肺腺癌预后相关microRNA风险模型的构建及miR-101-3p抑癌机制的初步研究

发布时间：2018-04-19 14:27

本文选题：肺腺癌 + miRNAs　；参考：《南京医科大学》2017年硕士论文

【摘要】：第一部分基于TCGA数据库构建肺腺癌预后相关的microRNA风险模型目的:肺癌是全球范围内肿瘤致死的首要因素,尽管近年来诊断方法及治疗手段有了较大的提高,其预后仍然没有得到显著的改善。miRNAs(microRNAs,微小RNAs)是一类短小非编码RNAs,能够在转录后水平发挥重要的调控作用,其异常表达参与了肿瘤的发生与发展。越来越多的证据表明,miRNAs有望成为新的肿瘤标志物,包括肿瘤的预后。本研究旨在寻找LUAD(lung adenocarcinoma,肺腺癌)特异性的预后相关miRNAs,为LUAD患者的预后预测及个性化治疗方案的制定奠定一定的工作基础。方法:1.下载TCGA肿瘤数据库中522例LUAD患者的miRNA-Seq数据及详细的临床数据,并进行LUAD组织与癌旁正常组织中差异miRNAs分析。2.将患者随机分为训练集及测试集,在训练集中利用LASSOCOX回归模型进行LUAD预后相关miRNAs的筛选,并构建基于miRNAs表达谱的线性风险模型预测患者的预后。3.分别在测试集和总体样本中对风险模型预测患者预后的有效性进行验证。4.单因素及多因素COX回归分析miRNAs风险模型与其他临床变量的相关性。结果:1.相比于癌旁正常组织,LUAD组织中共筛出72个显著差异表达的miRNAs(差异表达倍数2,且调整后的P值0.05),其中表达量上调的miRNAs有45个,表达量下调的miRNAs有27个。2.在训练集中确定了 7个与LUAD患者总生存期相关的miRNAs,并基于7个miRNAs构建了预后风险模型,风险评分=(-0.109×miR-101-3p的表达量)+(-0.455×miR-148a-3p 的表达量)+(0.146×miR-192-5p 的表达量)+(0.179×miR-193b-3p 的表达量)+(0.383×miR-505-3p 的表达量)+(0.212×miR-584-5p 的表达量)+(-0.06×miR-99a-5p 的表达量)。3.在训练集、测试集及总体样本中,高风险组患者与低风险组患者相比总体生存时间均显著降低(所有P值0.05)。4.多因素COX回归分析显示,风险模型在训练集、测试集及总体样本中均是一个独立的预后因子(训练集:HR=1.97,P=0.02;测试集:HR=1.927,P=0.009;总体:HR=1.909,P=0.001)。结论:1.miR-101-3p、miR-148a-3p 及 miR-99a-5p 与 LUAD 患者的预后呈正相关;miR-192-5p、miR-193b-3p、miR-505-3p 及 miR-584-5p 与 LUAD 患者的预后呈负相关。2.基于以上7个miRNAs构建的风险模型能够很好地将LUAD患者分为预后不良的高风险组和低风险组,且独立于患者临床变量预测患者的预后。第二部分MiR-101-3p靶向TGFA在肺腺癌中发挥抑癌作用的初步研究目的:异常表达的miRNAs在肿瘤中发挥着双重作用,既能促进又能抑制肿瘤的发生与发展,同时与其靶基因共同构成了一个复杂的网络调控着肿瘤的进展。越来越多的证据表明miR-101-3p在肿瘤中呈异常表达的状态,包括LUAD。然而,miR-101-3p在LUAD中的功能及其潜在的分子机制仍需进一步阐明。方法:1.下载TCGA及GEO数据库中LUAD相关的miRNAs测序及芯片数据,整合分析miR-101-3p在LUAD组织中的表达水平。2.采用荧光定量PCR技术在细胞水平验证miR-101-3p的表达水平。3.采用CCK-8、克隆形成、细胞划痕、Transwell细胞侵袭实验检测过表达miR-101-3p后LUAD细胞增殖、迁移及侵袭等生物学功能的变化。4.采用TargetScan、miRanda、PITA及RNA22四个靶基因预测数据库对miR-101-3p的靶基因进行预测,并对靶基因进行功能及通路的富集分析,以确定miR-101-3p的最佳下游靶基因。5.运用荧光定量PCR技术及western blot技术在RNA及蛋白水平检测过表达miR-101-3p后TGFA的表达水平变化。6.利用TCGA及Kaplan-Meier plotter两个数据库分别分析TGFA在LUAD组织中的表达水平,及其与LUAD患者预后的相关性。结果:1.在 TCGA 及 3 个 GEO 数据集(GSE74190、GSE51853、GSE48414)中,与正常肺组织相比,LUAD组织中的miR-101-3p均呈显著低表达状态(所有P值0.001)。2.与正常肺上皮细胞BEAS-2B相比,LUAD细胞株A549及H1299中的miR-101-3p均呈显著低表达状态(所有P值0.001)。3.过表达miR-101-3p后LUAD细胞A549的增殖、迁移及侵袭能力均受到了显著的抑制(P0.05)。4.TGFA作为miR-101-3p的最佳预测靶标,在过表达miR-101-3p后,其RNA水平及蛋白水平均显著下调(P0.01)。5.LUAD组织中TGFA呈高表达状态,且高表达的TGFA与患者的不良预后相关(HR:1.44;P=0.0023)。结论:miR-101-3p在LUAD中能够通过靶向调节TGFA抑制LUAD细胞的增殖、迁移及侵袭。
[Abstract]:The first part is based on the TCGA database to construct the microRNA risk model related to the prognosis of lung adenocarcinoma. Lung cancer is the leading factor in cancer death worldwide. Although the diagnostic methods and treatment methods have been greatly improved in recent years, the prognosis is still not significantly improved,.MiRNAs (microRNAs, small RNAs) is a class of short and non coding. RNAs can play an important regulatory role at post transcriptional levels, and its abnormal expression is involved in the occurrence and development of tumors. More and more evidence suggests that miRNAs is expected to become a new tumor marker, including the prognosis of the tumor. This study aims to find the specific prognosis associated miRNAs for LUAD (lung adenocarcinoma, lung adenocarcinoma), for LUAD patients Prognosis prediction and the formulation of individualized treatment plan lay a certain work basis. Methods: 1. download the miRNA-Seq data and detailed clinical data of 522 cases of LUAD patients in the TCGA tumor database, and carry out the difference miRNAs analysis between the LUAD tissue and the normal tissue adjacent to the cancer, and randomly divide the patients into training set and test set, and use LA in training to concentrate on the training set. The SSOCOX regression model was used to screen the prognosis related miRNAs for LUAD and to construct a linear risk model based on miRNAs expression to predict the prognosis of patients..3. was used to verify the effectiveness of the prognosis of patients in the test set and the overall sample, respectively. The.4. single factor and the multiple factor COX regression analysis of the miRNAs risk model and other clinical changes were carried out. Results: 1. compared with para cancerous normal tissue, 72 significant differentially expressed miRNAs were screened in LUAD tissue (differential expression multiple 2, and adjusted P 0.05), of which 45 expressed miRNAs, and 27.2. in miRNAs expression determined 7 miRNAs associated with the total survival of LUAD patients in the training set. And based on 7 miRNAs, the prognosis risk model was constructed, the risk score = (-0.109 x miR-101-3p expression) + (-0.455 x miR-148a-3p expression) + (0.146 x miR-192-5p expression) + (the expression of 0.179 * miR-193b-3p) + + (0.212 x miR-584-5p expression) + (-0.06 * miR-99a-5p expression).3. is In the training set, the test set and the overall sample, the overall survival time of the patients in the high risk group was significantly lower than the low risk group (all P value 0.05).4. multiple factor COX regression analysis showed that the risk model was an independent prefactor in the training set, the test set and the overall sample (training set: HR=1.97, P=0.02; test set: HR=1.927, P=0 .009; overall: HR=1.909, P=0.001). Conclusion: 1.miR-101-3p, miR-148a-3p and miR-99a-5p are positively correlated with the prognosis of patients with LUAD; miR-192-5p, miR-193b-3p, miR-505-3p and miR-584-5p are negatively correlated with the prognosis of patients with LUAD. The risk group and the low risk group are independent of the patient's clinical variables to predict the prognosis of the patient. Second part second the preliminary study on the inhibitory effect of TGFA on lung adenocarcinoma: the abnormal expression of miRNAs plays a double role in the tumor, which can both promote and inhibit the occurrence and development of the tumor, and co construct with its target gene. A complex network regulates the progress of cancer. More and more evidence shows that miR-101-3p is abnormal in tumor, including LUAD., however, the function of miR-101-3p in LUAD and its potential molecular mechanism still need to be further clarified. Method: 1. download LUAD related miRNAs sequencing and chip data in TCGA and GEO database. Integrated analysis of the expression level of miR-101-3p in LUAD tissues.2. using fluorescence quantitative PCR technique to verify the expression level of miR-101-3p at the level of miR-101-3p using CCK-8, cloned formation, cell scratch, and Transwell cell invasion test to detect the proliferation of LUAD cells after miR-101-3p, migration and invasion of LUAD cells,.4. adopt Targe Four target gene prediction databases of tScan, miRanda, PITA and RNA22 were used to predict the target genes of miR-101-3p, and the function and pathway of the target genes were enriched and analyzed to determine the best downstream target gene of miR-101-3p.5. using fluorescence quantitative PCR technology and Western blot technology to detect the expression of miR-101-3p after RNA and protein levels. .6. using two databases of TCGA and Kaplan-Meier plotter to analyze the expression level of TGFA in LUAD tissue and the correlation with the prognosis of LUAD patients. Results: 1. in TCGA and 3 GEO datasets (GSE74190, GSE51853, GSE48414), compared with normal lung tissue, the expressions in the tissues were significantly lower. The state (all P 0.001).2. compared with the normal lung epithelial cell BEAS-2B, the miR-101-3p in the LUAD cell line A549 and H1299 showed significant low expression (all P value 0.001).3. overexpression miR-101-3p after LUAD cell A549 proliferation, migration and invasion ability were significantly inhibited as the best prediction target, After overexpression of miR-101-3p, the level of RNA and protein decreased significantly (P0.01) in.5.LUAD tissues, and the high expression of TGFA was associated with the poor prognosis of the patients (HR:1.44; P=0.0023). Conclusion: miR-101-3p in LUAD can inhibit the proliferation, migration and invasion of LUAD cells by targeting regulation TGFA.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

【参考文献】