Using RNA sequencing to identify a putative lncRNA-associated ceRNA network in laryngeal squamous cell carcinoma

Accumulating evidence indicates that lncRNAs can interact with miRNAs to regulate target mRNAs through competitive interactions. However, this mechanism remains largely unexplored in laryngeal squamous cell carcinoma (LSCC). In this study, transcriptome-wide RNA sequencing was performed on 3 pairs of LSCC tissues and adjacent normal tissues to investigate the expression profiles of lncRNAs, miRNAs and mRNAs, with differential expression of 171 lncRNAs, 36 miRNAs and 1709 mRNAs detected. Seven lncRNAs, eight mRNAs and three miRNAs were identified to be dysregulated in patients’ tissues by using qRT-PCR. GO and KEGG pathway enrichment analyses were performed to elucidate the potential functions of these differentially expressed genes in LSCC. Subsequently, a ceRNA (lncRNA-miRNA-mRNA) network including 4631 ceRNA pairs was constructed based on predicted miRNAs shared by lncRNAs and mRNAs. Cis- and transregulatory lncRNAs were analysed by bioinformatics-based methods. Importantly, mRNA-related ceRNA networks (mRCNs) were further obtained based on potential cancer-related coding genes. Coexpression between lncRNAs and downstream mRNAs was used as a criterion for the validation of mRCNs, with the ZNF561-AS1-miR217-WNT5A and SATB1-AS1-miR1299-SAV1/CCNG2/SH3 KBP1/JADE1/HIPK2 ceRNA regulatory interactions determined, followed by experimental validation after siRNA transfection. Moreover, ceRNA activity analysis revealed that different activities of ceRNA modules existing in specific pathological environments may contribute to the tumorigenesis of LSCC. Consistently, both downregulated SATB1-AS1 and ZNF561-AS1 significantly promoted laryngeal cancer cell migration and invasion, indicating their important roles in LSCC via a ceRNA regulatory mechanism. Taken together, the results of this investigation uncovered and systemically characterized a lncRNA-related ceRNA regulatory network that may be valuable for the diagnosis and treatment of LSCC.

越来越多的研究证据表明，长链非编码RNA（long non-coding RNAs, lncRNAs）可通过竞争性相互作用与微小RNA（microRNAs, miRNAs）结合，进而调控靶信使RNA（messenger RNAs, mRNAs）的表达。然而，该调控机制在喉鳞状细胞癌（laryngeal squamous cell carcinoma, LSCC）中的研究仍未得到充分阐明。本研究对3对喉鳞状细胞癌组织及配对癌旁正常组织开展全转录组RNA测序，以分析长链非编码RNA、微小RNA及信使RNA的表达谱，共检测到171个差异表达长链非编码RNA、36个差异表达微小RNA及1709个差异表达信使RNA。通过实时定量逆转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR），本研究在患者组织中鉴定出7个异常表达的长链非编码RNA、8个异常表达的信使RNA及3个异常表达的微小RNA。本研究通过基因本体（Gene Ontology, GO）及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析，阐明了上述差异表达基因在喉鳞状细胞癌中的潜在功能。随后，基于长链非编码RNA与信使RNA共享的预测微小RNA，本研究构建了包含4631对互作对的内源竞争RNA（competing endogenous RNA, ceRNA）网络。本研究通过生物信息学方法分析了顺式调控与反式调控长链非编码RNA的功能。重要的是，基于潜在癌症相关编码基因，本研究进一步获取了信使RNA相关内源竞争RNA网络（mRNA-related competing endogenous RNA network, mRCNs）。以长链非编码RNA与下游信使RNA的共表达作为信使RNA相关内源竞争RNA网络的验证标准，最终确定了ZNF561-AS1-miR217-WNT5A及SATB1-AS1-miR1299-SAV1/CCNG2/SH3KBP1/JADE1/HIPK2这两组内源竞争RNA调控互作，并通过小干扰RNA（small interfering RNA, siRNA）转染实验完成了实验验证。此外，内源竞争RNA活性分析显示，特定病理微环境中存在的不同内源竞争RNA模块活性，可能参与喉鳞状细胞癌的肿瘤发生过程。一致性实验结果表明，表达下调的SATB1-AS1与ZNF561-AS1均可显著促进喉癌细胞的迁移与侵袭，提示二者通过内源竞争RNA调控机制在喉鳞状细胞癌中发挥重要作用。综上，本研究揭示并系统表征了一个与长链非编码RNA相关的内源竞争RNA调控网络，该网络有望为喉鳞状细胞癌的诊断与治疗提供潜在应用价值。