METHODS: High-throughput RNA sequencing was performed to assess genome-wide lncRNA expression in CA and paired adjacent mucosal tissue. The expression of candidate lncRNAs and their target genes in larger CA specimens was validated using real-time quantitative reverse transcriptase polymerase chain reaction (RT‒qPCR). Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for the functional enrichment analysis of these candidate lncRNAs and differential mRNAs. The coexpressed mRNAs of the candidate lncRNAs, calculated by Pearson\'s correlation coefficient, were also analysed using GO and KEGG analysis. In addition, the interactions among differentially expressed lncRNAs (DElncRNAs)-cis-regulatory transcription factors (cisTFs)-differentially expressed genes (DEGs) were analysed and their network was constructed.
RESULTS: A total of 546 lncRNAs and 2553 mRNAs were found to be differentially expressed in CA compared to the paired control. Functional enrichment analysis revealed that the DEGs coexpressed with DElncRNAs were enriched in the terms of cell adhesion and keratinocyte differentiation, and the pathways of ECM-receptor interaction, local adhesion, PI3K/AKT and TGF-ß signaling. We further constructed the network among DElncRNAs-cisTFs-DEGs and found that these 95 DEGs were mainly enriched in GO terms of epithelial development, regulation of transcription or gene expression. Furthermore, the expression of 3 pairs of DElncRNAs and cisTFs, EVX1-AS and HOXA13, HOXA11-AS and EVX1, and DLX6-AS and DLX5, was validated with a larger number of specimens using RT‒qPCR.
CONCLUSIONS: CA has a specific lncRNA profile, and the differentially expressed lncRNAs play regulatory roles in mRNA expression through cis-acting TFs, which provides insight into their regulatory networks. It will be useful to understand the pathogenesis of CA to provide new directions for the prevention, clinical treatment and efficacy evaluation of CA.
方法:进行高通量RNA测序以评估CA和配对的邻近粘膜组织中的全基因组lncRNA表达。使用实时定量逆转录酶聚合酶链反应(RT-qPCR)验证了较大CA标本中候选lncRNAs及其靶基因的表达。此外,基因本体论(GO)和京都基因和基因组百科全书(KEGG)分析用于这些候选lncRNA和差异mRNA的功能富集分析。候选lncRNAs的共表达mRNA,由皮尔逊相关系数计算,还使用GO和KEGG分析进行了分析。此外,分析了差异表达lncRNAs(DElncRNAs)-顺式调节转录因子(cisTFs)-差异表达基因(DEGs)之间的相互作用,并构建了它们的网络。
结果:发现与配对对照相比,总共546个lncRNA和2553个mRNA在CA中差异表达。功能富集分析显示,与DElncRNAs共表达的DEGs在细胞粘附和角质形成细胞分化方面被富集,和ECM-受体相互作用的途径,局部附着力,PI3K/AKT和TGF-β信号传导。我们进一步构建了DElncRNAs-cisTFs-DEGs之间的网络,发现这95个DEGs主要富集在上皮发育的GO方面,转录或基因表达的调节。此外,3对DElncRNAs和cisTFs的表达,EVX1-AS和HOXA13,HOXA11-AS和EVX1以及DLX6-AS和DLX5使用RT-qPCR对大量标本进行了验证。
结论:CA具有特定的lncRNA谱,差异表达的lncRNAs通过顺式作用TFs在mRNA表达中发挥调节作用,这提供了深入了解他们的监管网络。为了解CA的发病机制提供新的预防方向,CA的临床治疗及疗效评价。