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

doi:10.13523/j.cb.2204018

China Biotechnology

2022, Vol. 42

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

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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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

Received: 08 April 2022 Published: 10 October 2022

ZTFLH:

R739

Corresponding Authors: Yong ZHU E-mail: yongz59@cqmu.edu.cn

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Cite this article:

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.

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

Table 1 Primer sequence

Fig.1 Expression of CPSF6 was upregulated in GBM (a) Analysis of CPSF6 mRNA levels in different subtypes of glioma from CGGA (b)Analysis of CPSF6 mRNA levels of GBM (n = 10) and non-tumor tissues (n = 528) from TCGA (c) Increased expression of CPSF6 with increasing histological grade of GBM (d) The immunohistochemistry staining assay of CPSF6 in GBM and adjacent tissues of the tumor (scale bar: 100 μm) (e) Western blot assay for detecting CPSF6 expression in 8 paired surrounding non-tumor (N) and GBM tumor (T) tissues. ^* P<0.05, n>3

Fig.2 Copy number variation and survival analysis of CPSF6 gene in GBM (a) Mutations of CPSF6 gene in GBM in TCGA GBM database (b)TCGA, Cell 2013 database of CPSF6 copy number variants (c) Effect of CPSF6 mRNA expression on overall survival in GBM patients (TCGA database) (d) Effect of CPSF6 mRNA expression on overall survival in glioblastoma patients (CGGA database)

Fig.3 Knockdown efficiency of CPSF6 in U87 and U251 cells (a),(b) The expression levels of CPSF6 in U87 and U251 cells transfected with shRNA-CPSF6 were detected by qRT-PCR (c),(d) The expression levels of CPSF6 in U87 and U251 cells transfected with shRNA-CPSF6 were detected by western blot. ^*** P<0.001 ( NC vs. shCPSF6) ; ^**** P<0.000 1 (NC vs. shCPSF6), n=3

Fig.4 CPSF6 promotes the proliferation and ATP production in GBM cells (a),(b) The effects of CPSF6 on proliferation after knocking down CPSF6 in U87 and U251 cells were determined by CCK8 assay 72 h: ^*** P<0.001 (NC vs shCPSF6) (c),(d) Intracellular ATP production level after knocking down CPSF6 in U87 and U251 cells were determined by ATP assay ^** P<0.05 ; ^**** P<0.000 1, n=3 (NC vs. shCPSF6)

Fig.5 CPSF6 promoted the migration and invasion of GBM cells (a),(b) The effects of CPSF6 on migration after knocking down CPSF6 in U87 and U251 cells were determined by Transwell migration assay (c),(d) The effects of CPSF6 on invasion after knocking down CPSF6 in U87 and U251 cells were determined by Transwell invasion assay. Scale bars, 100 μm. ^**** P<0.000 1; n=3 (NC vs. shCPSF6)

Fig.6 Volcano map of knockdown CPSF6 in GBM cells (a) U87 shCPSF6-1 vs. U87 shCtrl (b) U87 shCPSF6-2 vs. U87 shCtrl (c) U251 shCPSF6-1 vs. U251 shCtrl (d) U251 shCPSF6-2 vs. U251 shCtrl

Fig.7 Signaling pathway associated with differential gene KEGG enrichment (a) The KEGG analysis of U87 cells (b) The KEGG analysis of U251 cells

Fig.8 Knockdown of CPSF6 activates autophagy in U87 and U251 cells (a),(b) Transmission electron microscopy (TEM) analysis showing autophagosome (arrowed) after knocking down of CPSF6 in U87 and U251 cells (c),(d) The protein expression levels of LC3 and Beclin-1 in U87 and U251 cells after knocking down of CPSF6 were detected by western blot (n=3)


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