M6a reader igf2bp2-stabilized casc9 accelerates glioblastoma aerobic glycolysis by enhancing hk2 mrna stability

ABSTRACT N6-methyladenosine (m6A) has been identified to exert critical roles in human cancer; however, the regulation of m6A modification on glioblastoma multiforme (GBM) and long

non-coding RNA (lncRNA) CASC9 (cancer susceptibility 9) is still unclear. Firstly, MeRIP-Seq revealed the m6A profile in the GBM. Moreover, the m6A-related lncRNA CASC9 expression was

significantly elevated in the GBM tissue and its ectopic high expression was associated with poor survival, acting as an independent prognostic factor for GBM patients. Functionally, the

aerobic glycolysis was promoted in the CASC9 overexpression transfection, which was inhibited in CASC9 knockdown in GBM cells. Mechanistically, m6A reader IGF2BP2 (insulin-like growth factor

2 mRNA binding protein 2) could recognize the m6A site of CASC9 and enhance its stability, then CASC9 cooperated with IGF2BP2, forming an IGF2BP2/CASC9 complex, to increase the HK2

(Hexokinase 2) mRNA stability. Our findings reveal that CASC9/IGF2BP2/HK2 axis promotes the aerobic glycolysis of GBM. SIMILAR CONTENT BEING VIEWED BY OTHERS LIN28A PROMOTES IRF6-REGULATED

AEROBIC GLYCOLYSIS IN GLIOMA CELLS BY STABILIZING SNHG14 Article Open access 11 June 2020 M6A-MODIFIED CIRCFOXK2 TARGETS GLUT1 TO ACCELERATE ORAL SQUAMOUS CELL CARCINOMA AEROBIC GLYCOLYSIS

Article 20 September 2022 CIRCRNA CIRC-PDCD11 PROMOTES TRIPLE-NEGATIVE BREAST CANCER PROGRESSION VIA ENHANCING AEROBIC GLYCOLYSIS Article Open access 21 August 2021 INTRODUCTION Glioblastoma

multiforme (GBM) is one of the most aggressive tumors of the central nervous system while representing 80% of all malignant brain tumors [1, 2]. Since decades GBM is characterized by a

median overall survival of 15 months, suggesting the tremendous hazard of GBM [3, 4]. Although GBM is very dangerous, it is hardly excised by surgical treatment or intensive treatments,

leading to a low survival rate [5]. GBM is characterized by a median overall survival around 15 months for decades, suggesting an urgent need for an accurate underlying mechanism of GBM.

N6-methyladenosine (m6A) is the most abundant modification in mRNA mediated by m6A methyltransferases, demethylases and readers [6]. M6A methyltransferases install the m6A modification on

RNA, especially methyltransferase-like3 (METTL3) and its auxiliary partners METTL14 and WTAP. Currently, m6A modification is found to be involved in most pivotal biological processes,

including stem cells differentiation, spermatogenesis, RNA metabolism and so on. For example, m6A demethylase ALKBH5 is highly expressed in GBM stem-like cells and ALKBH5 mediated the

demethylation of FOXM1 nascent transcripts. Moreover, lncRNA FOXM1-AS accelerates the interaction of ALKBH5 and FOXM1 nascent transcripts, leading to the FOXM1 expression increasing [7].

Moreover, researchers found that the m6A level is decreased in glioma tissue and the high level of m6A results in a decreased migration and proliferation ability [8]. Therefore, these

findings reveal the potential roles of m6A on GBM. More than the DNA methylation, histone and chromatin modifications, m6A is one of the most researched hotspots in epigenetics. IGF2BP2 is

one of the main RNA reader regulating human tumorigenesis. For example, upregulation of IGF2BP2 is correlated to pancreatic cancer patients’ poor outcomes. Moreover, IGF2BP2 positively

regulates lncRNA DANCR expression [9]. Here, our research paid close attention to the coordination within m6A and lncRNA CASC9 (cancer susceptibility 9) based on the MeRIP-Seq (methylated

RNA immunoprecipitation sequencing) in GBM. CASC9 cooperated with IGF2BP2 (insulin-like growth factor 2 mRNA binding protein 2) to increase the HK2 (Hexokinase 2) mRNA stability, thereby

promoting aerobic glycolysis. RESULTS MERIP-SEQ REVEALED THE M6A PROFILE IN THE GBM To explore the m6A profile in the GBM, MeRIP-Seq was performed and detected. It is found that the m6A

peaks frequency was mainly located in the 5-end untranslated regions (5ʹ-UTR), coding region (CDS) and 3ʹ-UTR (Fig. 1A). The pie chart demonstrated the distribution of m6A peaks in the GBM

genome (Fig. 1B). The volcano plot displayed the upregulated or downregulated targets labeled with m6A peaks (Fig. 1C). Among the m6A motif, the GGACU motif occupied the main ingredient

(Fig. 1D). Collectively, the above results implied that MeRIP-Seq revealed the m6A profile in the GBM. M6A MODIFIED LNCRNA CASC9 INDICATED THE UNFAVORABLE PROGNOSIS OF GBM According to the

data analysis of MeRIP-Seq, we found that several lncRNAs owned the m6A modified site in the RRACH motif. After screening, several m6A modified lncRNAs were selected and displayed (Fig. 2A).

In these candidate RNAs, we focused on the lncRNA CASC9 and investigated its functions in the GBM tumorigenesis. Firstly, in the clinically collected GBM tissue specimens, CASC9 expression

elevated when compared with the adjacent normal tissue (Fig. 2B). On the basis of median value, the clinical tissue specimens were divided into high expression group and low expression group

(Fig. 2C). Overall survival (OS) curve calculated by the Kaplan−Meier method and survival for GBM patients showed that the patients who had high levels of CASC9 within tissues had

remarkably shorter overall survival rate as compared to low levels of CASC9 (Fig. 2D). Clinically, CASC9 may serve as an independent factor to predict the prognosis of GBM patients.

Collectively, the above results indicated that m6A modified lncRNA CASC9 indicated the unfavorable prognosis of GBM. M6A READER IGF2BP2 ENHANCED THE STABILITY OF LNCRNA CASC9 In the GBM

cohort, IGF2BP2 was found to be upregulated according to the GEPIA dataset (http://gepia.cancer-pku.cn/index) (Fig. 3A). To evaluate the interaction of IGF2BP2 and CASC9, IGF2BP2

overexpression was established by plasmids transfection (Fig. 3B). RNA stability assay demonstrated that IGF2BP2 overexpression could enhance the stability of lncRNA CASC9 in U87MG cells

when treated with Act D (Fig. 3C). MeRIP-Seq data were displayed by IGV and there were two m6A modification sites in the 3ʹ-UTR of CASC9 (Fig. 3D). Accurately, the two sites’ sequences were

shown in the genomic location (Fig. 3E). MeRIP-qPCR demonstrated that CASC9 was enriched in the GBM cell lines (U87MG) using anti-m6A antibody (Fig. 3F). Moreover, RIP

(RNA-immunoprecipitation)-qPCR showed that CASC9 could interact with the IGF2BP2 in U87MG cells (Fig. 3G). In conclusion, these findings suggested that m6A reader IGF2BP2, probably via its

interaction with CASC9, enhanced the stability of lncRNA CASC9, thereby promoting CASC9 expression. LNCRNA CASC9 ACCELERATED THE AEROBIC GLYCOLYSIS OF GBM Firstly, CASC9 expression was

elevated in the GBM cells (U251, U87MG) as compared to normal cells (Fig. 4A). To test whether CASC9 was essential for GBM cellular phenotype, we silenced the expression of CASC9 in GBM

cells through shRNA-expressing lentiviruses in U87MG cells, and CASC9 overexpression was constructed in U251 cells through plasmids transfection (Fig. 4B). Glucose uptake analysis, lactate

production and ATP generation analysis demonstrated that CASC9 overexpression promoted the glycolytic capacity, including glucose uptake (Fig. 4C), lactate production (Fig. 4D) and ATP

generation (Fig. 4E). Meanwhile, CASC9 knockdown repressed the glycolytic capacity. In further research, extracellular acidification rate (ECAR) analysis found that CASC9 overexpression

promoted the ECAR in U251 cells, and CASC9 knockdown reduced ECAR in U87MG cells (Fig. 4F). Besides, oxygen consumption rate (OCR) assay revealed that CASC9 overexpression promoted

respiratory rate in U251 cells, and CASC9 knockdown reduced respiratory rate in U87MG cells (Fig. 4G). Taken together, the data suggest that CASC9 accelerated the aerobic glycolysis of GBM.

CASC9/IGF2BP2 COMPLEX CONTRIBUTED TO THE STABILITY OF HK2 MRNA In the correlation analysis, we found that the expression of IGF2BP2 was positively correlated to the expression of HK2 in the

GBM group (Fig. 5A) based on the public database data (GEPIA, http://gepia.cancer-pku.cn/). According to the MeRIP-Seq, there was a remarkable m6A site in the 3ʹ-UTR of HK2 mRNA (Fig. 5B).

Consistent with our original hypothesis, the m6A site in the 3ʹ-UTR of HK2 mRNA was identified to be AGGGACU (Fig. 5C). RT-PCR assay demonstrated that the HK2 mRNA level was increased in the

IGF2BP2 overexpression transfection (U251 cells) and in the CASC9 overexpression transfection (U87MG cells) (Fig. 5D). MeRIP-PCR assay illustrated that HK2 mRNA was enriched in the anti-m6A

antibody group as compared to the Input group (Fig. 5E). RIP-qPCR unveiled that CASC9 overexpression promoted the interaction within IGF2BP2 and HK2 mRNA, while CASC9 silencing reduced the

combination within IGF2BP2 and HK2 mRNA (Fig. 5F). RNA stability assay demonstrated that CASC9 overexpression promoted the stability of HK2 mRNA in U251 cells. Moreover, IGF2BP2

overexpression could enhance the stability of HK2 mRNA in U87MG cells when treated with Act D (Fig. 5G). In conclusion, these results suggested that the function of the CASC9/IGF2BP2 complex

in regulating HK2 mRNA depended on its binding to the HK2 m6A modification site. DISCUSSION Recent evidence demonstrated that m6A is an intense research field and exerts critical functions

on human cancers, encompassing GBM [10,11,12]. It is known that m6A could regulate the metabolism of RNA, including mRNA, miRNA and lncRNAs [13, 14]. However, the potential role of m6A and

lncRNA in GBM is still hazy. In the data of MeRIP-Seq, we found that hundreds of potential RNA owned the m6A modification sites. Moreover, m6A peaks frequency was mainly located in the 5-end

untranslated regions (5ʹ-UTR), coding region (CDS) and 3ʹ-UTR. Upregulated or downregulated targets labeled with m6A peaks were identified. Among the m6A motif, the GGACU motif occupied the

main ingredient. Interestingly, we found that there was an m6A site in the lncRNA CASC9. On account of the regulation of m6A on RNA metabolism, m6A key enzyme may function as a regulator

for CASC9. In the clinical analysis, we found that the m6A modified lncRNA CASC9 was upregulated in GBM and indicated the unfavorable prognosis of GBM [15]. Clinically, CASC9 may serve as an

independent factor to predict the prognosis of GBM patients. In the functional assays, using CASC9 overexpression transfection and CASC9 silencing, we found that CASC9 could positively

regulate the aerobic glycolysis in GBM cells. Under basal conditions, there was no difference on glucose uptake, lactate production and ECAR. Aerobic glycolysis, also known as the Warburg

effect, functions as an essential initiating factor for GBM [16,17,18]. Aerobic glycolysis not only supports energy production but also provides the carbon skeletons for the cellular

synthesis of nucleic acids in tumor cells. Besides, fatty acids act as the energetic substrates or materials for lipid membrane construction. Therefore, the energy and outcomes of aerobic

glycolysis could remarkably promote the development and progression of GBM. Another valuable finding is that the m6A-modified CASC9 expression was mediated by the specific m6A reader

IGF2BP2. IGF2BP2 has been found to be upregulated in glioma and indicates a poor prognosis [19]. MeRIP-Seq displayed that there were two m6A modification sites in the 3ʹ-UTR of CASC9. RNA

stability assay and RT-PCR demonstrated that IGF2BP2 overexpression could enhance the stability of lncRNA CASC9 and promote its expression. Moreover, RIP illustrated that m6A reader IGF2BP2

significantly interacted with CASC9, highlighting a molecular interaction within IGF2BP2 and CASC9. Given that CASC9 could regulate aerobic glycolysis, we tried to investigate the potential

downstream targets of CASC9. After screening, we found that there was an obvious m6A site in the 3ʹ-UTR of HK2 mRNA. Glycolytic enzyme hexokinase 2 (HK2) is a crucial regulator for the GBM

Warburg effect [20, 21]. Functional analysis found that the overexpression of CASC9 could enhance the interaction within IGF2BP2 and HK2 mRNA. However, depleting CASC9 disrupted the

interaction within IGF2BP2 and HK2 mRNA. RNA stability assay demonstrated that CASC9 overexpression promoted the stability of HK2 mRNA. Moreover, IGF2BP2 overexpression could enhance the

stability of HK2 mRNA when treated with Act D. In public database data (GEPIA, http://gepia.cancer-pku.cn/), IGF2BP2 expression is positively correlated to HK2; however, the correlation

coefficient of IGF2BP2 and HK2 is not so significant (0.2). In conclusion, these results suggested that the function of the CASC9/IGF2BP2 complex in regulating HK2 mRNA depended on its

binding to the HK2 m6A modification site (Fig. 6). In conclusion, we describe the function of m6A and lncRNA in the GBM aerobic glycolysis. CASC9/IGF2BP2 complex positively regulates the HK2

mRNA, which depended on its binding to the HK2 m6A modification site. Our findings uncover a critical function for m6A-modified lncRNA CASC9 and provide insight into a critical role of m6A

methylation in GBM. MATERIALS AND METHODS BRAIN TISSUE COLLECTION Both normal brain tissue and GBM tissue were collected during surgical specimens at The Second Hospital of Hebei Medical

University (Table 1). All these processes were performed in accordance with the protocols approved by The Second Hospital of Hebei Medical University Ethics Committee in compliance with the

Declaration of Helsinki. Written informed consents were obtained from all study participants before the procedure with an explanation of experimental details. CELL CULTURE Normal human

astrocytes (NHA) and human glioblastoma cell lines (U87MG, U251) were purchased from the American Type Culture Collection (ATCC, USA). Cells were cultured in 1640 medium (Gibco, USA)

supplemented with 10% FBS (Gibco, USA), 100 U/ml penicillin and streptomycin (Gibco, USA) in humidified 37 °C and 5% CO2 incubator (Thermo, Germany). SHRNA-LENTI-VIRAL CONSTRUCTION AND

INFECTION Independent recombinant lentiviruses (pCDH-CMV-MCS-GFP) shRNA targeting CASC9 for stable knockdown were constructed by Shanghai Genechem Co., Ltd., (Shanghai, China). Stably

transfected cells were chosen with 1 μg/ml puromycin (Calbiochem, USA) for 2 weeks. Overexpression CASC9 full length was synthesized and cloned into a pLKO.1-derived plasmid vector. The

other siRNAs were bought by Riobo Bio (Guangzhou, China) and transfected into GBM cells using Lipofectamine 2000 (Invitrogen) when cells were grown to 70% confluent. DATA AVAILABILITY

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Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by Youth Grant from the Science Foundation of Hebei Province (grant number: H2021206008), Key Research and

Development Plan Program of Hebei Province (20377703D), Youth Fund for Scientific and Technological Research in Higher Education Institutions of Hebei Province (QN2018020), Key Research and

Development Program of Science and Technology Department of Hebei Province (19277723D), The Scientific Research Foundation of the Second Hospital of Hebei Medical University (grant number:

2h2019008). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Neurosurgery, The Second Hospital of Hebei Medical University, 050000, Shijiazhuang, Hebei, China Hongjiang Liu, Chen

Li, Jiankai Yang, Shunyao Zhang, Zhongjie Yan, Xiaopeng Liu, Jipeng Yang & Xiaofeng Sun * Department of Cardiology, The Second Hospital of Hebei Medical University, 050000, Shijiazhuang,

Hebei, China Shan Qin * Medical Records Room, The Second Hospital of Hebei Medical University, 050000, Shijiazhuang, Hebei, China Changqi Liu * Office of Academic Research, The Second

Hospital of Hebei Medical University, 050000, Shijiazhuang, Hebei, China Le Jiang Authors * Hongjiang Liu View author publications You can also search for this author inPubMed Google Scholar

* Shan Qin View author publications You can also search for this author inPubMed Google Scholar * Changqi Liu View author publications You can also search for this author inPubMed Google

Scholar * Le Jiang View author publications You can also search for this author inPubMed Google Scholar * Chen Li View author publications You can also search for this author inPubMed Google

Scholar * Jiankai Yang View author publications You can also search for this author inPubMed Google Scholar * Shunyao Zhang View author publications You can also search for this author

inPubMed Google Scholar * Zhongjie Yan View author publications You can also search for this author inPubMed Google Scholar * Xiaopeng Liu View author publications You can also search for

this author inPubMed Google Scholar * Jipeng Yang View author publications You can also search for this author inPubMed Google Scholar * Xiaofeng Sun View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS Conception and design by XS, SQ, Jiankai Y. Writing, review and revision of the manuscript by HL, C Li, C Liu, LJ. Experiments

assisted by SZ, ZY, XL, Jipeng Y. The author (s) read and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Xiaofeng Sun. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing interests. ETHICS STATEMENT All experiments were approved by the Ethics Committee of The Second Hospital of Hebei Medical University. ADDITIONAL INFORMATION

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THIS ARTICLE CITE THIS ARTICLE Liu, H., Qin, S., Liu, C. _et al._ m6A reader IGF2BP2-stabilized CASC9 accelerates glioblastoma aerobic glycolysis by enhancing HK2 mRNA stability. _Cell Death

Discov._ 7, 292 (2021). https://doi.org/10.1038/s41420-021-00674-y Download citation * Received: 11 June 2021 * Revised: 14 August 2021 * Accepted: 20 September 2021 * Published: 13 October

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