背景： 肺癌是全球范围内发病率和癌症致死率均排第一位的恶性肿瘤，其中以非小细胞肺癌最为常见，占到肺癌的80％左右，肺腺癌占到非小细胞肺癌70％。尽管出现多种治疗方式，但肺癌患者的总生存率并没有显著提高，仅15％左右。因此，寻找特异敏感的肺腺癌潜在诊断和预后标志分子，对于提高患者手术切除率，改善患者预后都具有十分积极的作用。 长链非编码RNA(long noneoding RNAs，lncRNAs)是一类序列长度>200核苷酸的非编码RNA，不具备编码蛋白能力的RNA。大量研究证实lncRNAs可作为癌症诊断或预后的重要生物标志分子。小核仁RNA宿主基因6(small nucleolar RNA host gene 6， SNHG6)位于U87HG内含子内，具有高度保守的序列，其主要是作为一种促癌lncRNA参与到肿瘤发生发展的过程中。人们还发现lncRNA SNHG6主要是通过竞争性结合对应的miRNA来参与下游靶基因的调控，这种竞争结合的关系被叫作竞争性内源性RNA机制。竞争性内源性RNA(competing endogenous RNAs，ceRNAs)是一种转录本，通过竞争共享的miRNAs，在转录后水平上相互调节。lncRNA SNHG6作为ceRNA参与到多种恶性肿瘤细胞多个方面的生物功能调控。虽然大量的研究报道了SNHG6作为ceRNA在多种常见恶性肿瘤细胞中的作用，但是有关其在肺腺癌中的研究却很少。 有关肺腺癌的TCGA数据显示，SNHG6在肺腺癌组织中的表达显著高于相应正常组织中的表达。结合以上的数据分析和文献报道，我们推测SNHG6也可能在调控肺腺癌中起关键作用。 目的： 本研究旨在探究SNHG6表达在肺腺癌患者中临床意义，以及细胞水平分析SNHG6对肺腺癌细胞系生物学功能和分子机制，为寻找肺腺癌患者的诊断及治疗的新靶点提供理论依据和研究基础。 方法： 1.利用TCGA数据和qRT-PCR实验分析SNHG6在肺腺癌组织和对应癌旁组织间的差异表达，利用qRT-PCR检测SNHG6在淋巴结转移组和无转移组间的表达差异。 2.利用TCGA数据和随访信息分析SNHG6对肺癌预后的影响。 3.qRT-PCR检测SNHG6在肺腺癌细胞系和正常肺上皮细胞中的表达；利用慢病毒技术上调或下调肺腺癌细胞中SNHG6的表达；利用MTT和平板克隆形成实验检测SNHG6表达改变对肿瘤细胞增殖能力的影响。细胞周期检测试剂盒评估SNHG6表达改变对细胞周期的影响。 4.Transwell实验和细胞划痕实验评估SNHG6表达改变对肺腺癌细胞系迁移和侵袭的影响；western blot检测SNHG6表达改变对EMT关键标志蛋白的表达影响。 5.利用starbase v2.0软件分析TCGA数据中miR-26a-5p在肺腺癌组织和正常肺组织中的表达；qRT-PCR分析miR-26a-5p在肺腺癌组织和对应正常肺组织中的表达； Peason系数分析miR-26a-5p与SNHG6之间的表达相关性；qRT-PCR分析SNHG6表达改变对miR-26a-5p表达的影响；双荧光素报告基因检测SNHG6与miR-26a-5p的直接关系。 6.利用TCGA和qRT-PCR分析E2F7mRNA在肺腺癌组织和对应正常肺组织中的表达；Peason系数分析E2F7与miR-26a-5p或SNHG6之间的表达相关性。双荧光素酶报告基因实验评估miR-26a-5p与E2F7的直接关系。Western blot检测SNHG6或miR-26a-5p表达改变对E2F7蛋白的影响。 7.Transwell实验和划痕实验评估SNHG6对细胞迁移的影响是否依赖miR-26a-5p／E2F7轴。 8.Western blot实验评估SNHG6对肺腺癌细胞EMT关键蛋白的影响是否依赖于miR-26a-5p／E2F7轴。 9.裸鼠皮下移植瘤实验评估SNHG6对体内肿瘤生长的影响。 结果： 1.SNHG6在收集的肺腺癌组织中的表达也明显高于对应癌旁组织中的表达； TCGA数据分析也得到了类似的结果；SNHG6在肺腺癌转移组中的表达明显高于非转移组中的表达。 2.SNHG6高表达组患者的生存期显著短于SNHG6低表达组患者的生存期。 3.SNHG6在肺腺癌细胞中的表达明显高于正常支气管上皮细胞中的表达；利用慢病毒技术显著上调或下调了肺腺癌细胞中SNHG6的表达。 4.过表达SNHG6促进肺腺癌细胞增殖、周期、迁移、侵袭及EMT，而敲低SNHG6发挥相反的作用。 5.miR-26a-5p在肺腺癌组织中的表达明显低于对应癌旁组织中的表达：miR-26a-5p在肺腺癌细胞系中的表达明显低于正常支气管上皮细胞BEAS-2B的表达；SNHG6在肺腺癌组织中的表达与miR-26a-5p呈负相关；上调SNHG6的表达降低了miR-26a-5p；双荧光素报告基因检测证实SNHG6与miR-26a-5p直接结合。 6.E2F7mRNA在肺腺癌中的表达明显高于对应癌旁组织中的表达，并且与miR-26a-5p的表达负相关，与SNHG6的表达正相关；双荧光素报告基因实验证实miR-26a-5p可以直接结合E2F7：western blot实验证明SNHG6和或miR-26a-5p均能影响E2F7的蛋白水平。 7.SNHG6对肺腺癌细胞系迁移和EMT的影响依赖于miR-26a-5p／E2F7轴。 8.SNHG6的敲低可以抑制移植瘤的生长。 结论： 1.长链非编码RNA SNHG6在肺腺癌组织中高表达，并且其与肺腺癌患者的不良预后密切相关。 2.SNHG6在肺腺癌细胞中发挥促癌作用，过表达SNHG6可以促进肺腺癌细胞增殖、周期、迁移及EMT，敲低SNHG6后发挥相反作用。 3.SNHG6表达改变对肺腺癌细胞生物学功能的影响依赖于miR-26a-5p／E2F7轴。 4.敲低SNHG6抑制裸鼠体内肿瘤的生长。 关键词：肺腺癌；SNHG6；eeRNA；miR-26a-5p；E2F7 论文类型：应用基础

 Introduction: Lung cancer is a malignant tumor with the highest morbidity and mortality in the world, and non-small cell lung cancer is the most common lung cancer, accounting for about 80% of lung cancer. Lung adenocarcinoma accounts for 70% of non-small cell lung cancer. Despite various treatments, the overall survival rate of patients with lung cancer has not significantly improved, only about 15%. Therefore, the search for specific and sensitive potential diagnostic and prognostic indicators of lung adenocarcinoma (LUAD) is of great significance for increasing surgical resection rate, improving prognosis. Long strand noncoding RNA (lncRNAs) is a kind of RNA whose length is more than 200 nucleotides with lack of functional protein-coding capacity. Numerous studies confirmed that lncRNAs can be used as an important biomarker for cancer diagnosis or prognosis. The small nucleolar RNA host gene 6 (small nucleolar RNA host gene 6, SNHG6) is located in the intron of U87HG and has a highly conserved sequence. It is mainly involved in the process of tumorigenesis and development. In vitro studies, lncRNA SNHG6 mainly participates in the regulation of downstream target genes by competitively binding the corresponding miRNA, which is known as the competitive endogenous RNA mechanism. Competitive endogenous RNA (ceRNAs) is a transcript that regulates each other at the post-transcriptional level through competitively shared miRNAs. As a ceRNA, lncRNA SNHG6 is involved in many aspects of biological function regulation of many kinds of malignant tumor cells. Although a large number of studies have reported the role of SNHG6 as ceRNA in a variety of common malignant tumor cells, but little research has been done on the role of ceRNA in lung adenocarcinoma. The TCGA data of lung adenocarcinoma showed that SNHG6 expression in lung adenocarcinoma was significantly higher than that in corresponding paracancerous tissues. Combined with the above data analysis and literature reports. We speculate that SNHG6 may also play a key role in regulating lung adenocarcinoma. Objectives: The aim is to investigate the clinical significance of SNHG6 in patients with lung adenocarcinoma, to explore the biological function and molecular mechanism of SNHG6 in vitro, to provide theoretical basis and research basis for finding specific and sensitive biomarkers for diagnosis and therapy. Methods: 1.The differential expression of SNHG6 between lung adenocarcinoma tissue and corresponding paracancerous tissue was detected by qRT-PCR and TCGA datas. qRT-PCR was used to detect the differential expression of SNHG6 between metastatic group and non-metastatic group in lung adenocarcinoma. 2.TCGA and follow-up data were used to analyze the effect of SNHG6 on the prognosis of lung cancer patients. 3.qRT-PCR was used to detect the expression of SNHG6 in lung adenocarcinoma cell lines and normal lung epithelial cells. Up-regualtion or down-regualtion of SNHG6 in LUAD cells were constructed by lentivirus technique, and the effects of SNHG6 expression change on cell proliferation were detected by MTT and plate colony formation assay. Cell cycle detection kit was used to evaluate the effect of SNHG6 expression change on cell cycle. 4.Transwell assay and wound healing assay were used to evaluate the effect of SNHG6 expression change on cell migration and invasion. Western blot was used to detect the expression of EMT key marker proteins after changing SNHG6 expression. 5.The expression of miR-26a-5p in lung adenocarcinoma tissue and normal lung tissue was analyzed by TCGA data and qRT-PCR. The correlation between the expression of miR-26a-5p and SNHG6 was analyzed by Peason coefficient. The effect of SNHG6 expression change on miR-26a-5p level was analyzed by qRT-PCR; The direct relationship between SNHG6 and miR-26a-5p was evaluated by luciferase reporter assays and RIP assay. 6.The expression of E2F7mRNA in lung adenocarcinoma tissue and normal lung tissue was analyzed by TCGA data and qRT-PCR. The correlation between E2F7 expression and miR-26a-5p or SNHG6 was analyzed by Peason coefficient. The direct relationship between miR-26a-5p and E2F7 was evaluated by luciferase reporter assays. Western blot was used to detect the effect of SNHG6 or miR-26a-5p expression change on E2F7 protein. 7.Transwell assay and wound healing assay were used to evaluate whether the effects of SNHG6 on the cell migration was dependent on the miR-26a-5p/E2F7 axis. 8.Western blot assay was used to evaluate whether the effect of SNHG6 on EMT marker protein in lung adenocarcinoma cells was dependent on the miR-26a-5p/E2F7 axis. 9.Evaluation of the effect of SNHG6 expression on tumor growth in vivo after subcutaneous tumor transplantation in nude mice. Results: 1.TCGA data showed that the expression of SNHG6 in lung adenocarcinoma tissue was significantly higher than that in normal lung tissue. The expression of SNHG6 in collected lung adenocarcinoma tissue was also significantly higher than that in corresponding paracancerous tissue, and the expression of SNHG6 in lung adenocarcinoma metastasis group was also significantly higher than that in corresponding paracancerous tissue. SNHG6's expression level in the metastatic group was significantly higher than that in the non-metastatic group. 2.The survival time of patients with high expression of SNHG6 was significantly shorter than that of patients with low expression of SNHG6. 3.The expression of SNHG6 in lung adenocarcinoma cells was significantly higher than that in normal bronchial epithelial cells; After 48 h of transfection, SNHG6 expression was dramatically lower in HCC827 and H1299 cells and higher in A549 cells than in the control cells. 4.Overexpression of SNHG6 promoted cell proliferation, cell cycle, cell migration and invasion, and EMT of lung adenocarcinoma cells, while knockdown of SNHG6 played an opposite role. 5.The expression of miR-26a-5p in lung adenocarcinoma tissues was significantly lower than that in corresponding paracancerous tissues. The expression of miR-26a-5p in lung adenocarcinoma cell lines was significantly lower than that in normal bronchial epithelial cells. The expression of SNHG6 in lung adenocarcinoma tissues was negatively correlated with miR-26a-5p. The upregulation of SNHG6 expression decreased the expression of miR-26a-5p; luciferase reporter assays and RNA immunoprecipitation assay confirmed the direct binding of SNHG6 to miR-26a-5p. 6.The expression of E2F7mRNA in lung adenocarcinoma was significantly higher than that in paracancerous tissues, and it was negatively correlated with the expression of miR-26a-5p and positively correlated with the expression of SNHG6. Double fluorescein reporter gene assay confirmed that miR-26a-5p could directly bind to E2F7. Western blot experiments showed that both SNHG6 and / or miR-26a-5p could affect the protein level of E2F7. 7.The effect of SNHG6 on the migration and EMT of lung adenocarcinoma cell line depends on the miR-26a-5p/E2F7 axis. 8.SNHG6 knockdown can inhibit the growth of transplanted tumor. Conclusions: 1.Long chain noncoding RNA SNHG6 is highly expressed in lung adenocarcinoma, and it is closely related to the poor prognosis of patients with lung adenocarcinoma. 2.SNHG6 plays a carcinogenic role in lung adenocarcinoma cells. Over-expression of SNHG6 can promote proliferation, cycle, migration and EMT of lung adenocarcinoma cells, while knockdown SNHG6 have a reverse effect. 3.The effect of SNHG6 expression change on the Biological function of Lung Adenocarcinoma cells depends on the miR-26a-5p/E2F7 axis. 4.Knockdown of SNHG6 inhibits tumor growth in nude mice. KEY WORDS: Lung adenocarcinoma; SNHG6; ceRNA; miR-26a-5p; E2F7 TYPE OF DISSERTATION: Application Fundamentals