【作者】 李娟；

【导师】 张国俊；

【作者基本信息】 郑州大学 ， 内科学（呼吸病学）， 2016， 博士

【摘要】 背景和目的在全球范围内,肺癌是发病率和死亡率均居于首位的恶性肿瘤,每年有160万患者被诊断为肺癌,同时有140余万的患者被肺癌夺取生命,其中80%的肺癌为非小细胞肺癌(non-small cell lung cancer,NSCLC)。目前治疗NSCLC的常规方法有手术治疗、铂二联化疗、放疗和靶向治疗等,但是这些常规治疗方法对中晚期肿瘤患者来说不能达到治愈的目的,因此,多数患者治疗后生存期仍很短,5年生存率不到15%。由于多数NSCLC患者早期无特异临床表现,在确诊时已至中晚期,多数患者已出现转移和局部侵犯。因此,探讨NSCLC的发病调控机制,早期干预肿瘤细胞的生长、侵袭和迁移能力,有助于提高NSCLC病人的生存率,降低其死亡率。长链非编码RNA(long non-codingRNA,lncRNA)是一类转录本长度大于200核苷酸,不能编码蛋白质的功能性RNA分子。LncRNA参与了生命进程中一系列的重要进程,包括生长发育、骨髓造血、细胞凋亡、细胞增殖等。但是人类对lncRNA在肿瘤中的调控机制却了解很少。已有的研究资料显示许多lncRNA在不同肿瘤组织中的表达发生了改变。因此,其在肿瘤发生、发展过程中的作用备受关注。虽然大多数lncRNA的详细作用机制尚不明确,但lncRNA已成为继mi RNA之后的新的肿瘤研究热点。LncRNA DLX6-AS1定位于7q21.3,查阅国内外文献目前未见其他研究者关于lncRNA DLX6-AS1在肺癌中的报道。为了探索lncRNA DLX6-AS1在肺腺癌细胞的表达、生物学作用及其对肿瘤的调控的初步机制,本课题进行了三个部分的研究。第一部分:肺腺癌组织中lncRNA表达分析;第二部分:下调肺腺癌细胞中lncRNA DLX6-AS1的表达对细胞增殖、凋亡和侵袭的影响;第三部分:lncRNA DLX6-AS1作用机理的初步分析。第一部分肺腺癌组织中lncRNA表达分析方法:1.收集标本,包括72例肺腺癌组织及相对应的癌旁正常组织标本。2.采用lncRNA芯片检测3例肺腺癌组织及相对应的癌旁正常组织标本中lncRNA表达情况,分析差异化表达的lncRNA。3.运用q RT-PCR检测72例样本中lncRNA DLX6-AS1表达水平,分析其表达水平与肺腺癌患者性别、年龄、肿瘤分化程度、淋巴结转移情况及TNM分期的相关性。结果:1.LncRNA芯片检测分析筛选出18个lncRNAs在肺腺癌组织中表达异常,其中9个lncRNAs表达上调,9个lncRNAs表达下调。LncRNA DLX6-AS1在肺腺癌组织中呈高表达(P<0.05)。2.q RT-PCR检测显示在肺腺癌组织,lncRNA DLX6-AS1表达水平较正常组织显著升高(P<0.05),lncRNA基因芯片结果和q RT-PCR的结果保持一致,这也说明了基因芯片结果的可靠性。3.肺腺癌组织中lncRNA DLX6-AS1的表达水平与患者的肿瘤分化程度和TNM分期有关(P<0.05),而与其性别、年龄、淋巴结转移情况无关(P>0.05)。第二部分下调肺腺癌细胞中lncRNA DLX6-AS1的表达对细胞增殖、凋亡和侵袭的影响方法:1.制备和包装沉默lncRNA DLX6-AS1表达的重组慢病毒,感染对数期生长的肺腺癌细胞系A549、H1650,建立下调lncRNA DLX6-AS1表达的肺腺癌细胞株。2.设立三组:实验组(lncRNA DLX6-AS1si RNA慢病毒感染组)、NC组(lncRNA-NC无关序列对照组)和Blank组(空白细胞)。3.CCK-8法、平板克隆形成实验检测三组细胞增殖和生长能力。4.Anncxin V-FITC/PI双标记流式细胞术、Hoechst染色、Caspase-3/7活性检测检测三组细胞凋亡率。5.Western-blot检测细胞中凋亡相关蛋白的表达情况。6.Transwell小室实验、划痕实验检测三组细胞侵袭迁移能力。7.荷瘤小鼠实验检测体内水平沉默lncRNA DLX6-AS1的表达对肺腺癌影响。结果:1.成功制备了重组慢病毒lncRNA DLX6-AS1si RNA和无关序列对照lncRNA DLX6-AS1 NC,慢病毒滴度分别为1.8×108TU/ml和2.3×108TU/ml。对比空白组和无关序列对照组,si Lnc DLX6-AS1组lncRNA DLX6-AS1表达水平显著下调(P<0.05)。2.CCK8法检测结果显示在A549和H1650细胞中,si Lnc DLX6-AS1组细胞较空白组和无关序列对照组吸光度显著减少,且随着时间的持续,差异越来越大,差异具有统计学意义(P<0.05)。平板克隆形成实验结果显示在A549和H1650细胞中,si Lnc DLX6-AS1组细胞较空白组和无关序列对照组细胞克隆形成数显著降低,差异有统计学意义(P<0.05)。3.Anncxin V-FITC/PI双标记流式细胞术和Hoechst染色检测显示在A549和H1650细胞中,si Lnc DLX6-AS1组细胞凋亡率较空白组和无关序列对照组显著升高,对比空白组和无关序列对照组,si Lnc DLX6-AS1组Caspase-3/7细胞活性显著升高,差异具有统计学意义(P<0.05)。4.Western blot结果显示在A549和H1650细胞中,si Lnc DLX6-AS1组细胞与空白组和无关序列对照组细胞相比,Cleaved caspase3和Cleaved caspase9蛋白表达水平呈升高状态,而Pro-capase-9与Bcl-2的蛋白表达水平呈明显降低状态,差异具有统计学意义(P<0.05)。5.Transwell小室实验结果显示在A549和H1650细胞中,si Lnc DLX6-AS1组细胞与空白组和无关序列对照组细胞相比,穿膜细胞数显著减少,差异有统计学意义(P<0.05)。划痕实验显示在A549和H1650细胞中,si Lnc DLX6-AS1组细胞与空白组和无关序列对照组细胞相比,划痕愈合速度明显下降,差异有统计学意义(P<0.05)。6.裸鼠移植瘤体内实验结果显示对比空白组和无关序列对照组,si Lnc DLX6-AS1组移植瘤显著减小,差异有统计学意义(P<0.05)。第三部分lncRNA DLX6-AS1作用机理的初步分析方法:1.通过生物信息学分析推测lncRNA DLX6-AS1潜在相互作用的mi RNA。2.构建lncRNA DLX6-AS1野生型和Seed Region突变型表达载体(pc DNA3.1-Wt DLX6 AS1、pc DNA3.1-Mt DLX6 AS1),并转染A549细胞。q RT–PCR方法检测转染细胞中lncRNA DLX6-AS1和mi R-497的表达。3.构建报告基因重组载体pmir GLO-Wt3’UTR-Bcl2和pmir GLO-Mt3’UTR-Bcl2。采用双荧光素酶报告实验验证Bcl-2是mi R-497的靶基因及lncRNA DLX6-AS1通过负调控mi R-497调控Bcl-2的表达。4.CCK-8法、Anncxin V-FITC/PI双标记流式细胞术、Transwell小室实验检测上调mi R-497和下调lncRNA DLX6-AS1的表达对肺腺癌细胞的作用。5.构建无3’UTR区Bcl-2的表达载体,单独转入或与mi R-497 mimics共转染入A549细胞或si Lnc DLX6-AS1转染的A549细胞中,通过restore方法分析mi R-429调控Bcl-2表达的作用机制。结果:1.生物信息学分析推测ln RNA DLX6-AS1和mi R-497存在潜在的相互作用的seed region区有特异结合位点。2.LncRNA DLX6-AS1的对A549细胞株中mi R-497的表达起负调控作用,该负调控作用是通过seed region区特异结合而实现的。3.双荧光素酶报告实验显示Bcl-2是mi R-497一个靶基因。4.在A549细胞中过表达mi R-497和沉默lncRNA DLX6-AS1的表达可以起到相似的生物学效应。5.血清饥饿诱导凋亡实验结果显示将不含Bcl-2 3’UTR区的重组载体pc DNA3.1-Bcl2转染至A549细胞后,可以回复上调mi R-497和下调lncRNA DLX6-AS1促进细胞凋亡的能力。结论:1.芯片筛选出18个lncRNAs在肺腺癌组织中表达异常,其中9个lncRNAs表达上调,9个lncRNAs表达下调。LncRNA DLX6-AS1在肺腺癌组织中呈高表达,其表达水平与患者的肿瘤分化程度和TNM分期有关。2.体外下调肺腺癌细胞中的lncRNA DLX6-AS1表达可有效抑制细胞的增殖、降低细胞侵袭迁移能力,并且促进细胞的凋亡。动物实验表明下调lncRNA DLX6-AS1的表达能有效抑制裸鼠移植瘤生长。3.LncRNA DLX6-AS1通过负调控mi R-497调控Bcl-2的表达,从而发挥相应的生物学功能。4.LncRNA DLX6-AS1发挥促癌作用,有望成为肺腺癌治疗新的靶点。更多还原

【Abstract】 Lung cancer is one of the most common malignant tumor in the world, and its morbidity and mortality in a variety of tumors, every year there are 1.6 million patients with newly diagnosed worldwide, which kills more than 1.4 million patients at the same time, 80% of lung cancer for non-small cell lung cancer(NSCLC). The main causes of high mortality for NSCLC patients were that patients with no obvious clinical symptoms in the early stage were diagnosised in the late stage with tumor cell invasion and metastasis. Current standard treatment of NSCLC include surgery,radiation therapy and chemotherapy based on platinum, but these treatments are hard to reach the purpose of cure NSCLC, therefore, the effect of these treatments in NSCLC patients is not beautiful, the prognosis is poor, the 5-year survival rate is still less than 15%. In order to raise survival rate of the NSCLC patients must further study of its pathogenesis, and inhibit the proliferation and invasion and metastasis of malignant tumor as soon as possible.Long non-coding(lncRNAs), is a diverse class of RNA transcripts>200nucleotides in length with limited protein coding potential, due to the lack of significant open reading frame. LncRNA involved in tumor biology function through regulating the expression and transcription of gene in epigenetics, and the transcription and posttranslation levels. So far, people know a bit about he regulatingmechanism of lncRNAs in the cancer.Several research datas show that expression levels of many lncRNAs changed in different tumor tissues.Therefore, its role in the tumorigenesis and development process. So people focused on its role in the tumorigenesis and development process. Although the specific mechanism of most lncRNAs are unclear, but the lncRNAs have become the new research hot spot and front after mi RNAs.LncRNA DLX6-AS1 is located on human chromosomal band 7q21.3. No report was found about lncRNA DLX6-AS1 in lung cancer at home and abroad. In order to discovery the function of lncRNA DLX6-AS1 in the proliferation and apoptosis of lung adenocarcinoma lines, and the contribution of lncRNA DLX6-AS1 to lung adenocarcinoma malignancy and the molecular mechanisms, the research includes three parts.The first part is expression and analysis of lncRNA DLX6-AS1 in lung adenocarcinoma tissues; the second part is biological effects of downregulating lncRNA DLX6-AS1 expression in lung adenocarcinoma cell; the third part is preliminary study of mechanism of lncRNA DLX6-AS1.Part One: Expression and analysis of lncRNA DLX6-AS1 in lung adenocarcinoma tissues.Methods:1. Seventy-two pairs of primary lung adenocarcinoma tissues and corresponding adjacent normal lung tissues were used.2. LncRNA chip was used to detect expression of lncRNA in 3 cases of lung adenocarcinoma tissue and corresponding normal adjacent tissue samples and analysis the differential expression of lncRNA.3. The q RT–PCR method was used to detect expressions of lncRNA DLX6-AS1 in seventy-two cases of lung adenocarcinoma tissue and Bivariate correlation analysis was used to verify the correlation between lncRNA DLX6-AS1 expression and gender, age, differentiation status, lymph node metastases and TNM stage.Results:1. Compared with adjacent normal lung tissues, tumor tissues exhibited 18 differentially expressed ln RNAs, including 9 lncRNAs upregulated and 9lncRNAs downregulated. LncRNA DLX6-AS1 is upregulated in lung adenocarcinoma tissue(P < 0.05).2. qRT-PCR detection showed that compared to normal tissues, expression level of lncRNA DLX6-AS1 has a significant upregulation in lung adenocarcinoma tissues(P < 0.05). The results of ln RNA chip and q RT-PCR are consistent.3. LncRNA DLX6-AS1 expression level in lung adenocarcinoma tissues was associated with tumor location and TNM stage(P>0.05). There was no statistically significant correlation between lncRNA DLX6-AS1 expression and either gender, age, lymph node metastases or differentiation status(P<0.05).Part Two Biological effects of downregulating lncRNA DLX6-AS1 expression in lung adenocarcinoma cell.Methods:1. Construct lentiviral vector with lncRNA DLX6-AS1 si RNA and non-sense si RNA, which were transfected into A549 and H1650 cells, screen a stable infected cell line that downregulation the expression level of lncRNA DLX6-AS1.2. The cells are divided into three groups: control group(recombined lncRNA DLX6-AS1 si RNA lentivirus infected cells), negative control(NC) group(lncRNA DLX6-AS1 non-sense si RNA lentivirus infected cells) and blank group(uninfected cells).3. Using CCK8 method and plate clone formation experiment to detect proliferation and growth ability of cells in each group.4. Using AnncxinV-FITC/PI double marker flow cytometry, Hoechst staining and Caspase-3/7 to test apoptosis ability of cells in each group.5. Using Western- blot to test the expression of apoptosis related proteins of cells in each group.6. Using transwell chambers experiment and scratch to test invasion and metastasis ability of cells in each group.7. Using Tumor xenograft model to detect the influence of downregulating lncRNA DLX6-AS1 expression in A549 cells on BALB/c nude mice.Results:1. Lentiviruses with lncRNA DLX6-AS1 si RNA and lncRNA DLX6-AS1 NC were successfully constructed. The titer were respectively 1.8×108TU/ml and2.3×108TU/ml. Compared with Blank and NC groups,the expression of lncRNA DLX6-AS1 was significantly decreased in si Lnc DLX6-AS1 group(P < 0.05).2. CCK8 results showed that compared to Blank and NC groups, OD450 value of si Lnc DLX6-AS1 group has a significant reduce in A549 and H1650 cells, which was more obvious with time passing. The difference has statistically significant(P < 0.05). Plate clone formation experiment results showed that compared to Blank and NC groups, the colony formation number of si Lnc DLX6-AS1 group has a significant reduce in A549 and H1650 cells. The difference has statistically significant(P < 0.05).3. AnncxinV-FITC/PI double marking flow cytometry assay and Hoechst staining method results showed that compared to Blank and NC groups, the apoptosis rate of si Lnc DLX6-AS1 group was significantly raised after infected in A549 and H1650 cells. Besides, the Caspase-3/7 activity also has a significantly raised when compared to Blank and NC groups in A549 and H1650 cells. The difference has statistically significant(P < 0.05).4. Western blot results showed that compared to Blank and NC groups,Pro-capase-9 and Bcl-2 in si Lnc DLX6-AS1 group were significantly reduce in A549 and H1650 cells, when Cleaved caspase3 and Cleaved caspase9 were significantly increased. The difference has statistically significant(P < 0.05).5. Transwell invasion experiment results showed that compared to Blank and NC groups, the cell number went through membrane of si Lnc DLX6-AS1 group has a significant reduce. The difference has statistically significant(P < 0.05). Scratch experiments results showed that compared to Blank and NC groups, after recombination of lentivirus infected cells, scratch healing speed after 48 h of si Lnc DLX6-AS1 group was significantly lower in A549 and H1650 cells. The difference was statistically significant(P < 0.05).6. To confirm the growth inhibitory effect of lncRNATP73-AS1 si RNA on lung adenocarcinoma in vivo, tumor xenograft transplant experiment was performed.Tumor size was significantly reduced in the si Lnc DLX6-AS1 mice group(A549cells transfected with lncRNA DLX6-AS1 si RNA) as compared to control mice(NC and Blank groups) at the fourth week(P<0.05).Part Three Preliminary study of mechanism of lncRNA DLX6-AS1Methods:1. Predict the potential interaction mi RNA of lncRNA DLX6-AS1 by bioinformatics analysis.2. Construct wildtype and seed region mutation expressional vector of lncRNA DLX6-AS1(pc DNA3.1-Wt DLX6 AS1、pc DNA3.1-Mt DLX6 AS1), which were transfected into A549 cells. Using q RT –PCR to test the expreesion of lncRNA DLX6-AS1 and mi R-497 in infected cells.3. Construct reporter gene recombinant vectors pmirGLO-Wt3’UTR-Bcl2 and pmir GLO-Mt3’UTR-Bcl2. Use dual-luciferase report experimental to validate that Bcl-2 is the target genes of mi R-497 and lncRNA DLX6-AS1 regulate Bcl-2expression by negative regulating mi R-497.4. Using CCK8, AnncxinV-FITC/PI double marker flow cytometry and Transwell invasion experiment to test the effect of upregulating mi R-497 and downregulating lncRNA DLX6-AS1 in lung adenocarcinoma cell.5. We constructed expression vectors containing Bcl-2 lacking the 3’UTR sequence.Transfected expression vectors alone or co-transfected with mi R-497 mimics into A549 and A549 infected si Lnc DLX6-AS1 cells. Restore assay was used to analyse mechanism that mi R-497 regulated the expression of Bcl-2.Results:1. We predicted the 3’ untranslated region(3’UTR) of mi R-497 contains one seed region for lncRNA DLX6-AS1 by the bioinformatics algorithms.2. Ln RNA DLX6-AS1 regulate negatively mi R-497 by binding the seed region in A549 cells.3. Dual luciferase report experiments show that Bcl-2 is the target gene of mi R-497.4. Overexpression of mi R-497 had similar function with lncRNA DLX6-AS1 si RNA in A549 cells.5. Serum starvation induced apoptosis experiments showed transfected the recombination vector pc DNA3.1-Bcl2 without 3’UTR regions of Bcl-2 into A549 cells led to the restoration of the negative function of mi R-497 and lncRNA DLX6-AS1. At the same time, the ability promote the apoptosis of cells had been restored.Conclusion:1. A total of 18 lncRNAs in lung adenocarcinoma tissues have abnormal expression,including 9 up-regulation and 9 down-regulation. LncRNA DLX6-AS1 expression level increased significantly when compared to corresponding adjacent normal lung tissues. LncRNA DLX6-AS1 expression level in lung adenocarcinoma tissues was associated with tumor location and TNM stage(P <0.05).2. Down-regulation of lncRNA DLX6-AS1 in lung adenocarcinoma cells in vitro can significantly inhibit cells growth, reduce the invasion ability of cells and promote cells apoptosis. The animal experiment indicated that the down-regulation of mi R-335 can significantly inhibit the transplant tumor growth in nude mouse models.3. LncRNA DLX6-AS1 regulate Bcl-2 expression by negative regulating mi R-497,thus exert its biological function.4. LncRNA DLX6-AS1 can play a role in cancer promotion and is expected to become new targets for lung adenocarcinoma gene therapy.更多还原