基于全转录组测序分析多形性胶质母细胞瘤增殖及侵袭的相关机制

doi:10.3969/j.issn.1673-8640.2022.08.014

检验医学 ›› 2022, Vol. 37 ›› Issue (8): 772-781.DOI: 10.3969/j.issn.1673-8640.2022.08.014

基于全转录组测序分析多形性胶质母细胞瘤增殖及侵袭的相关机制

王仕超¹, 刘斌², 赵星¹, 任晓敏¹, 潘园园¹, 李霞¹, 徐淑英¹

1.内蒙古自治区呼和浩特市第一医院医学遗传实验室,内蒙古呼和浩特 010030
2.内蒙古自治区人民医院神经内科,内蒙古呼和浩特 010030

收稿日期:2021-03-04 修回日期:2022-05-17 出版日期:2022-08-30 发布日期:2022-09-16
作者简介:王仕超,女,1980年生,博士,副主任技师,主要从事肿瘤分子生物学研究。
基金资助:
呼和浩特市科技局资助项目(2019-社-5)

Mechanism of glioblastoma multiforme proliferation and invasion analyzed by whole-transcriptome sequencing

WANG Shichao¹, LIU Bin², ZHAO Xing¹, REN Xiaomin¹, PAN Yuanyuan¹, LI Xia¹, XU Shuying¹

1. Medical Genetics Laboratory,the First Hospital of Hohhot,Hohhot 010030,Inner Mongolia,China
2. Department of Neurology,Inner Mongolia People's Hospital,Hohhot 010030,Inner Mongolia,China

Received:2021-03-04 Revised:2022-05-17 Online:2022-08-30 Published:2022-09-16

摘要/Abstract

摘要：

目的通过对多形性胶质母细胞瘤（GBM）组织及癌旁组织的转录组测序及生物信息数据进行分析,挖掘导致GBM发生的关键基因并明确其功能,深入探讨GBM细胞增殖、侵袭及迁移的分子调控机制。方法收集4例GBM患者的癌组织及癌旁组织进行全转录组测序分析,筛选差异表达的mRNA、微小RNA（miRNA）、长链非编码RNA（lncRNA）和环状RNA（circRNA）,并对其进行生物信息学分析[基因本体（GO）富集分析、京都基因与基因组数据库（KEGG）通路分析和转录本富集分析等]。构建mRNA-miRNA-lncRNA/circRNA的关联网络,寻找与脑胶质瘤发生、迁移和侵袭密切相关的目标分子。结果筛选出3 088个癌组织与癌旁组织差异表达的mRNA,其中表达上调1 375个、表达下调1 713个;差异表达的lncRNA 106个,其中表达上调52个、表达下调54个;差异表达的circRNA 82个,其中表达上调32个、表达下调50个;差异表达的miRNA 15个,其中表达上调8个、表达下调7个。差异表达的mRNA转录本富集共得到显著性通路19条,涉及Shh（Sonic hedgehog）信号途径、胶质瘤信号途径、胰腺癌信号途径、轴突导向、肿瘤坏死因子（TNF）信号途径等通路。全转录组多元关联分析结果显示,BOC位于调控网络关键节点位置,与has-miR-4763-5p、has-miR-6848-5p、has-miR-6860 miRNA具有潜在的靶向结合位点。胶质瘤患者癌组织BOC mRNA水平明显高于癌旁组织（P<0.05）。结论 BOC可能受相关miRNA、circRNA、lncRNA等非编码RNA的调控,影响下游靶基因的表达,进而影响GBM细胞的增殖、侵袭等生物学行为。

关键词: 全转录组测序, 多形性胶质母细胞瘤, Hedgehog信号通路

Abstract:

Objective To analyze the transcriptome sequencing and biological information data of glioblastoma multiforme（GBM） tissues and adjacent tissues,to dig out the key genes that cause GBM and identify their functions,and to investigate the molecular regulatory mechanism of malignant proliferation,invasion and migration of GBM. Methods Totally,4 patients with GBM were enrolled,and whole-transcriptome sequencing for tumor tissues and adjacent tissues was performed. The mRNA,microRNA（miRNA）,long non-coding RNA（lncRNA） and circular RNA（circRNA） with different expressions were screened. Bio-informatics [Gene Ontology（GO） enrichment analysis,Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway analysis,transcript enrichment analysis and so on] was performed. The mRNA-miRNA-lncRNA/circRNA associated network was established,and glioma development,migration and invasion related to target molecules were found. Results There were 3 088 different mRNA between tumor tissues and adjacent tissues,of which 1 375 cases were up-regulated,and 1 713 cases were down-regulated. There were 106 different lncRNA,of which 52 cases were up-regulated,and 54 cases were down-regulated. There were 82 different circRNA,of which 32 cases were up-regulated,and 50 cases were down-regulated. There were 15 different miRNA,of which 8 cases were up-regulated,and 7 cases were down-regulated. Differentially expressed mRNA transcripts were enriched to obtain 19 significant pathways. These pathways included Shh（Sonic hedgehog） signaling pathway,glioma signaling pathway,pancreatic cancer signaling pathway,axon guidance pathway,tumor necrosis factor（TNF） signaling pathway and other related pathways. The multivariate association analysis of whole-transcriptome sequencing found that BOC was located at the key node of its regulatory pathway（has-miR-4763-5p,has-miR-6848-5p and has-miR-6860 miRNA）. The mRNA expression level of BOC in tumor tissues was higher than that in adjacent tissues. Conclusions BOC may affect the expression of downstream target genes by the regulation of related miRNA,circRNA,lncRNA and other non-coding RNA,leading to the proliferation and invasion of GBM.

Key words: Whole-transcriptome sequencing, Glioblastoma multiforme, Hedgehog signaling pathway

中图分类号:

Q781

王仕超, 刘斌, 赵星, 任晓敏, 潘园园, 李霞, 徐淑英. 基于全转录组测序分析多形性胶质母细胞瘤增殖及侵袭的相关机制[J]. 检验医学, 2022, 37(8): 772-781.

WANG Shichao, LIU Bin, ZHAO Xing, REN Xiaomin, PAN Yuanyuan, LI Xia, XU Shuying. Mechanism of glioblastoma multiforme proliferation and invasion analyzed by whole-transcriptome sequencing[J]. Laboratory Medicine, 2022, 37(8): 772-781.

图/表 11

表1 引物序列

基因名称	引物序列（5'~3'）	产物长度/bp
BOC	F：CGGACGAGGGCTCTTTCTT	81
	R：GTTGGCAGCAGTCGTGATG
GAPDH	F：AGAAGGCTGGGGCTCATT	158
	R：TGCTAAGCAGTTGGTGGTG

图1 mRNA测序分析结果

图2 lncRNA测序分析结果

图3 circRNA测序分析结果

图4 miRNA测序分析结果

图5 mRNA-miRNA-circRNA的关联分析注：□ 基因;○ miRNA;△ circRNA;红色为表达上调;绿色为表达下调。

图6 mRNA-miRNA-lncRNA的关联分析注：□ 基因;○ miRNA;△ circRNA;红色为表达上调;绿色为表达下调。

图7 mRNA-miRNA-lncRNA/circRNA的关联分析注：□ 基因;○ miRNA;◇ lncRNA;△ circRNA;红色为表达上调;绿色为表达下调。

图8 BOC与ncRNA、基因的关联分析注：节点大小与其互作的分子数目呈正相关。

图9 BOC-蛋白互作分析图

图10 5例GBM患者癌组织与癌旁组织BOC mRNA相对表达量比较注：■ 肿瘤组织;■ 癌旁组织。

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基于全转录组测序分析多形性胶质母细胞瘤增殖及侵袭的相关机制

Mechanism of glioblastoma multiforme proliferation and invasion analyzed by whole-transcriptome sequencing

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