CircTTBK2 Contributes to the Progression of Glioma Through Regulating miR-145-5p/CPEB4 Axis

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Abstract

Background

Currently, circular RNAs (circRNAs) have been demonstrated to play vital roles in malignant tumors, including glioma. Nevertheless, the functions of circTTBK2 in glioma are largely unclear.

Materials and Methods

Quantitative real-time polymerase chain reaction (qRT-PCR) was applied for the expression levels of circTTBK2, TTBK2 mRNA, miR-145-5p and cytoplasmic polyadenylation element binding protein 4 (CPEB4) mRNA. Actinomycin D and RNase R digestion assays were utilized for the characteristics of circTTBK2. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry analysis and transwell assay were conducted for cell proliferation, apoptosis and metastasis, respectively. The glycolysis level was estimated with specific kits. Western blot assay was adopted for the protein levels of hexokinase2 (HK2) and CPEB4. The targeting relationship between miR-145-5p and circTTBK2 or CPEB4 was verified by Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Murine xenograft assay was used for the role of circTTBK2 in tumorigenesis in vivo.

Results

CircTTBK2 was upregulated in glioma tissues and cells, and its level was associated with poor survival of glioma patients. CircTTBK2 knockdown suppressed glioma cell proliferation, migration, invasion and glycolysis and accelerated apoptosis in vitro and hampered tumor growth in vivo. CircTTBK2 functioned as a sponge of miR-145-5p, and miR-145-5p inhibition restored the effects of circTTBK2 knockdown on the malignant behaviors of glioma cells. Moreover, CPEB4 was the direct target gene of miR-145-5p, and miR-145-5p inhibition facilitated glioma cell progression by targeting CPEB4.

Conclusion

CircTTBK2 functioned as a tumor promoter in glioma by modulating miR-145-5p/CPEB4 axis, which might offer a new sight for glioma therapy.

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Most cited references 18

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Advances in the molecular genetics of gliomas — implications for classification and therapy

Guido Reifenberger, Hans-Georg Wirsching, Christiane B Knobbe-Thomsen … (2017)

In 2016, a revised WHO classification of glioma was published, in which molecular data and traditional histological information are incorporated into integrated diagnoses. Herein, the authors highlight the developments in our understanding of the molecular genetics of gliomas that underlie this classification, and review the current landscape of molecular biomarkers used in the classification of disease subtypes. In addition, they discuss how these advances can promote the development of novel pathogenesis-based therapeutic approaches, paving the way to precision medicine.

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circPTN sponges miR-145-5p/miR-330-5p to promote proliferation and stemness in glioma

Jiansheng Chen, Taoliang Chen, Yubo Zhu … (2019)

Background Growing evidences indicate that circular RNAs (circRNAs) play an important role in the regulation of biological behavior of tumor. We aim to explore the role of circRNA in glioma and elucidate how circRNA acts. Methods Real-time PCR was used to examine the expression of circPTN in glioma tissues and normal brain tissues (NBT). Assays of dual- luciferase reporter system, biotin label RNA pull-down and FISH were used to determine that circPTN could sponge miR-145-5p and miR-330-5p. Tumor sphere formation assay was performed to determine self- renewal of glioma stem cell (GSCs). Cell counting Kit-8 (CCK8), EdU assay and flow cytometry were used to investigate proliferation and cell cycle. Intracranial xenograft was established to determine how circPTN impacts in vivo. Tumor sphere formation assay was performed to determine self- renewal of glioma stem cell (GSCs). Results We demonstrated circPTN was significantly higher expression in glioma tissues and glioma cell lines, compared with NBT and HEB (human astrocyte). In gain- and loss-of-function experiments, circPTN significantly promoted glioma growth in vitro and in vivo. Furthermore, we performed dual-luciferase reporter assays and RNA pull-down assays to verify that circPTN acts through sponging miR-145-5p and miR-330-5p. Increasing expression of circPTN rescued the inhibition of proliferation and downregulation of SOX9/ITGA5 in glioma cells by miR-145-5p/miR-330-5p. In addition, we found that circPTN promoted self-renewal and increased the expression of stemness markers (Nestin, CD133, SOX9, and SOX2) via sponging miR-145-5p. Moreover, this regulation was disappeared when circPTN binding sites in miR-145-5p were mutated. Conclusions Our results suggest that circPTN is an oncogenic factor that acts by sponging miR-145-5p/miR-330-5p in glioma. Electronic supplementary material The online version of this article (10.1186/s13046-019-1376-8) contains supplementary material, which is available to authorized users.

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TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1β/Derlin-1 pathway

Jian Zheng, Xiaobai Liu, Yixue Xue … (2017)

Background Circular RNAs are a subgroup of non-coding RNAs and generated by a mammalian genome. Herein, the expression and function of circular RNA circ-TTBK2 were investigated in human glioma cells. Methods Fluorescence in situ hybridization and quantitative real-time PCR were conducted to profile the cell distribution and expression of circ-TTBK2 and microRNA-217 (miR-217) in glioma tissues and cells. Immunohistochemical and western blot were used to determine the expression of HNF1β and Derlin-1 in glioma tissues and cells. Stable knockdown of circ-TTBK2 or overexpression of miR-217 glioma cell lines (U87 and U251) were established to explore the function of circ-TTBK2 and miR-217 in glioma cells. Further, luciferase reports and RNA immunoprecipitation were used to investigate the correlation between circ-TTBK2 and miR-217. Cell Counting Kit-8, transwell assays, and flow cytometry were used to investigate circ-TTBK2 and miR-217 function including cell proliferation, migration and invasion, and apoptosis, respectively. ChIP assays were used to ascertain the correlations between HNF1β and Derlin-1. Results We found that circ-TTBK2 was upregulated in glioma tissues and cell lines, while linear TTBK2 was not dysregulated in glioma tissues and cells. Enhanced expression of circ-TTBK2 promoted cell proliferation, migration, and invasion, while inhibited apoptosis. MiR-217 was downregulated in glioma tissues and cell lines. We also found that circ-TTBK2, but not linear TTBK2, acted as miR-217 sponge in a sequence-specific manner. In addition, upregulated circ-TTBK2 decreased miR-217 expression and there was a reciprocal negative feedback between them in an Argonaute2-dependent manner. Moreover, reintroduction of miR-217 significantly reversed circ-TTBK2-mediated promotion of glioma progression. HNF1β was a direct target of miR-217, and played oncogenic role in glioma cells. Remarkably, circ-TTBK2 knockdown combined with miR-217 overexpression led to tumor regression in vivo. Conclusions These results demonstrated a novel role circ-TTBK2 in the glioma progression. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0422-2) contains supplementary material, which is available to authorized users.

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Author and article information

Journal

Journal ID (nlm-ta): Cancer Manag Res

Journal ID (iso-abbrev): Cancer Manag Res

Journal ID (publisher-id): cmar

Journal ID (pmc): cancmanres

Title: Cancer Management and Research

Publisher: Dove

ISSN (Electronic): 1179-1322

Publication date (Electronic): 08 September 2020

Publication date Collection: 2020

Volume: 12

Pages: 8183-8195

Affiliations

[1 ]Department of Neurosurgery, General Hospital of Tianjin Medical University , Tianjin 300052, People’s Republic of China

[2 ]International Medical Center, Tianjin Hospital , Tianjin 300052, People’s Republic of China

Author notes

Correspondence: Weidong Yang Email Liuxingyu20070803@163.com

[*]

These authors contributed equally to this work

Article

Publisher ID: 263586

DOI: 10.2147/CMAR.S263586

PMC ID: 7490103

PubMed ID: 32982415

SO-VID: daea5fb9-5522-4605-bb8b-10bd0af6fd5e

License:

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

History

Date received : 28 May 2020

Date accepted : 12 August 2020

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Figures: 8, References: 25, Pages: 13

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There is no funding to report.

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CircTTBK2 Contributes to the Progression of Glioma Through Regulating miR-145-5p/CPEB4 Axis

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Center for Diffuse Intrinsic Pontine Glioma (DIPG) & Diffuse Midline Glioma (DMG)

Most cited references 18

Advances in the molecular genetics of gliomas — implications for classification and therapy

circPTN sponges miR-145-5p/miR-330-5p to promote proliferation and stemness in glioma

TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1β/Derlin-1 pathway

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