CPSF6在胶质母细胞瘤进展中的作用及相关调控机制研究<sup>*</sup>

doi:10.13523/j.cb.2204018

中国生物工程杂志

2022, Vol. 42

Issue (9): 1-16 DOI: 10.13523/j.cb.2204018

研究报告

CPSF6在胶质母细胞瘤进展中的作用及相关调控机制研究^*

黄进,娄哲琦,朱勇^**(

)

重庆医科大学生命科学研究院重庆 400016

The Role of CPSF6 in the Progression of Glioblastoma and Related Regulatory Mechanism

HUANG Jin,LOU Zhe-qi,ZHU Yong^**(

)

Institute of Life Sciences, Chongqing Medical University, Chongqing 400016, China

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摘要：

目的: 研究mRNA前体切割和多聚腺苷酸化特异性因子6(polyadenylation specific factor 6,CPSF6)对人胶质母细胞瘤(glioblastoma,GBM)细胞系U87和U251的增殖、迁移、侵袭以及ATP水平的影响,进一步探究其相关调控机制。方法: 通过Western blot和免疫组化检测CPSF6在GBM组织中的表达水平,利用在线数据库分析CPSF6在GBM组织和配对的非肿瘤组织中的表达水平,同时分析CPSF6与GBM的组织学级别和患者预后的关系。构建敲低CPSF6的U87和U251稳定表达细胞株,并采用RT-qPCR和Western blot方法分别验证U87和U251细胞中CPSF6的敲低效率;利用CCK8和Transwell实验分别检测CPSF6敲降对细胞增殖、迁移和侵袭能力的影响;ATP实验检测细胞内的ATP水平变化,确定CPSF6在GBM中的致癌作用。通过RNA-seq分析敲低CPSF6后GBM内mRNA 3'UTR变化情况,KEGG富集分析差异靶基因相关的信号通路。在富集出的信号通路指示下,利用透射电镜和Western blot实验进一步验证敲低CPSF6后GBM自噬的发生情况。 结果: CPSF6在GBM组织中呈现出高表达,其表达水平随组织学级别的增加而升高,且与患者不良预后相关。在U87和U251中敲低CPSF6后,细胞的增殖、迁移及侵袭能力均明显降低,细胞内ATP水平下降。对RNA-seq结果分析表明,敲低CPSF6后发生3'UTR缩短事件的基因远多于3'UTR延长事件的基因;KEGG富集到自噬信号通路与肿瘤进展密切相关,透射电镜和Western blot实验验证敲低CPSF6可以促进自噬通路的激活。结论: CPSF6在GBM中高表达,且与GBM的组织学级别和患者不良预后呈正相关,CPSF6可能通过抑制自噬通路的激活来促进U87和U251细胞的增殖、迁移、侵袭以及ATP的生成,进而促进GBM发生、发展。

关键词： 切割和多聚腺苷酸化特异性因子6; 胶质母细胞瘤; ATP; 自噬; 细胞增殖; 细胞迁移; 细胞侵袭

Abstract:

Objective: To investigate the effects of cleavage and polyadenylation specific factor 6 (CPSF6) on the proliferation, migration, invasion and ATP production of human glioblastoma (GBM) cell lines U87 and U251, and to further investigate the related regulatory mechanism. Methods: First, the expression levels of CPSF6 in GBM tissues and paired non-tumor tissues were detected by western blot and immunohistochemistry and analyzed by an online database, and the relationship between CPSF6 and the histological grade of GBM and patient prognosis was also analyzed. CPSF6 was knocked down in U87 and U251 cells with short hairpin RNA (shCPSF6). The expression of CPSF6 in U87 and U251 cells was detected by real-time quantitative PCR and western blot, respectively. After knocking down CPSF6, the proliferation ability of GBM cells was tested by CCK8 assay, and the migration and invasion ability of GBM cells was detected by Transwell assay. ATP assay was performed to detect changes in intracellular ATP levels and to determine the oncogenic role of CPSF6 in GBM. RNA-seq was used to analyze mRNA 3'UTR changes in GBM after CPSF6 knockdown, and KEGG enrichment was used to analyze signal pathways related to different target genes. Under the instructions of the enriched signal pathway, the protein expression levels of LC3 and Beclin-1, which are related markers in the autophagy signaling pathway, were detected by western blot assay after knocking down CPSF6. Transmission electron microscopy was used to observe the occurrence of intracellular autophagy in GBM cells in the experimental and control groups. Results: CPSF6 was significantly up-regulated in GBM tissues compared with the paired non-tumor tissues, and the high expression of CPSF6 was associated with poor prognosis in patients. After CPSF6 was knocked down, the proliferation, migration and invasion of GBM cells were significantly reduced, and the intracellular ATP level was decreased. Bioinformatics analysis, transmission electron microscopy and western blot assay demonstrated that CPSF6 potentially promoted the activation of autophagy pathway. Conclusion: CPSF6 was upregulated in GBM. The high expression of CPSF6 is associated with poor prognosis in patients and is positively correlated with histological grade of GBM. CPSF6 exerts oncogenic effects in GBM, and potentially promotes the proliferation, migration, invasion and ATP production of U87 and U251 cells. Knockdown of CPSF6 potentially activates the autophagy pathway in GBM.

Key words: Cleavage and polyadenylation specific factor 6 (CPSF6) Glioblastoma ATP Autophagy Cell proliferation Cell migration Cell invasion

收稿日期: 2022-04-08 出版日期: 2022-10-10

ZTFLH:

R739

基金资助: * 重庆市留学人员回国创业创新支持计划(cx2019003)

通讯作者: 朱勇 E-mail: yongz59@cqmu.edu.cn

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引用本文:

黄进,娄哲琦,朱勇. CPSF6在胶质母细胞瘤进展中的作用及相关调控机制研究^*[J]. 中国生物工程杂志, 2022, 42(9): 1-16.

HUANG Jin,LOU Zhe-qi,ZHU Yong. The Role of CPSF6 in the Progression of Glioblastoma and Related Regulatory Mechanism. China Biotechnology, 2022, 42(9): 1-16.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2204018 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I9/1

表1 引物序列

图1 CPSF6在胶质母细胞瘤组织中表达上调

图2 CPSF6基因在GBM中拷贝数变异与生存分析

图3 CPSF6 在U87和U251细胞中的敲降效率检测

图4 CPSF6促进GBM细胞增殖和ATP生成

图5 CPSF6促进GBM细胞迁移和侵袭

图6 GBM细胞中敲低CPSF6的火山图

图7 差异基因KEGG富集的相关信号通路

图8 敲低CPSF6激活U87和U251细胞自噬

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