿瘤最大直径和分化程度无明显相关(P(29)0.05)。4.ADAR2蛋白定位于细胞核,呈棕黄色或棕褐色颗粒。10.4%的肺腺癌组织ADAR2高表达,而癌旁正常组织中8.3%为高表达,两组差异无统计学意义(P(29)0.05)。5.ODC蛋白主要定位于细胞质,细胞浆呈棕黄色。56.3%的肺腺癌组织ODC高表达,而癌旁正常组织仅12.5%为高表达,两组差异有统计学意义(P(27)0.05)。ODC蛋白的表达与肺腺癌患者的TNM分期明显相关(P(27)0.05);而与性别、年龄、吸烟史、胸膜侵犯情况、肿瘤最大直径、分化程度和淋巴结转移情况无明显相关(P(29)0.05)。6.经过秩相关性分析,肺腺癌患者中ADAR1与ODC的表达呈正相关(r=0.832,P(27)0.01)。生存分析显示ADAR1与ODC的表达与预后相关。Cox比例风险回归模型进行多因素生存分析显示,ADAR1蛋白表达水平、TNM分期是影响肺腺癌患者预后的独立因素。7.通过XTT实验测试A549、A549-edt/AZIN1和A549-wt/AZIN1的增殖能力。第一天,三组细胞增殖未出现明显的差异。第三天,A549-edt/AZIN1与A549-wt/AZIN1的增殖速度明显高于A549(P(27)0.05)。第六天,A549-edt/AZIN1的增殖速度明显高于A549-wt/AZIN1(P(27)0.05)。8.通过Transwell细胞侵袭实验,比较A549,A549-edt/AZIN1和A549-wt/AZIN1穿透Matrigel Invasion Chamber的侵袭能力,结果显示:A549-edt/AZIN1(477±68)穿过小室的细胞数量明显多于A549-wt/AZIN1(327±42)(P(27)0.05),即A549-edt/AZIN1的侵袭能力比A549-wt/AZIN1的侵袭能力强。9.通过Western blot检测A549-edt/AZIN1和A549-wt/AZIN1中ODC的表达量,发现A549-edt/AZIN1和A549-wt/AZIN1中ODC的蛋白表达量高于A549(P(27)0.05),且ODC在A549-edt/AZIN1的表达量高于A549-wt/AZIN1,差异具有统计学意义,P(27)0.05。结论:1.相对于癌旁正常组织,肺腺癌组织的RNA编辑水平升高。在肺腺癌肿瘤组织编辑水平最高的20个基因中,在基因编码区的共涉及14个基因位点,包括:COG3,SRP9,COPA,SRP9,CACNA1D,RICTOR,ZNF669,METTL6,FLNA,AZIN1,C20orf30,PODXL,FLNB,PDZD7。2.肺腺癌患者肿瘤组织中ADAR1与ODC的表达明显高于癌旁正常组织,二者在肺腺癌中的表达呈正相关。肺腺癌患者肿瘤组织与癌旁正常组织中ADAR2的表达无明显差异,主要为低表达。ADAR1蛋白的表达与肺腺癌患者的TNM分期和淋巴结转移情况明显相关。ODC蛋白的表达与肺腺癌患者的TNM分期明显相关。生存分析显示ADAR1与ODC的表达与预后相关。Cox比例风险回归模型进行多因素生存分析显示,ADAR1蛋白表达水平、TNM分期是影响肺腺癌患者预后的独立因素。3.编辑型的AZIN1可以促进A549的增殖与转移,同时AZIN1的高编辑状态可以上调ODC蛋白的表达。
[Abstract]:Objective: To study the early 1. lung adenocarcinoma patients and the gene encoding RNA.2. editing of Ada (Adenosine Deaminases that Act 1 on RNA 1, ADAR1 2 (Adenosine), adenosine deaminase Deaminases that Act on RNA 2, ADAR2) and ornithine decarboxylase (Ornithine Decarboxylase, ODC) on the clinical significance and prognostic value of.3. expression in lung adenocarcinoma antizyme inhibitor 1 (Antizyme 1 inhibitor, AZIN1) RNA editing effect on A549 cell proliferation and metastasis. Methods: 1. application of microfluidic chip multiplex polymerase chain reaction and RNA sequencing (microfluidics-based multiplex PCR and RNA sequencing) for early detection of lung adenocarcinoma gene encoding region of RNA edit. Application of fluorescent quantitative PCR (RT-q PCR) ADAR1 detection of cancer and lung cancer adjacent normal tissues, ADAR2 and ODC gene expression of.2. by immunohistochemical staining ADAR1 color detection technology, the expression of ADAR2 and ODC protein in cancer tissues and lung cancer adjacent normal tissues. The expression of ADAR1 and ODC in lung adenocarcinoma correlation and prognostic value of.3. to build a stable expression editing type AZIN1 gene (A549-edt/AZIN1) and A549 cells stably expressing wild-type AZIN1 gene in A549 cells (A549-wt/AZIN1). Application of XTT assay and Transwell invasion assay of AZIN1 cells, RNA editing effects on the proliferation and invasion of A549, the expression level of ODC protein was detected by Western blotting. Results: 1. compared with normal tissues, increased lung adenocarcinoma RNA editing level. In lung adenocarcinoma tumor tissue level editor 20 the highest gene, including in involving a total of 14 loci, the gene encoding COG3, SRP9, COPA, SRP9, CACNA1D, RICTOR, ZNF669, METTL6, FLNA, AZIN1, C20orf30, PODXL, FLNB, PDZD7.2.ADAR1 and ODC gene Gene expression in lung cancer tissues was significantly higher than that in normal tissues, P (27) and no significant difference between the expression of 0.05.ADAR2 gene in human lung adenocarcinoma in tumor tissue and adjacent normal tissues, including P (29) 0.05.3.ADAR1 protein located in the nucleus, brownish yellow or brown granules of.60.4% lung adenocarcinoma high ADAR1 expression, and normal tissues only 20.8% high expression, there was significant difference between two groups (P (27) 0.05) the expression of.ADAR1 protein and lung cancer TNM staging and lymph node metastasis was significantly correlated (P (27) 0.05); and gender, age, smoking history, pleural invasion, was not related to tumor size and degree of differentiation (P (29) 0.05).4.ADAR2 protein located in the nucleus, brownish yellow or brown granules of.10.4% lung adenocarcinoma with high expression of ADAR2, and 8.3% adjacent normal tissues showed high expression, no statistical difference between the two groups The meaning of (P (29) 0.05).5.ODC protein was mainly localized in the cytoplasm, brownish yellow cytoplasm showed high expression of.56.3% in lung adenocarcinoma tissues and normal tissues ODC, only 12.5% high expression, there was significant difference between two groups (P (27) 0.05) the expression of.ODC protein and lung cancer patients. TNM stage was significantly correlated (P (27) 0.05); and gender, age, smoking history, pleural invasion, tumor diameter, was not related to the degree of differentiation and lymph node metastasis (P (29) 0.05) by.6. rank correlation analysis, a positive correlation between the expression of ADAR1 and ODC in patients with lung adenocarcinoma (r=0.832, P (27) 0.01). Survival analysis showed that ADAR1 and ODC.Cox expression and prognosis of regression model for multivariate survival analysis showed that the expression level of ADAR1 protein, TNM.7. stage were independent prognostic factors in patients with lung adenocarcinoma by XTT test A549, A549-edt/AZIN1 and A549-wt/AZIN1 The proliferation ability. The first day, three groups of cell proliferation showed no obvious difference. The third day, A549-edt/AZIN1 and A549-wt/AZIN1 proliferation rate was significantly higher than that of A549 (P (27) 0.05). For sixth days, the proliferation rate of A549-edt/AZIN1 was significantly higher than that of A549-wt/AZIN1 (P (27).8. 0.05) by Transwell invasion assay, A549, A549-edt/AZIN1 Matrigel Invasion and A549-wt/AZIN1 penetrate the invasion ability of Chamber, the results showed that A549-edt/AZIN1 (477 + 68) the number of cells through the cell was higher than A549-wt/AZIN1 (327 + 42) (P (27) 0.05), namely the invasion ability of A549-edt/AZIN1 than the invasion ability of A549-wt/AZIN1 strong.9. expression by Western blot detection of A549-edt/AZIN1 and A549-wt/AZIN1 in the amount of ODC A549-edt/AZIN1 and A549-wt/AZIN1, found in the protein expression of ODC was higher than that of A549 (P (27) 0.05), and the ODC expression of A549-wt/AZIN1 in A549-edt/ is higher than the amount of AZIN1, the difference is Statistical significance, P (27) 0.05. conclusion: 1. compared with normal tissues, increased lung adenocarcinoma RNA editing level. In lung adenocarcinoma tumor tissue editing level 20 genes included in the highest, involving a total of 14 loci, the gene encoding COG3, SRP9, COPA, SRP9. CACNA1D, RICTOR, ZNF669, METTL6, FLNA, AZIN1, C20orf30, PODXL, FLNB, ADAR1 and ODC PDZD7.2. expression in lung cancer tissues was significantly higher than that in normal tissues, two positive expression in lung adenocarcinoma. No significant difference between the expression of ADAR2 in tumor tissues of patients with cancer and lung adenocarcinoma by in normal tissues, mainly for the low expression of.ADAR1 protein expression in lung cancer patients with TNM staging and lymph node metastasis was significantly related to the expression of.ODC protein in lung cancer patients with TNM stage were significantly related. Survival analysis showed that ADAR1 and ODC expression of.Cox and prognosis risk ratio Multivariate analysis of survival showed that ADAR1 protein expression and TNM stage were independent prognostic factors in patients with lung adenocarcinoma..3. editing AZIN1 could promote the proliferation and metastasis of A549. Meanwhile, the high editing status of AZIN1 could upregulate the expression of ODC protein.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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