口腔疾病防治 ›› 2021, Vol. 29 ›› Issue (5): 296-305.DOI: 10.12016/j.issn.2096-1456.2021.05.002

• 基础研究 • 上一篇    下一篇

口腔白斑病组织中环状RNA的差异表达谱分析

徐思明1(),宋雨翰1,邵彦雄1,陶岚2(),周海文1()   

  1. 1.上海交通大学医学院附属第九人民医院·口腔医学院口腔黏膜病科,国家口腔疾病临床研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海(200011)
    2.上海交通大学医学院附属第九人民医院·口腔医学院口腔综合科,国家口腔疾病临床研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海(200011)
  • 收稿日期:2020-09-25 修回日期:2020-12-04 出版日期:2021-05-20 发布日期:2021-03-08
  • 通讯作者: 陶岚,周海文
  • 作者简介:徐思明,硕士研究生,Email: xusimingmm@163.com
  • 基金资助:
    国家自然科学基金项目(81170968)

Differential expression profile of circRNAs in oral leukoplakia

XU Siming1(),SONG Yuhan1,SHAO Yanxiong1,TAO Lan2(),ZHOU Haiwen1()   

  1. 1. Department of Oral Mucosal Diseases, Shanghai Ninth People′s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China
    2. Department of General Dentistry, Shanghai Ninth People′s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China
  • Received:2020-09-25 Revised:2020-12-04 Online:2021-05-20 Published:2021-03-08
  • Contact: Lan TAO,Haiwen ZHOU
  • Supported by:
    The National Natural Science Foundation of China(81170968)

摘要:

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(normal 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circRNA,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。

关键词: 环状RNA, 口腔白斑病, 癌前病变, 高通量测序, 差异表达谱, ceRNA, miRNA, circHLA-C, 上皮异常增生

Abstract:

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 miRanda 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.

Key words: circRNA, oral leukoplakia, premalignant lesion, high-throughput sequencing, differential expression profile, competing endogenous RNA, miRNA, circHLA-C, epithelial dysplasia

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