Supplementary Material for: Circular RNA Expression Profile in Laryngeal Squamous Cell Carcinoma Revealed by Microarray

Background/Aims: A growing body of evidence has suggested that circular RNAs (circRNAs) have crucial functions in the regulation of gene expression, and their dysregulation has been implicated in various types of cancers. However, the roles of circRNAs in laryngeal cancer remain largely unknown. This study investigated the global changes in the expression pattern of circRNAs in laryngeal squamous cell carcinoma (LSCC) to identify potential differentially expressed circRNAs. Methods: Microarray-based circRNA expression was determined in LSCC and paired normal laryngeal tissues. Pathway analyses of the genes producing differentially expressed circRNAs were performed to predict the function of circRNAs using standard enrichment computational methods. Expression levels of candidate circRNAs and microRNAs (miRNAs) were detected by quantitative real-time PCR. The circRNA/ miRNA interactions were constructed using bioinformatics methods to predict the binding of miRNA with circRNA. Results: We identified 506 differentially expressed circRNAs from human LSCC and normal laryngeal mucosa tissues. We confirmed that hsa_circ_0044520 and hsa_circ_0044529 were significantly upregulated in LSCC tissues. The most likely potential target miRNAs for hsa_ circ_0044520 and hsa_circ_0044529 were hsa-miR-4726-5p and hsa-miR-4640-5p, respectively. Functional analysis showed that hsa_circ_0044520 and hsa_circ_0044529 were involved in the process of collagen synthesis. Conclusion: Competitive endogenous RNA network prediction and bioinformatics functional analysis revealed that hsa_circ_0044520 and hsa_circ_0044529 play important regulatory roles by sponging hsa-miR-4726-5p and hsa-miR-4640-5p, thereby providing novel insights into the tumorigenesis of LSCC.

研究背景与目的：越来越多的研究证据表明，环状RNA（circular RNAs, circRNAs）在基因表达调控中发挥关键作用，其表达失调与多种癌症的发生密切相关。然而，环状RNA在喉癌中的作用目前仍不甚明确。本研究旨在分析喉鳞状细胞癌（laryngeal squamous cell carcinoma, LSCC）中环状RNA表达谱的整体变化，以筛选差异表达的潜在环状RNA。
方法：采用基于微阵列的表达检测技术，对喉鳞状细胞癌组织及其配对正常喉组织中的环状RNA表达谱进行测定。通过标准富集分析计算方法，对转录差异表达环状RNA的宿主基因进行通路富集分析，以预测环状RNA的功能。采用实时荧光定量PCR检测候选环状RNA及微小RNA（microRNAs, miRNAs）的表达水平。通过生物信息学方法构建环状RNA与微小RNA的互作网络，以预测二者的结合情况。
结果：本研究从人喉鳞状细胞癌组织及正常喉黏膜组织中筛选得到506个差异表达环状RNA。经验证，hsa_circ_0044520与hsa_circ_0044529在喉鳞状细胞癌组织中显著上调。hsa_circ_0044520与hsa_circ_0044529的潜在最优靶微小RNA分别为hsa-miR-4726-5p与hsa-miR-4640-5p。功能富集分析显示，hsa_circ_0044520与hsa_circ_0044529参与胶原蛋白合成过程。
结论：竞争性内源RNA网络预测及生物信息学功能分析表明，hsa_circ_0044520与hsa_circ_0044529可通过海绵吸附hsa-miR-4726-5p与hsa-miR-4640-5p发挥重要的调控作用，为喉鳞状细胞癌的肿瘤发生机制研究提供了新的见解。