口腔白斑病组织中环状RNA的差异表达谱分析

Differential expression profile of circRNAs in oral leukoplakia

目的探讨环状RNA(circRNA)在口腔白斑(oral leukoplakia,OLK)组织及正常口腔黏膜组织(nor⁃mal oral mucosal,NOM)中表达谱的差异及临床意义。方法采用高通量测序技术检测6对OLK和NOM组织中差异表达circRNA,利用qRT⁃PCR验证所筛选的10个circRNA在6对OLK与NOM组织中的表达情况。通过酶耐受实验和sanger测序验证目标circRNA成环情况,对20对OLK与NOM组织中目标circHLA⁃C进行qRT⁃PCR验证。分别使用UCSC基因组浏览器对circHLA⁃C进行可视化分析;利用GO和KEGG富集分析对差异表达circRNA进行功能分析;TargetScan、MiRanda预测目标circRNA下游miRNA、mRNA,并构建竞争性内源RNA(competing endogenous RNA,ceRNA)网络。结果测序数据显示OLK组织中共有366个显著差异表达的cir⁃cRNA,包括65个上调和301个下调circRNA。经qRT⁃PCR验证,筛选的10个circRNA中有7个表达结果同测序一致。且经酶耐受和sanger测序验证,确定上调的circHLA⁃C是具有反式剪接位点的真正的circRNA。相关性分析显示circHLA⁃C与OLK的异常增生程度呈正相关。ROC曲线分析提示circHLA⁃C具有诊断OLK的潜在价值,且具有较高的准确性和特异性。结论circRNA在OLK组织中异常表达,上调的circHLA⁃C表达可能与OLK异常增生程度相关,对OLK诊断具有指导价值。

Objective To investigate the differences and clinical significance of circRNA expression profiles in oral leukoplakia(OLK)tissues and normal oral mucosal(NOM)tissues.Methods High⁃throughput sequencing was used to detect differentially expressed circRNAs in 6 pairs of OLK and NOM tissues,and qRT⁃PCR was used to verify the expression of 10 circRNAs screened in 6 pairs of OLK and NOM tissues.The ring formation of circRNA was verified by RNase R digestion and Sanger sequencing,and the target circHLA⁃C was further verified by qRT⁃PCR in 20 pairs of OLK and NOM tissues.CircHLA⁃C was visualized using the UCSC genome browser(genome.ucsc.edu).The function of differentially expressed circRNAs was analyzed by GO and KEGG enrichment analyses.TargetScan and mi⁃Randa predicted the downstream miRNAs and mRNAs of the target circRNAs,and a ceRNA network related to the identified circRNAs was constructed in Cytoscape.Results Sequencing analysis showed that 366 circRNAs were significantly differentially expressed in OLK tissues,including 65 upregulated and 301 downregulated circRNAs.After qRT⁃PCR verification,7 of the 10 screened circRNAs were expressed consistent with the sequencing results.The upregulated circHLA⁃C was confirmed to be a real circRNA with back⁃splice junction sites by RNase R digestion and Sanger sequencing.Correlation analysis showed a positive correlation between circHLA⁃C and the degree of OLK dysplasia.ROC curve analysis suggested that circHLA⁃C had potential value in diagnosing OLK with high accuracy and specificity.Conclusion CircRNA was significantly abnormally expressed in OLK tissues,and the upregulation of circHLA⁃C may be related to the degree of OLK dysplasia,providing guiding value for the diagnosis of OLK in the future.