口腔白斑病组织中环状RNA的差异表达谱分析

doi:10.12016/j.issn.2096-1456.2021.05.002

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

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.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. 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/Abstract

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

中图分类号:

R781.5

徐思明,宋雨翰,邵彦雄,陶岚,周海文. 口腔白斑病组织中环状RNA的差异表达谱分析[J]. 口腔疾病防治, 2021, 29(5): 296-305.

XU Siming,SONG Yuhan,SHAO Yanxiong,TAO Lan,ZHOU Haiwen. Differential expression profile of circRNAs in oral leukoplakia[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(5): 296-305.

图/表 10

表1 高通量测序口腔白斑组织的临床特点

Table 1 The clinical characteristics of patients with oral leukoplakia for sequencing

Number	Age (year)	Sex	Location	Dysplasia degree	Smoking history (No/Yes)	Alcohol history (No/Yes)
1	47	Female	Dorsal of the tongue	Mild	No	No
2	61	Female	Ventral of the tongue	Mild	No	No
3	41	Male	Ventral of the tongue	Mild	No	No
4	51	Female	Ventral of the tongue	Mild-moderate	No	No
5	69	Male	Ventral of the tongue	Mild-moderate	No	Yes
6	52	Female	Buccal	Moderate-severe	No	No

表2 qRT-PCR引物信息

Table 2 The primers used for qRT-PCR experiments

Circular RNA	Primer type	Primer sequence (5’-3’)
CircHLA-C (chr6:31238920-31324013-)	Forward	CGGCAAGGATTACATCGC
CircHLA-C (chr6:31238920-31324013-)	Reverse	CCTTCCCGTTCTCCAGGT
CircPLIN4 （chr19:4511523-4511918-）	Forward	TGTGTGCAGTGGGGTGAC
CircPLIN4 （chr19:4511523-4511918-）	Reverse	CCCTTTGGCGACATTCAC
CircMTX2 (chr2:177161588-177202305+)	Forward	TGACGCACTGGTATTTGGC
CircMTX2 (chr2:177161588-177202305+)	Reverse	GCATTTTCAGGCCAAGGTT
CircRNF13 （chr3:149613260-149639014+）	Forward	TGGGCATCTGTCTCATCTTG
CircRNF13 （chr3:149613260-149639014+）	Reverse	GTCGTTGGATCCCATGCT
CircSENP2 （chr3:185293003-185344181+）	Forward	CCTCAACAGCTGAATGGGA
CircSENP2 （chr3:185293003-185344181+）	Reverse	CCCACATCTCCCCCTTCT
CircPLEKHM2 （chr1:16044388-16047883+）	Forward	ACTCCGTCACCTCCACCA
CircPLEKHM2 （chr1:16044388-16047883+）	Reverse	CCGCAGGTAGCTCTCCAA
CircEMB （chr5:49694941-49707217-）	Forward	TGCTCAGCAGGAGCTTCA
CircEMB （chr5:49694941-49707217-）	Reverse	CTGCATTCAAATCCCCAGA
CircERICH1 （chr8:618598-624047-）	Forward	AAAACGCTGCTGCTCCTG
CircERICH1 （chr8:618598-624047-）	Reverse	TCTTTGGCTGGTCATGAGG
CircALDH3A2 （chr17:19554860-19575269+）	Forward	TCAAAGGTGGATTGGGGA
CircALDH3A2 （chr17:19554860-19575269+）	Reverse	CATCCAGCATGGTGAGCA
CircZNF720 （chr16:31733947-31734674+）	Forward	CGCTGTCTCTAAGCCGGA
CircZNF720 （chr16:31733947-31734674+）	Reverse	GGTGTTCCCACTCCTCCC

图1 OLK与NOM组织中circRNA的表达谱

Figure 1 Expression profiles of circRNAs in OLK versus NOM tissues a: hierarchical clustering analysis showed circRNA expression profles that were different between OLK tissues and NOM tissues; red represents upregulated circRNAs, and green represents downregulated circRNAs; b: scatter plot of differences in circRNA expression between OLK and NOM tissues; red represents upregulated circRNAs with FC ≥ 2.0 in OLK tissues; green represents downregulated circRNAs with FC ≥ 2.0; c: volcano plot of signifcantly dysregulated circRNAs in OLK tissues; red represents eligible circRNAs (FC ≥ 2.0; P < 0.05). OLK: oral leukoplakia; NOM: normal oral mucosal

图2 显著表达差异circRNA分布特点

Figure 2 Distribution of the characteristics of significantly dysregulated circRNAs a: significantly differentially expressed circRNAs were divided into 4 types according to the host gene structure（exon, intron, antisense, and sense overlapping circRNA）; b: filtered circRNAs were classified by whether they were newly discovered; c: classification according to the circRNA length; d: chromosome distribution of upregulated and downregulated signifcantly differentially expressed circRNAs

表3 OLK组织10个显著表达差异circRNA

Table 3 Ten circRNAs differentially expressed in OLK

CircRNA ID	FC	P	Chromosome	Strand	Gene name	Sequence length（bp）
CircHLA-C（chr6:31238920-31324013-）	7.719	< 0.001	chr6	-	HLA-C	85 094
CircPLIN4（chr19:4511523-4511918-)	5.992	0.001	chr19	-	PLIN4	396
CircMTX2（chr2:177161588-177202305+)	5.980	0.003	chr2	+	MTX2	40 718
CircRNF13（chr3:149613260-149639014+)	5.735	0.002	chr3	+	RNF13	379
CircSENP2（chr3:185293003-185344181+)	-8.583	< 0.001	chr3	+	SENP2	51 179
CircPLEKHM2（chr1:16044388-16047883+)	-6.122	< 0.001	chr1	+	PLEKHM2	435
CircERICH1（chr8:618598-624047-)	-5.917	< 0.001	chr8	-	ERICH1	954
CircEMB（chr5:49694941-49707217-)	-5.722	< 0.001	chr5	-	EMB	12 277
CircALDH3A2（chr17:19554860-19575269+）	-5.532	0.006	chr17	+	ALDH3A2	1 290
CircZNF720（chr16:31733947-31734674+）	-5.258	0.001	chr16	+	ZNF720	223

图3 OLK组织10个显著差异表达circRNA

Figure 3 Ten circRNAs differentially expressed in OLK a: ten selected circRNAs were subjected to qRT-PCR validation in 6 NOM and 6 OLK tissues; b: comparison of the results of high-throughput sequencing and qRT-PCR. c: RNase R analysis showed that circHLA-C, circPLIN4 and circRNF13 resisted digestion by RNase R; *：P < 0.05. OLK: oral leukoplakia; FC: fold change; HLA-C: human leukocyte antigen-C; PLIN4: perilipin-4; MTX2: metaxin 2; RNF13: ring finger protein 13; SENP2: sentrin/SUMO-specific proteases; PLEKHM2: pleckstrin homology and RUN domain containing M2; ERICH1: glutamate rich 1; EMB: embigin; ALDH3A2: aldehyde dehydrogenase 3 family member A2; ZNF720: zinc finger protein 720

图4 circHLA-C的成环机制及反剪接位点

Figure 4 Ring formation mechanism and back spliced site of circHLA-C HLA-C: human leukocyte antigen-C

图5 KEGG分析OLK中差异表达circRNA

Figure 5 KEGG analysis of the differential expression of circRNA in OLK a: top 10 in the KEGG analysis of upregulated circRNAs; b: top 10 in the KEGG analysis of downregulated circRNAs. The terms in the red frame indicate important pathways in OLK; OLK：oral leukoplakia

图6 circHLA-C在OLK中的ROC曲线分析

Figure 6 ROC curve analysis of circHLA-C in OLK OLK: oral leukoplakia

图7 CeRNA网络图

Figure 7 CeRNA interaction ceRNA：competing endogenous RNA

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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

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