小鼠高脂血症模型的竞争性内源RNA调控网络研究

目的基于高通量测序数据,探讨小鼠高脂血症模型中长链非编码RNA(long non-coding RNA,lncRNA)的表达特征及竞争性内源RNA(competitive endogenous RNA,ceRNA)调控网络,筛选lncRNA介导的参与血脂异常发生发展的关键调控通路。方法应用正常饮食和高脂饮食分别诱导ApoE-/-小鼠12周,建立高脂血症模型;进行肝脏全转录组测序,筛选差异表达的lncRNAs、微小RNAs(microRNAs,miRNAs)和信使RNAs(messenger RNAs,mRNAs);对差异表达的mRNAs进行基因功能(Gene Ontology,GO)分析和信号通路(KEGG)注释;结合并分析lncRNA-mRNA共表达关系、miRNA-mRNA靶向关系、lncRNA-miRNA共表达和靶向关系,构建lncRNA介导的ceRNA网络并筛选关键ceRNA调控轴。结果高脂血症小鼠肝脏全转录组测序筛选出117个差异表达lncRNAs、53个差异表达miRNAs和1689个差异表达mRNAs;差异表达的mRNAs主要参与脂质代谢过程、脂肪酸代谢过程和类固醇代谢过程等生物学功能,涉及PPAR信号通路和不饱和脂肪酸合成等途径;构建的ceRNA调控网络包括16个lncRNAs节点、18个miRNAs节点和33个mRNAs节点,其中lnc-Dubr介导的ceRNA调控轴可能对血脂异常具有重要调控作用。结论成功绘制出高脂血症小鼠肝脏lncRNA介导的ceRNA调控网络,并筛选出具有潜在生物学作用的ceRNA调控轴,有望为高脂血症及其他心血管疾病的发生发展和治疗提供新的线索和思路。     

甲基丙烯酸环氧丙酯诱导16HBE细胞恶性转化过程中LncRNA表达特征分析及ceRNA调控网络预测

目的： 筛选甲基丙烯酸环氧丙酯（GMA）诱导人支气管上皮16HBE细胞恶性转化过程中不同时期差异表达的LncRNA并以此为基础构建ceRNA调控网络,分析相关信号通路和生物学功能。方法： 收集用GMA和溶剂DMSO分别处理的16HBE细胞。采用LncRNA芯片检测并分析两组细胞中LncRNA表达量的差异,运用TargetScan和MiRanda数据库筛选出关键的差异表达LncRNA,使用Cytoscape构建以差异表达LncRNA为核心的ceRNA调控网络,之后对差异表达LncRNA的靶基因和通路进行分析预测,通过实时荧光定量PCR （qPCR）对上述LncRNA相对表达量进行检测验证。结果： 芯片筛选结果发现,与DMSO对照组相比,GMA处理组共16个LncRNA在细胞恶性转化过程中表达上调。在此基础上筛选构建了由3个LncRNA,32个mRNA和204个miRNA组成的LncRNA相关ceRNA调控网络,其中有3个调控轴（LncRNA G013234-hsa-miR-378b-TMEM129、LncRNA CTA-384D8.35-hsa-miR-486-3p-LYPD、LncRNA G087116-miR-29b-3p-NAV1）与GMA诱导16HBE细胞恶性转化过程相关（P<0.05）,提示这3个调控轴是与此恶性转化过程关联较为密切的ceRNA三元组。qPCR验证结果表明在细胞恶性转化过程的不同时期LncRNA G013234,CTA-384D8.35和G087116的相对表达水平变化趋势与LncRNA芯片结果一致。结论： 在GMA诱导16HBE细胞发生恶性转化过程中,LncRNA G013234、CTA-384D8.35、G087116在恶性转化不同时期均发生上调,以此为基础构建的ceRNA调控网络及预测得到细胞恶性转化相关的关键mRNA,为GMA诱导16HBE细胞恶性转化机制研究提供了支持数据。     

Bioinformatics analysis of the key potential ceRNA biomarkers in human thymic epithelial tumors

Background:Thymic epithelial tumors (TETs), originating from the thymic epithelial cells, are the most common primary neoplasms of the anterior mediastinum. Emerging evidence demonstrated that the competing endogenous RNAs (ceRNAs) exerted a crucial effect on tumor development. Hence, it is urgent to understand the regulatory mechanism of ceRNAs in TETs and its impact on tumor prognosis.Methods:TETs datasets were harvested from the UCSC Xena as the training cohort, followed by differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and miRNAs (DEmiRNAs) at different pathologic type (A, AB, B, and TC) identified via DESeq2 package. clusterProfiler package was utilized to carry out gene ontology and Kyoto encyclopedia of genes and genomes functional analysis on the DEmRNAs. Subsequently, the lncRNA-miRNA-mRNA regulatory network was constructed to screen the key DEmRNAs. After the key DEmRNAs were verified in the external cohort from Gene Expression Omnibus database, their associated-ceRNAs modules were used to perform the K-M and Cox regression analysis to build a prognostic significance for TETs. Lastly, the feasibility of the prognostic significance was validated by receiver operating characteristic (ROC) curves and the area under the curve.Results:Finally, a total of 463 DEmRNAs, 87 DElncRNAs, and 20 DEmiRNAs were obtained from the intersection of differentially expressed genes in different pathological types of TETs. Functional enrichment analysis showed that the DEmRNAs were closely related to cell proliferation and tumor development. After lncRNA-miRNA-mRNA network construction and external cohort validation, a total of 4 DEmRNAs DOCK11, MCAM, MYO10, and WASF3 were identified and their associated-ceRNA modules were significantly associated with prognosis, which contained 3 lncRNAs (lncRNA LINC00665, lncRNA NR2F1-AS1, and lncRNA RP11-285A1.1), 4 mRNAs (DOCK11, MCAM, MYO10, and WASF3), and 4 miRNAs (hsa-mir-143, hsa-mir-141, hsa-mir-140, and hsa-mir-3199). Meanwhile, ROC curves verified the accuracy of prediction ability of the screened ceRNA modules for prognosis of TETs.Conclusion:Our study revealed that ceRNAs modules might exert a crucial role in the progression of TETs. The mRNA associated-ceRNA modules could effectively predict the prognosis of TETs, which might be the potential prognostic and therapeutic markers for TETs patients.     

Construction of ceRNA network and identification of two differentially expressed circRNAs in hepatocellular carcinoma by bioinformatic analysis

Covalently closed circular RNAs (circRNAs) display dysregulated expression in several types of cancer. However, their functions remain largely unclear. In this work, datasets {"type":"entrez-geo","attrs":{"text":"GSE125469","term_id":"125469"}}GSE125469 and {"type":"entrez-geo","attrs":{"text":"GSE128274","term_id":"128274"}}GSE128274 of hepatocellular carcinoma (HCC) were selected from Gene Expression Omnibus (GEO) database. To identify differentially expressed genes (DEGs) in HCC and adjacent tissues, we used R package DESeq for analysis. Then, 15 DEcircRNAs, 65 DEmiRNAs, and 2084 DEmRNAs were identified comparing HCC and normal tissues. Next, to predict the target relationship of circRNA-miRNA and miRNA-mRNA in DEGs, we use the databases CircInteractome and starBase v2.0 for analysis. Finally, the ceRNA network of circRNA-miRNA-mRNA was established by Cytoscape software based on 2 DEcircRNAs (hsa_circ_0007813 and hsa_circ_0089372), 2 DEmiRNAs, and 98 DEmRNAs. In addition, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of DEGs to explore the function of DEGs in HCC. Functional enrichment analyses indicated DEmRNAs might be associated with HCC occurrence and progression. In general, our research reveals an important role of ceRNA’s molecular mechanism in HCC.     

Long non‐coding RNA TUG1 promotes migration and invasion by acting as a ceRNA of miR‐335‐5p in osteosarcoma cells

Long non‐coding RNA (lncRNA) have been the focus of increasing attention due to the role they play in many diseases, including osteosarcoma. The function of taurine upregulated gene 1 (TUG1) and its mechanism in osteosarcoma remain unclear. In our research, we found that TUG1 was elevated and correlated with a poor prognosis in osteosarcoma patients. In addition, the following functional experiment showed that decreased TUG1 could remarkably inhibit osteosarcoma cell migration and invasion, indicating that TUG1 functioned as an oncogene in osteosarcoma. Moreover, we revealed that TUG1 and Rho‐associated coiled‐coil‐containing protein kinase 1 (ROCK1), a metastasis‐related gene targeted by microRNA‐335‐5p (miR‐335‐5p), had the same miR‐335‐5p combining site. The subsequent luciferase assay verified TUG1 was a target of miR‐335‐5p. Furthermore, the results of a real‐time quantitative PCR showed that TUG1 and miR‐335‐5p could affect each other's expression. respectively. Finally, we affirmed that TUG1 affected ROCK1 expression and ROCK1‐mediated migration/invasion by working as a competitive endogenous RNA (ceRNA) via miR‐335‐5p. In summary, the findings of this study, based on ceRNA theory, combining the research foundation of miR‐335‐5p and ROCK1, and taking TUG1 as a new study point, provide new insight into molecular‐level reversing migration and invasion of osteosarcoma.     

AC016405.3, a novel long noncoding RNA,acts as a tumor suppressor through modulation of TET2 by microRNA‐19a‐5p sponging in glioblastoma

Long non‐coding RNAs (lncRNAs) are crucial regulators in various malignancies including glioblastoma multiforme (GBM). In the present study, we screened out a new lncRNA, AC016405.3, through a previous genome‐wide lncRNA microarray analysis in GBM. It showed that AC016405.3 was downregulated in GBM tissue specimens and cell lines, and it also illustrated that the downregulated AC016405.3 was closely correlated with several aggressive features of patients with GBM. Functionally, we found that overexpression of AC016405.3 suppressed GBM cells’ proliferation and metastasis using a gain of function experiment. We further showed that microRNA (miR)‐19a‐5p, a carcinogenic miRNA, was a downstream miRNA of AC016405.3. AC016405.3 was revealed as a target of miR‐19a‐5p, and overexpression of miR‐19a‐5p reversed the inhibitive effect of AC016405.3 on GBM cell proliferation and metastasis. Furthermore, a novel downstream gene of miR‐19a‐5p, TET2, was identified through a constructed microarray analysis. We showed that TET2 was downregulated in GBM and was involved in miR‐19a‐5p‐mediated proliferation and metastasis by directly being targeted. Finally, through a western blot assay and a series of functional CCK‐8 and metastatic assays, we showed that AC016405.3 suppressed proliferation and metastasis through modulation of TET2 by sponging of miR‐19a‐5p in GBM cells. In summary, the findings of the current study identified a novel lncRNA and illustrated that AC016405.3, acting as an anti‐oncogene, suppressed GBM cell proliferation and metastasis by regulating TET through miR‐19a‐5p sponging. Our present study might provide a new axis in the molecular treatment of GBM.     

LncRNA ZEB1⁃AS1对B细胞非霍奇金淋巴瘤增殖和凋亡的影响及其下游调控网络的分析

目的 探讨长链非编码 RNA（lncRNA） ZEB1-AS1对B 细胞非霍奇金淋巴瘤（B-cell non-Hodgkin's lymphoma,B-NHL）细胞增殖和凋亡的影响,构建lncRNA、miRNA、mRNA相关竞争性内源RNA（ceRNAs）网络。方法 采用Cox回归模型筛选GEO数据集（GSE31312）中与弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)预后相关的lncRNAs。采用慢病毒转染技术建立ZEB1-AS1敲低的B-NHL细胞模型,采用CCK-8法和流式细胞术分别检测敲低ZEB1-AS1及不同浓度（50 ng/mL、100 ng/mL、200 ng/mL、400 ng/mL）多柔比星（doxorubicin,Dox）处理后的细胞增殖与凋亡情况。构建以ZEB1-AS1为节点的 lncRNA-miRNA-mRNA ceRNA调控网络,利用GO、KEGG和PPI分析该调控网络下游相关的核心mRNA及其功能。结果 共筛选了20个高风险lncRNAs。预测与ZEB1-AS1有相互结合的miRNA 4个,与ZEB1-AS1呈共表达关系又与miRNA相结合的潜在下游mRNA 1 208个,以此构建lncRNA-miRNA-mRNA调控网络及PPI调控网络。在体外实验中,与空载组相比,敲低ZEB1-AS1可显著抑制细胞增殖和促进细胞凋亡（均P<0.05）,敲低ZEB1-AS1+Dox作用对抑制淋巴瘤细胞增殖和促进细胞凋亡有协同作用。结论  ZEB1-AS1敲低可抑制B-NHL细胞增殖和促进细胞凋亡,且可能增强Dox的药物敏感性;以ZEB1-AS1为节点构建的ceRNA调控网络,可能是DLBCL重要的调控机制和诊疗靶点。     

乳腺癌竞争内源性RNA网络构建与分析

目的:通过对癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中的乳腺癌数据进行综合分析,寻找在乳腺癌中差异表达的长链非编码RNA(long non-coding RNA,lncRNA）、微小RNA（microRNA,miRNA）和信使RNA（messenger RNA,mRNA）,构建乳腺癌竞争内源性RNA（competing endogenous RNA,ceRNA）网络,识别和预测ceRNA网络在乳腺癌中的作用。方法:下载TCGA数据库中乳腺癌组织样本基因表达谱数据,寻找差异表达的lncRNA、miRNA和mRNA,通过miRcode、miRTarBase、TargetScan和miRDB四个数据库对差异表达的RNA之间的关系进行分析,构建以上调和下调的miRNA为中心的ceRNA网络。采用Kaplan-Meier法分析乳腺癌ceRNA网络中的lncRNA、miRNA和mRNA表达量与患者生存预后的关系,采用基因富集分析法对乳腺癌ceRNA网络中mRNA基因功能和调控通路进行分析。结果:在乳腺癌中异常表达的350个lncRNA、185个miRNA和2 928个mRNA中,分别有23个lncRNA、27个miRNA、70个mRNA参与构建乳腺癌的ceRNA网络。Kaplan-Meier生存分析显示,lncRNA MAGI2-AS3（P=0.01）、GRIK1-AS1（P=0.03）以及miRNA hsa-miR-301b（P=0.01）、hsa-miR-503（P=0.04）和mRNA CCNE1（P=0.01）、KPNA2（P=0.02）、FLI1（P=0.02）、TGFBR2（P=0.02）这8个基因与乳腺癌患者的生存预后显著相关。70个mRNA主要被富集到20条通路上,其中有15条通路与肿瘤有关,最显著的通路为“Pathways in cancer”。结论:ceRNA网络在乳腺癌中发挥着重要的作用,多种差异表达基因与乳腺癌预后相关,可能成为潜在的肿瘤诊断标志物和治疗靶点。     

前列腺癌PC3细胞的增殖和侵袭

目的：探究长链非编码RNA（long non-coding RNA, lncRNA）LINC00308对前列腺癌细胞的增殖、侵袭及迁移的影响及其相关作用机制。方法：利用基因芯片在前列腺癌组织与癌旁对照组织中筛选差异表达的 lncRNA 及 mRNA, 并确定LINC00308 及甲状腺激素受体因子 13（thyroid hormone receptor interactor13,TRIP13）为研究对象。MTT 实验、平板克隆及Transwell和划痕实验检测LINC00308对前列腺癌细胞增殖、侵袭及迁移能力的影响,应用裸鼠移植瘤在体内验证上述影响,应用Western blotting及免疫组化实验在瘤组织和癌细胞中探究LINC00308对TRIP13表达的影响。生物信息学分析技术与RNA免疫共沉淀（RNA immunoprecipitation, RIP）及qPCR和双荧光素酶基因报告实验预测并探究miR-361-5p与LINC00308及TRIP13之间的相互作用机制,并利用平板克隆、Transwell侵袭实验对癌细胞恶性生物行为进行验证。结果：芯片结果及qPCR共同证实LINC00308（P<0.01）与 TRIP13（P<0.05）在前列腺癌组织及 4 种细胞系中均异常高表达 ;细胞功能实验结果表明过表达LINC00308可以促进前列腺癌细胞PC3的增殖、侵袭及迁移能力（均P<0.05）,而下调前列腺癌细胞中LINC00308表达起相反作用。裸鼠移植瘤实验证实,LINC00308能够在体内促进前列腺癌PC3细胞的成瘤（P<0.05或P<0.01）,且能够在体内体外促进TRIP13 表达（P<0.05）。生物信息学分析与 RIP 及 qPCR 和双荧光素酶基因报告实验结果证实 miR-361-5p 能够分别与LINC00308与TRIP13的3''-UTR靶向结合,且LINC00308能够通过吸附miR-361-5p而作为内源性竞争RNA（competing endoge‐nous RNA,ceRNA）调控TRIP13的表达,MTT、平板克隆及Transwell实验检测癌细胞的增殖、克隆形成和侵袭能力变化证实了三者之间的调控作用。结论：在前列腺癌组织和细胞中异常高表达的LINC00308通过发挥ceRNA的功能抑制miR-361-5p表达而增强TRIP13表达,从而促进前列腺癌的增殖、侵袭及迁移能力。