基于高通量测序的结直肠癌组织中环状RNA功能研究与预后分析

doi:10.3969/j.issn.1674-8115.2021.02.010

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (2): 187-195.doi: 10.3969/j.issn.1674-8115.2021.02.010

基于高通量测序的结直肠癌组织中环状RNA功能研究与预后分析

傅中懋(), 罗再, 戎泽印, 章建明, 李腾飞, 余志龙, 黄陈()

上海交通大学附属第一人民医院普外科，上海 200080

收稿日期:2020-04-26 出版日期:2021-02-28 发布日期:2021-02-28
通讯作者: 黄陈 E-mail:fzmaoalb@163.com;richard-hc@hotmail.com
作者简介:傅中懋（1996—），男，硕士生；电子信箱：fzmaoalb@163.com。
基金资助:
国家自然科学基金(81772526);上海市教育委员会高峰高原学科建设计划(20161425);上海交通大学医工交叉项目(YG2017MS28);上海市松江区科技攻关项目(18SJKJGG23)

Study on the function and prognosis of circular RNA in colorectal cancer tissues based on high-throughput sequencing

Zhong-mao FU(), Zai LUO, Ze-yin RONG, Jian-ming ZHANG, Teng-fei LI, Zhi-long YU, Chen HUANG()

Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, China

Received:2020-04-26 Online:2021-02-28 Published:2021-02-28
Contact: Chen HUANG E-mail:fzmaoalb@163.com;richard-hc@hotmail.com
Supported by:
National Natural Science Foundation of China(81772526);Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(20161425);Shanghai Jiao Tong University Medical Engineering Cross Fund(YG2017MS28);Shanghai Songjiang Science and Technology Project Fund(18SJKJGG23)

摘要/Abstract

摘要：

目的·通过分析结直肠癌组织中环状RNA（circular RNA，circRNA）的差异表达谱，揭示结直肠癌的发生机制及差异表达circRNA与结直肠癌患者预后之间的关系。方法·收集上海交通大学附属第一人民医院普外科行结直肠癌根治术12例患者的肿瘤与癌旁正常组织，使用高通量测序技术检测结直肠癌及癌旁组织中circRNA的表达情况。通过基因本体（gene ontology，GO）注释分析及京都基因和基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析，对差异表达的circRNA进行功能预测。在显著表达上调与下调的circRNA中各挑选2个circRNA，采用实时荧光定量PCR（real-time fluorescent quantitative PCR，RT-qPCR）技术在44对结直肠癌组织中验证测序结果的准确性，通过搜索公共数据库构建这4种circRNA与微RNA（microRNA，miRNA）特异性结合网络图。最后结合患者预后资料判断差异表达circRNA与预后的关系。结果·经高通量测序共检测出20 924个circRNA。与癌旁正常组织比较在结直肠癌组织中异常显著表达的circRNA共有373个（︱log₂FC︱>1，P<0.05），其中显著上调的有243个，显著下调的有130个。在44对结直肠癌样本中，RT-qPCR验证结果与高通量测序数据筛选的circRNA表达趋势基本一致。通过绘制circRNA-miRNA-mRNA互作网络后发现，与circRNA相关的miRNA与mRNA在结直肠癌患病过程中可能起着关键作用。结合患者预后信息，hsa_circ_0023984、hsa_circ_0008192高表达的患者预后较差；hsa_circ_0020093、 hsa_circ_0069922高表达的患者预后较好。结论·结直肠癌组织中存在异常表达的circRNA，这对进一步研究circRNA在结直肠癌发生和发展中的作用机制奠定了基础，对未来新型临床诊断标志物及治疗的发展提供有价值信息。

关键词: 结直肠癌, 环状RNA, 预后, 调控网络

Abstract:

Objective·To reveal the mechanism of colorectal cancer and the association between differential expression of circular RNAs (circRNAs) and patients' prognosis.

Methods·Colorectal cancer tissues and normal tumor-adjacent tissues were harvested from 12 patients undergoing radical resection of colorectal cancer in the Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University, and the expressions of circRNAs were detected by high-throughput sequencing. Function predictions of differentially expressed circRNAs were carried out by gene ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Two circRNAs were selected from the significantly up-regulated and down-regulated circRNAs respectively, and the accuracy of sequencing results was verified in 44 sets of colorectal cancer tissues by real-time fluorescent quantitative PCR (RT-qPCR). The circRNA-miRNA targeting binding network map was constructed by searching the public database for the four circRNAs. Finally, the relationships between differential expression of circRNAs and prognosis were determined based on the patients' clinical data.

Results·A total of 20 924 differentially expressed circRNAs were detected after high-throughput sequencing. Compared with normal colorectal tissues, there were 373 circRNAs (︱log₂FC︱>1, P<0.05) abnormally and significantly expressed in colorectal cancer tissues, of which 243 were significantly up-regulated and 130 were significantly down-regulated. In 44 pairs of colorectal cancer samples, the results verified by RT-qPCR were basically consistent with the sequencing data. After the circRNA-miRNA-mRNA interactions being plotted, it was found that miRNA and target genes associated with circRNA may play an important role in the pathogenesis of colorectal cancer. Combined with the prognosis information, the patients with high expression of hsa_circ_0023984 or hsa_circ_0008192 had poorer prognosis while patients with high expression of hsa_circ_0020093 or hsa_circ_0069922 had better prognosis.

Conclusion·Abnormally expressed circRNAs in colorectal cancer tissues lay a foundation for further studies on the occurrence and development of colorectal cancer, and provide valuable information for the development of new clinical diagnostic markers and treatments in the future.

Key words: colorectal cancer, circular RNA (circRNA), prognosis, regulatory network

中图分类号:

R735.3

傅中懋, 罗再, 戎泽印, 章建明, 李腾飞, 余志龙, 黄陈. 基于高通量测序的结直肠癌组织中环状RNA功能研究与预后分析[J]. 上海交通大学学报(医学版), 2021, 41(2): 187-195.

Zhong-mao FU, Zai LUO, Ze-yin RONG, Jian-ming ZHANG, Teng-fei LI, Zhi-long YU, Chen HUANG. Study on the function and prognosis of circular RNA in colorectal cancer tissues based on high-throughput sequencing[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(2): 187-195.

图/表 6

表1 RT-qPCR 引物序列

Tab 1 Primer sequences of RT-qPCR

Gene	Primer sequence （5'→3'）
hsa_circ_0023984
Forward	5'-GTCAGTATATTACTCTACATTGTCCCAG-3'
Reverse	5'-TCTAAATTAGTTTGATACTTCAGCAGC-3'
hsa_circ_0008192
Forward	5'-TGTCAGTGCCGTCCAATC-3'
Reverse	5'-GCGAACAGGGAGGTCTAT-3'
hsa_circ_0020093
Forward	5'-CGGTTACTGTGACCTGACTGGAG -3'
Reverse	5'-TGGGCCATCTGTTAAATATCCAG-3'
hsa_circ_0069922
Forward	5'-GCTGATGAATCCAGCAGCAGC-3'
Reverse	5'-TCCAGGACAGCTTCATCCTCC-3'
GAPDH
Forward	5'-GTCTCCTCTGACTTCAACAGCG-3'
Reverse	5'-ACCACCCTGTTGCTGTAGCCAA-3'

图1 结直肠癌组织及癌旁组织中差异表达circRNA的可视化分析Note:A. Intersection between the predicted circRNA and the circBase database. B. Number of circRNAs differentially expressed between colorectal cancer tissues and adjacent normal tissues in the 12 pairs of samples. C. Volcanic map. Green dots represent significantly down-regulated circRNAs; red dots represent significantly up-regulated circRNAs; gray dots represent no significant difference in circRNAs. D. Cluster analysis. Red box represents low expression circRNAs and green represents high expression circRNAs.

Fig 1 Visual analysis of the differential expression of circRNAs in colorectal cancer and adjacent tissues

表2 circRNA测序结果中表达差异最显著的10种circRNA

Tab 2 Ten circRNAs with the most significant expression in the sequencing result

circRNA ID	FC	P value	Regulation	circRNA chromosome	Gene
hsa_circ_0079534	14.24	<0.001	Up	NC_000007.14:20154193_20161870_-	MACC1
hsa_circ_0023984	10.43	<0.001	Up	NC_000011.10:89400017_89402542_-	NOX4
hsa_circ_0008192	10.27	<0.001	Up	NC_000009.12:112262435_112297916_-	PTBP3
hsa_circ_0008694	8.55	<0.001	Up	NC_000020.11:51712966_51729978_-	ATP9A
hsa_circ_0004705	7.40	<0.001	Up	NC_000003.12:14443624_14447816_+	SLC6A6
hsa_circ_0020093	-8.27	<0.001	Down	NC_000010.11:115120185_115129535_+	ATRNL1
hsa_circ_0069922	-7.22	<0.001	Down	NC_000004.12:71236576_71255399_+	SLC4A4
hsa_circ_0073244	-6.54	<0.001	Down	NC_000005.10:84060300_84066606_-	EDIL3
hsa_circ_0005720	-5.31	0.001	Down	NC_000001.11:95143891_95173889_+	TMEM56
hsa_circ_0008676	-5.26	0.002	Down	NC_000004.12:71339370_71357187_+	SLC4A4

图2 差异表达circRNA 的GO分析和KEGG分析Note:A. GO analysis. X coordinate is the GO item name; Y coordinate is -log10 P value. B. KEGG analysis. The X axis is the enrichment score; The larger the bubble is, the more circRNA the item contains; The color of the bubble changes from purple to blue to green to red; The smaller the enrichment P value, the greater the significance.

Fig 2 GO analysis and KEGG analysis of differentially expressed circRNAs

图3 circRNA差异表达及circRNA-miRNA-mRNA网络图Note:A. RT-qPCR was performed for 4 different circRNAs—hsa_circ_0023984, hsa_circ_0008192, hsa_circ_0020093 and hsa_circ_0069922 in 44 colorectal cancer tumor and normal tissues. B. circRNA-miRNA-mRNA network plot. Red circles represent circRNA； green circles represent miRNA； yellow circles represent mRNA. ①P=0.017 1， ②P=0.034 0， ③P<0.000 1.

Fig 3 Differential expression of circRNA and circRNA-miRNA-mRNA network plot

图4 4种差异表达circRNA的临床预后分析Note: ①P=0.026 2， ②P=0.039 8， ③P=0.000 1， ④P=0.002 2.

Fig 4 Clinical prognosis analysis of four differentially expressed circRNAs

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基于高通量测序的结直肠癌组织中环状RNA功能研究与预后分析

Study on the function and prognosis of circular RNA in colorectal cancer tissues based on high-throughput sequencing

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