长链非编码RNA在人类疾病中的研究进展

人类基因组研究计划证实:在人类的基因序列中只有2/3的序列被反转录,其中仅有不到2%的核酸序列用于编码蛋白,非编码RNA数量远远大于编码RNA。而在非编码RNA中,绝大多数是长链非编码RNA(Long non-coding RNA,lncRNA),其在多个层面上参与基因表达和细胞分化,与人类疾病密切相关。现就长链非编码RNA的调节机制及其在人类疾病研究中的最新研究进展做一综述。

姜黄素调控长链非编码RNA AK125910的表达介导肝癌细胞凋亡

目的 检测姜黄素诱导肝癌细胞凋亡中lncRNA表达谱的改变及关键lncRNA的筛选鉴定。方法 使用不同浓度姜黄素刺激肝癌细胞株HepG2不同时间后,提取总RNA进行lncRNA芯片杂交检测lncRNA表达谱的改变,筛选出差异表达lncRNA;并利用MTT法和TUNEL染色观察HepG2细胞的凋亡,Real-time PCR验证差异表达的lncRNA AK125910在其中的作用。结果 与对照组细胞相比,姜黄素处理后的肝癌细胞中有5 432条lncRNA表达出现改变,而其中变化倍数大于3的lncRNA有8条,表达差异化最显著的是lncRNA AK058003,上调7.62倍。在姜黄素诱导肝癌细胞凋亡中,lncRNA AK125910表达上调7.16倍,与芯片杂交结果基本一致。结论 姜黄素诱导肝癌细胞凋亡过程中lncRNA表达谱出现显著变化,其中差异性表达最显著的lncRNA AK125910可能参与了肝癌细胞的凋亡进程。

长链非编码RNA HOTAIR在胃癌中的表达及临床意义

目的:探讨长链非编码RNA HOTAIR在胃癌及癌旁组织中的表达及其与临床病理特征之间的关系。方法:利用实时定量PCR法检测55例胃癌组织及其配对癌旁正常组织中长链非编码RNA HOTAIR的表达情况,并分析其与胃癌临床病理学特征之间的联系。结果:实时定量PCR法检测HOTAIR在胃癌组织中的相对表达量为0.0232±0.0073,其配对癌旁正常组织相对表达量为0.0071±0.0017,差异具有统计学意义(P<0.05)。长链非编码RNA HOTAIR的表达与胃癌患者肿瘤淋巴结转移、远处转移、TNM分期显著相关(P<0.05),与年龄、性别、分化程度等无关(P>0.05)。结论:长链非编码RNA HOTAIR在胃癌组织中表达显著高于癌旁组织,与胃癌转移、TNM分期等病理因素相关,可能在胃癌的侵袭、转移过程中起重要作用。

m^(6)A modification of lncRNA in middle ear cholesteatoma

Objective:Middle ear cholesteatoma is a non-tumorous condition that typically leads to hearing loss,bone destruction,and other severe complications.Despite surgery being the primary treatment,the recurrence rate remains high.Therefore,exploring the molecular mechanisms underlying cholesteatoma is crucial for discovering new therapeutic approaches.This study aims to explore the involvement of N6-methyladenosine(m^(6)A)methylation in long non-coding RNAs(lncRNAs)in the biological functions and related pathways of middle ear cholesteatoma.Methods:The m^(6)A modification patterns of lncRNA in middle ear cholesteatoma tissues(n=5)and normal post-auricular skin tissues(n=5)were analyzed using an lncRNA m^(6)A transcriptome microarray.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were conducted to identify potential biological functions and signaling pathways involved in the pathogenesis of middle ear cholesteatoma.Methylated RNA immunoprecipitation(MeRIP)-PCR was used to validate the m^(6)A modifications in cholesteatoma and normal skin tissues.Results:Compared with normal skin tissues,1525 lncRNAs were differentially methylated in middle ear cholesteatoma tissues,with 1048 showing hypermethylation and 477 showing hypomethylation[fold change(FC)≥3 or<1/3,P<0.05].GO enrichment analysis indicated that hypermethylated lncRNAs were involved in protein phosphatase inhibitor activity,neuron-neuron synapse,and regulation ofα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA)receptor activity.Hypomethylated lncRNAs were associated with mRNA methyltransferase activity,secretory granule membrane,and mRNA methylation.KEGG analysis revealed that hypermethylated lncRNAs were mainly associated with 5 pathways:the Hedgehog signaling pathway,viral protein interaction with cytokines and cytokine receptors,mitogen-activated protein kinase(MAPK)signaling pathway,cytokine-cytokine receptor interaction,and adrenergic signaling in cardiomyocytes.Hypomethylated lncRNAs were mainly involved in 4 pathways:Renal cell carcinoma,tumor necrosis factor signaling pathway,transcriptional misregulation in cancer,and cytokine-cytokine receptor interaction.Additionally,MeRIP-PCR confirmed the changes in m^(6)A methylation levels in NR_033339,NR_122111,NR_130744,and NR_026800,consistent with microarray analysis.Real-time PCR also confirmed the significant upregulation of MAPK1 and NF-κB,key genes in the MAPK signaling pathway.Conclusion:This study reveals the m^(6)A modification patterns of lncRNAs in middle ear cholesteatoma,suggests a direction for further research into the role of lncRNA m^(6)A modification in the etiology of cholesteatoma.The findings provide potential therapeutic targets for the treatment of middle ear cholesteatoma.

与放疗抵抗相关的长链非编码RNA在不同放疗敏感性结直肠癌细胞株中的表达

目的 筛选与结直肠癌细胞放疗敏感性相关的长链非编码RNA（lncRNA）.方法 将HT29、SW480、RKO、Lovo和HCT116等5株结直肠癌细胞梯度照光后行克隆形成实验,通过细胞存活率（SF2值）来检测5株细胞的放疗敏感性差异;利用高通量lncRNA芯片筛选在SW480、RKO和Lovo细胞株中两两比较表达差异均大于2倍的lncRNA,并通过实时定量PCR进一步检测所选lncRNA在5株结直肠癌细胞中的表达差异.结果 5株结直肠癌细胞放疗敏感性由低至高（即SF2值由高至低）依次为HT29 （0.83±0.03）、SW480 （0.69±0.02）、RKO （0.53±0.02）、Lovo （0.47±0.05）和HCT116（0.32±0.03）（P＜0.01）.lncRNA芯片筛选得到5种与结直肠癌细胞放疗敏感性相关的lncRNA基因,其中R05532、NR_015441和NR_033374基因表达水平与细胞放疗抵抗呈正相关（均P＜0.01）,而NR_073156和AA745020基因表达水平与细胞放疗抵抗无明显相关性（均P＞0.05）.结论 R05532、NR_015441和NR_033374三种lncRNA可能成为结直肠癌细胞放疗敏感性的预测分子,其高表达提示放疗抵抗。

长链非编码 RNA 在肾透明细胞癌组织中的表达

目的：分析长链非编码RNA（ long non－coding RNA，lncRNA）在肾透明细胞癌组织中的表达情况，并筛选出与肾透明细胞癌密切相关的lncRNA，探讨lncRNA在肾透明细胞癌诊断中的作用。方法2012年3月至2013年6月收集40例肾透明细胞癌患者的新鲜癌组织及癌旁组织，Trizol试剂提取总RNA，用Nanodrop 1000及变性琼脂糖凝胶电泳进行RNA检测。通过Arraystar Human LncRNA Microarray芯片筛选肾透明细胞癌组织与癌旁组织中的差异表达lncRNA，采用实时定量PCR方法验证所筛选的lncRNA在40对肾透明细胞癌组织与癌旁组织中的表达情况，采用受试者工作特征（ receiver－operating characteristic，ROC）曲线验证所筛选lncRNA的诊断效率。结果与癌旁组织相比，在肾透明细胞癌组织中差异表达2倍以上的lncRNA有1787个，其中941个表达上调、846个表达下调。实时定量PCR测定NR＿034095、NR＿038974、ENST00000571724及ENST00000566575的表达水平与基因芯片分析结果一致。 NR＿034095、NR＿038974、ENST00000571724及ENST00000566575诊断肾透明细胞癌的ROC曲线下面积分别为0．928（95％CI 0．873～0．984）、0．759（95％CI 0．647～0．871）、0．833（95％CI 0．747～0．919）及0．887（95％CI 0．815～0．959）。结论 NR＿034095、NR＿038974、ENST00000571724及ENST00000566575在肾透明细胞癌组织和癌旁组织中差异表达显著，可能与肾透明细胞癌的发生、发展密切相关，有可能成为肾透明细胞癌分子诊断和基因治疗的新靶点。

长链非编码RNA测序技术应用探讨

长链非编码RNA （ long non-coding RNA， LncRNA）是一类转录本长度超过200 nt的RNA分子，由RNA聚合酶Ⅱ（ RNA polymeraseⅡ）加工合成，是基因组序列转录生成的产物。近年来，随着高通量技术的不断发展，越来越多的LncRNA逐渐被发现并成为研究热点。 LncRNA作为新兴分子标志物与肿瘤的发生、发展机制密切相关。目前运用于功能性LncRNA筛查的分子高通量新一代测序方法高效且节源，并广泛应用于基因检测。 RNA-seq、 RIP-seq及CLIP-Seq此3种高通量测序在LncRNA中的研究运用已经报道，文章旨在深入探讨技术特点以帮助发掘肿瘤分子标志物。

长链非编码RNA在消化系统肿瘤中的研究进展

长链非编码RNA(lncRNA)是一系列广泛转录自人及其他哺乳动物基因组的长度超过200 nt的核苷酸片段,通常没有蛋白编码功能,一度被认为是基因组的转录"噪音"。近年来一些lncRNA被发现具有多种生物学功能,成为分子生物学领域研究的热点。已经在多种肿瘤中发现有lncRNA的异常表达,并且异常表达的lncRNA和肿瘤的复发、转移及预后有关。本文中将探讨近期发现的与消化系统肿瘤相关的lncRNA,部分着重于具有促癌作用的lncRNA,以期为消化系统肿瘤的诊断、预后及分子治疗提供线索。

Roles of miRNA and lncRNA in triple-negative breast cancer

Triple-negative breast cancer(TNBC)is currently the most malignant subtype of breast cancer without effective targeted therapies,which makes its pathogenesis an important target for research.A growing number of studies have shown that non-coding RNA(ncRNA),including microRNA(miRNA)and long non-coding RNA(lncRNA),plays a significant role in tumorigenesis.This review summarizes the roles of miRNA and lncRNA in the progression,diagnosis,and neoadjuvant chemotherapy of TNBC.Aberrantly expressed miRNA and lncRNA are listed according to their roles.Further,it describes the multiple mechanisms that lncRNA shows for regulating gene expression in the nucleus and cytoplasm,and more importantly,describes lnc RNA-regulated TNBC progression through complete combining with miRNA at the post-transcriptional level.Focusing on miRNA and lncRNA associated with TNBC can provide new insights for early diagnosis and treatment—they can be targeted in the future as a novel anticancer target of TNBC.

Exosomal long non-coding RNAs: biological properties and therapeutic potential in cancer treatment

Exosomes and long non-coding RNAs(lncRNAs)are emerging as important elements contributing to a more comprehensive understanding of cancer development and progression.The discovery of lncRNAs in exosomes further indicates their bona fide biological functional roles in cancer development and drug resistance.In this review,we describe the biogenesis of exosomes and summarize the function of exosomal lncRNAs in the field of cancer research.These findings strikingly advance current knowledge of exosomal lncRNAs and suggest that they may be promising diagnostic biomarkers and therapeutic targets for cancer.

Role of cytoplasmic lncRNAs in regulating cancer signaling pathways

Cancer remains a serious healthcare problem despite significant improvements in early detection and treatment approaches in the past few decades. Novel biomarkers for diagnosis and therapeutic strategies are urgently needed. In recent years, long noncoding RNAs(lncRNAs) have been reported to be aberrantly expressed in tumors and show crosstalk with key cancer-related signaling pathways. In this review, we summarized the current progress of research on cytoplasmic lncRNAs and their roles in regulating cancer signaling and tumor progression, further characterization of which may lead to effective approaches for cancer prevention and therapy.

Transcriptomic analysis reveals key lnc RNAs associated with ribosomal biogenesis and epidermis differentiation in head and neck squamous cell carcinoma

Objective： In this study, we aimed to expand current knowledge of head and neck squamous cell carcinoma （HNSCC）-associated long noncoding RNAs （IncRNAs）, and to discover potential IncRNA prognostic biomarkers for HNSCC based on next-generation RNA-seq. Methods： RNA-seq data of 546 samples from patients with HNSCC were downloaded from The Cancer Genome Atlas （TCGA）, including 43 paired samples of tumor tissue and adjacent normal tissue. An integrated analysis incorporating differential expression, weighted gene co-expression networks, functional enrichment, clinical parameters, and survival analysis was conducted to discover HNSCC-associated IncRNAs. The function of CYTOR was verified by cell-based experiments. To further identify IncRNAs with prognostic significance, a multivariate Cox proportional hazard regression analysis was performed. The identified IncRNAs were validated with an independent cohort using clinical feature relevance analysis and multivariate Cox regression analysis. Results： We identified nine HNSCC-relevant IncRNAs likely to play pivotal roles in HNSCC onset and development. By functional enrichment analysis, we revealed that CYTOR might participate in the multistep pathological processes of cancer, such as ribosome biogenesis and maintenance of genomic stability. CY-I-OR was identified to be positively correlated with lymph node metastasis, and significantly negatively correlated with overall survival （OS） and disease free survival （DFS） of HNSCC patients. Moreover, CYTOR inhibited cell apoptosis following treatment with the chemotherapeutic drug diamminedichloroplatinum （DDP）. HCG22, the most dramatically down-regulated IncRNA in tumor tissue, may function in epidermis differentiation. It was also significantly associated with several clinical features of patients with HNSCC, and positively correlated with patient survival. CYTOR and HCG22 maintained their prognostic values in- dependent of several clinical features in multivariate Cox hazards analysis. Notably, validation either based on an independent HNSCC cohort or by laboratory experiments confirmed these findings. Conclusions： Our transcriptomic analysis suggested that dysregulation of these HNSCC-associated IncRNAs might be involved in HNSCC oncogenesis and progression. Moreover, CYTOR and HCG22 were confirmed as two independent prognostic factors for HNSCC patient survival, providing new insights into the roles of these IncRNAs in HNSCC as well as clinical applications.

Transcriptome-wide identification of differentially expressed genes and long non-coding RNAs in nitroglycerin-tolerant rat aorta

Tolerance to nitroglycerin(GTN)greatly limits its long-time application,and the underlying mechanism remains largely unexplored.In the present study,we aimed to investigate the comprehensive changes in the transcriptome of rat aorta tolerant to GTN by analyzing lncRNA expression.We employed the RNA sequencing(RNA-seq)technique to identify mRNAs and lncRNAs.Ingenuity pathway analysis(IPA)was used for pathway and functional analysis.RT-qPCR was used to validate the RNA-seq results.We identified 22788 genes(reads per kilobase million[RPKM]>0.1,14720 protein-coding genes and 4408 lncRNAs),including 115 differentially expressed(DE)mRNAs(65 up-regulated and 50 down-regulated)and 104 DE lncRNAs(56 up-regulated and 48 down-regulated),in GTN-tolerant aortas.IPA revealed the inhibition of a canonical pathway“Signaling by Rho Family GTPases”and alteration in six upstream regulators.Functional analysis showed that 11 genes were related to“disorder of blood pressure”.We predicted the cis-target genes of DE lncRNAs by the analysis of their neighboring genes.The results revealed the 28 DE lncRNAs adjacent to the 26 protein-coding genes.Many DE mRNAs and cis-target genes of DE lncRNAs have been implicated in the regulation of blood pressure or cell contraction.These results suggested that the dysregulated mRNAs and lncRNAs contributed to the development of GTN tolerance and could serve as potential targets to prevent and reverse GTN tolerance.

Role of LINC00152 in non-small cell lung cancer

Non-small cell lung cancer(NSCLC)accounts for about 85%of all lung cancer cases.The pathogenesis of NSCLC involves complex gene networks that include different types of non-coding RNAs,such as long non-coding RNAs(lnc RNAs).The role of lnc RNAs in NSCLC is gaining an increasing interest as their function is being explored in various human cancers.Recently,a new oncogenic lnc RNA,LINC00152(cytoskeleton regulator RNA(CYTOR)),has been identified in different tumor types.In NSCLC,the high expression of LINC00152 in tumor tissue and peripheral blood samples has been shown to be associated with worse prognoses of NSCLC patients.Overexpression of LINC00152 has been confirmed to promote the proliferation,invasion,and migration of NSCLC cells in vitro,as well as increase tumor growth in vivo.This review discusses the role of LINC00152 in NSCLC.

Comparative transcriptomic analysis of vascular endothelial cells after hypoxia/re-oxygenation induction based on microarray technology

Objective: To provide comprehensive data to understand mechanisms of vascular endothelial cell(VEC) response to hypoxia/re-oxygenation. Methods: Human umbilical vein endothelial cells(HUVECs) were employed to construct hypoxia/re-oxygenation-induced VEC transcriptome profiling. Cells incubated under 5% O2, 5% CO2, and 90% N2 for 3 h followed by 95% air and 5% CO2 for 1 h were used in the hypoxia/re-oxygenation group. Those incubated only under 95% air and 5% CO2 were used in the normoxia control group. Results: By using a well-established microarray chip consisting of 58 339 probes, the study identified 372 differentially expressed genes. While part of the genes are known to be VEC hypoxia/re-oxygenation-related, serving as a good control, a large number of genes related to VEC hypoxia/re-oxygenation were identified for the first time. Through bioinformatic analysis of these genes, we identified that multiple pathways were involved in the reaction. Subsequently, we applied real-time polymerase chain reaction(PCR) and western blot techniques to validate the microarray data. It was found that the expression of apoptosis-related proteins, like pleckstrin homology-like domain family A member 1(PHLDA1), was also consistently up-regulated in the hypoxia/re-oxygenation group. STRING analysis found that significantly differentially expressed genes SLC38A3, SLC5A5, Lnc-SLC36 A4-1, and Lnc-PLEKHJ1-1 may have physical or/and functional protein–protein interactions with PHLDA1. Conclusions: The data from this study have built a foundation to develop many hypotheses to further explore the hypoxia/re-oxygenation mechanisms, an area with great clinical significance for multiple diseases.