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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.
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Received: 08 April 2022
Published: 10 October 2022
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Corresponding Authors:
Yong ZHU
E-mail: yongz59@cqmu.edu.cn
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[1] |
Majc B, Novak M, Kopitar-Jerala N, et al. Immunotherapy of glioblastoma: current strategies and challenges in tumor model development. Cells, 2021, 10(2): 265.
doi: 10.3390/cells10020265
|
|
|
[2] |
Lim M, Xia Y X, Bettegowda C, et al. Current state of immunotherapy for glioblastoma. Nature Reviews Clinical Oncology, 2018, 15(7): 422-442.
doi: 10.1038/s41571-018-0003-5
pmid: 29643471
|
|
|
[3] |
Wen P Y, Kesari S. Malignant gliomas in adults. New England Journal of Medicine, 2008, 359(17): 1850.
|
|
|
[4] |
Ostrom Q T, Gittleman H, Farah P, et al. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2006-2010. Neuro-oncology, 2013, 15(Suppl 2): ii1-56.
|
|
|
[5] |
Tian B, Manley J L. Alternative polyadenylation of mRNA precursors. Nature Reviews Molecular Cell Biology, 2017, 18(1): 18-30.
doi: 10.1038/nrm.2016.116
pmid: 27677860
|
|
|
[6] |
Lan Y L, Zhang J M. Modulation of untranslated region alternative polyadenylation in glioma tumorigenesis. Biomedicine & Pharmacotherapy, 2021, 137: 111416.
doi: 10.1016/j.biopha.2021.111416
|
|
|
[7] |
Xia Z, Donehower L A, Cooper T A, et al. Dynamic analyses of alternative polyadenylation from RNA-seq reveal a 3'-UTR landscape across seven tumour types. Nature Communications, 2014, 5: 5274.
doi: 10.1038/ncomms6274
pmid: 25409906
|
|
|
[8] |
Chang J W, Zhang W, Yeh H S, et al. mRNA 3'-UTR shortening is a molecular signature of mTORC1 activation. Nature Communications, 2015, 6: 7218.
doi: 10.1038/ncomms8218
|
|
|
[9] |
Lin Y F, Li Z H, Ozsolak F, et al. An in-depth map of polyadenylation sites in cancer. Nucleic Acids Research, 2012, 40(17): 8460-8471.
pmid: 22753024
|
|
|
[10] |
Xiang Y, Ye Y Q, Lou Y Y, et al. Comprehensive characterization of alternative polyadenylation in human cancer. Journal of the National Cancer Institute, 2018, 110(4): 379-389.
doi: 10.1093/jnci/djx223
pmid: 29106591
|
|
|
[11] |
Lembo A, di Cunto F, Provero P. Shortening of 3'UTRs correlates with poor prognosis in breast and lung cancer. PLoS One, 2012, 7(2): e31129.
doi: 10.1371/journal.pone.0031129
|
|
|
[12] |
Masamha C P, Xia Z, Yang J X, et al. CFIm25 links alternative polyadenylation to glioblastoma tumour suppression. Nature, 2014, 510(7505): 412-416.
doi: 10.1038/nature13261
|
|
|
[13] |
Mayr C, Bartel D P. Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell, 2009, 138(4): 673-684.
doi: 10.1016/j.cell.2009.06.016
pmid: 19703394
|
|
|
[14] |
Hardy J G, Norbury C J. Cleavage factor Im (CFIm) as a regulator of alternative polyadenylation. Biochemical Society Transactions, 2016, 44(4): 1051-1057.
doi: 10.1042/BST20160078
pmid: 27528751
|
|
|
[15] |
Rüegsegger U, Beyer K, Keller W. Purification and characterization of human cleavage factor im involved in the 3' end processing of messenger RNA precursors. Journal of Biological Chemistry, 1996, 271(11): 6107-6113.
doi: 10.1074/jbc.271.11.6107
pmid: 8626397
|
|
|
[16] |
Yang Q, Gilmartin G M, Doublié S. Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3' processing. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(22): 10062-10067.
|
|
|
[17] |
Brown K M, Gilmartin G M. A mechanism for the regulation of pre-mRNA 3' processing by human cleavage factor im. Molecular Cell, 2003, 12(6): 1467-1476.
pmid: 14690600
|
|
|
[18] |
Rüegsegger U, Blank D, Keller W. Human pre-mRNA cleavage factor im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits. Molecular Cell, 1998, 1(2): 243-253.
pmid: 9659921
|
|
|
[19] |
Zhu Y, Wang X Y, Forouzmand E, et al. Molecular mechanisms for CFIm-mediated regulation of mRNA alternative polyadenylation. Molecular Cell, 2018, 69(1): 62-74, e4.
doi: S1097-2765(17)30890-0
pmid: 29276085
|
|
|
[20] |
Gruber A R, Martin G, Keller W, et al. Cleavage factor Im is a key regulator of 3' UTR length. RNA Biology, 2012, 9(12): 1405-1412.
doi: 10.4161/rna.22570
pmid: 23187700
|
|
|
[21] |
Martin G, Gruber A R, Keller W, et al. Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length. Cell Reports, 2012, 1(6): 753-763.
doi: 10.1016/j.celrep.2012.05.003
pmid: 22813749
|
|
|
[22] |
Tan S, Zhang M, Shi X L, et al. CPSF6 links alternative polyadenylation to metabolism adaption in hepatocellular carcinoma progression. Journal of Experimental & Clinical Cancer Research, 2021, 40(1): 85.
|
|
|
[23] |
Shi X L, Ding K S, Zhao Q, et al. Suppression of CPSF 6 enhances apoptosis through alternative polyadenylation-mediated shortening of the VHL 3'UTR in gastric cancer cells. Frontiers in Genetics, 2021, 12: 707644.
doi: 10.3389/fgene.2021.707644
|
|
|
[24] |
Binothman N, Hachim I Y, Lebrun J J, et al. CPSF6 is a clinically relevant breast cancer vulnerability target: role of CPSF6 in breast cancer. EBioMedicine, 2017, 21: 65-78.
doi: S2352-3964(17)30258-X
pmid: 28673861
|
|
|
[25] |
Schonberg D L, Lubelski D, Miller T E, et al. Brain tumor stem cells: molecular characteristics and their impact on therapy. Molecular Aspects of Medicine, 2014, 39: 82-101.
doi: 10.1016/j.mam.2013.06.004
pmid: 23831316
|
|
|
[26] |
Gimple R C, Bhargava S, Dixit D, et al. Glioblastoma stem cells: lessons from the tumor hierarchy in a lethal cancer. Genes & Development, 2019, 33(11-12): 591-609.
doi: 10.1101/gad.324301.119
|
|
|
[27] |
Chu Y, Elrod N, Wang C J, et al. Nudt21 regulates the alternative polyadenylation of Pak1 and is predictive in the prognosis of glioblastoma patients. Oncogene, 2019, 38(21): 4154-4168.
doi: 10.1038/s41388-019-0714-9
pmid: 30705404
|
|
|
[28] |
Masamha C P, Wagner E J. The contribution of alternative polyadenylation to the cancer phenotype. Carcinogenesis, 2018, 39(1): 2-10.
doi: 10.1093/carcin/bgx096
pmid: 28968750
|
|
|
[29] |
Chen X, Zhang J X, Luo J H, et al. CSTF2-induced shortening of the RAC 1 3'UTR promotes the pathogenesis of urothelial carcinoma of the bladder. Cancer Research, 2018, 78(20): 5848-5862.
doi: 10.1158/0008-5472.CAN-18-0822
pmid: 30143523
|
|
|
[30] |
Kim S, Yamamoto J, Chen Y X, et al. Evidence that cleavage factor Im is a heterotetrameric protein complex controlling alternative polyadenylation. Genes to Cells, 2010, 15(9): 1003-1013.
doi: 10.1111/j.1365-2443.2010.01436.x
pmid: 20695905
|
|
|
[31] |
Brumbaugh J, di Stefano B, Wang X Y, et al. Nudt21 controls cell fate by connecting alternative polyadenylation to chromatin signaling. Cell, 2018, 172(1-2): 106-120, e21.
doi: S0092-8674(17)31370-3
pmid: 29249356
|
|
|
[32] |
Jafari Najaf Abadi M H, Shafabakhsh R, Asemi Z, et al. CFIm25 and alternative polyadenylation: conflicting roles in cancer. Cancer Letters, 2019, 459: 112-121.
doi: S0304-3835(19)30352-0
pmid: 31181319
|
|
|
[33] |
Tan S, Li H, Zhang W J, et al. NUDT21 negatively regulates PSMB2 and CXXC5 by alternative polyadenylation and contributes to hepatocellular carcinoma suppression. Oncogene, 2018, 37(35): 4887-4900.
doi: 10.1038/s41388-018-0280-6
pmid: 29780166
|
|
|
[34] |
Zhang L, Zhang W H. Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway. Cancer Management and Research, 2018, 10: 4311-4323.
doi: 10.2147/CMAR.S173496
pmid: 30349365
|
|
|
[35] |
Yang Q, Coseno M, Gilmartin G M, et al. Crystal structure of a human cleavage factor CFIm25/CFIm68/RNA complex provides an insight into poly(A) site recognition and RNA looping. Structure (London, England: 1993), 2011, 19(3): 368-377.
doi: 10.1016/j.str.2010.12.021
|
|
|
[36] |
Sowd G A, Serrao E, Wang H, et al. A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(8): E1054-E1063.
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