Jiang et al. J Hematol Oncol (2021) 14:54 https://doi.org/10.1186/s13045-021-01066-6

LETTER TO THE EDITOR

HNRNPA2B1 promotes multiple myeloma progression by increasing AKT3 expression via m6A-dependent stabilization of ILF3 mRNAFengjie Jiang1,2†, Xiaozhu Tang2†, Chao Tang2†, Zhen Hua2, Mengying Ke2, Chen Wang2, Jiamin Zhao2, Shengyao Gao2, Artur Jurczyszyn3, Siegfried Janz4, Meral Beksac5, Fenghuang Zhan6,7, Chunyan Gu1,2* and Ye Yang2,6*

Abstract

N6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic RNAs while accumulating studies suggest that m6A aberrant expression plays an important role in cancer. HNRNPA2B1 is a m6A reader which binds to nascent RNA and thus affects a perplexing array of RNA metabolism exquisitely. Despite unveiled facets that HNRNPA2B1 is deregulated in several tumors and facilitates tumor growth, a clear role of HNRNPA2B1 in multiple myeloma (MM) remains elusive. Herein, we analyzed the function and the regulatory mechanism of HNRNPA2B1 in MM. We found that HNRNPA2B1 was elevated in MM patients and negatively correlated with favorable prognosis. The depletion of HNRNPA2B1 in MM cells inhibited cell proliferation and induced apoptosis. On the contrary, the overex-pression of HNRNPA2B1 promoted cell proliferation in vitro and in vivo. Mechanistic studies revealed that HNRNPA2B1 recognized the m6A sites of ILF3 and enhanced the stability of ILF3 mRNA transcripts, while AKT3 downregulation by siRNA abrogated the cellular proliferation induced by HNRNPA2B1 overexpression. Additionally, the expression of HNRNPA2B1, ILF3 and AKT3 was positively associated with each other in MM tissues tested by immunohistochemistry. In summary, our study highlights that HNRNPA2B1 potentially acts as a therapeutic target of MM through regulating AKT3 expression mediated by ILF3-dependent pattern.

Keywords: M6A, HNRNPA2B1, Multiple myeloma, MeRIP-Seq, ILF3, RNA stability, RIP-seq, AKT3

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To the EditorN6-methyladenosine (m6A) modification is the most

frequent RNA modifications in eukaryotic RNAs affect-ing gene expression, which is seldom investigated in MM [1–3]. Therefore, we checked the m6A genes in MM compared to normal plasma cells and the correlation of these genes with patient outcome including HNRN-PA2B1, Mettl3, Mettl14, Wtap, etc., in MM patient

cohorts. Interestingly, HNRNPA2B1 was the exclusive gene, which was not only increased in MM samples but also associated with poor outcome in APEX, TT2 and HOVON65 patient cohorts (Fig. 1a, b, Additional file 1: Fig. S1a–h). HNRNPA2B1, RNA binding protein het-erogeneous nuclear ribonucleoprotein A2B1, is a nuclear reader of m6A [4] and highly expressed in several can-cers regulating the progression of cancer [5, 6] through multiple processes of mRNAs metabolism [7] including alternative splicing [8], cytoplasmic RNA trafficking [9], transcription and translation [10]. Here, we aimed to explore the potential functions and regulatory mecha-nism of HNRNPA2B1 in MM.

Open Access

*Correspondence: guchunyan@njucm.edu.cn; yangye876@sina.com†Fengjie Jiang, Xiaozhu Tang, and Chao Tang have contributed equally to this work.2 School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, ChinaFull list of author information is available at the end of the article

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Initially, we performed MTT assay that demonstrated the cellular proliferation was significantly increased in HNRNPA2B1 overexpression (HNRNPA2B1OE) cells and decreased in HNRNPA2B1 knockdown (HNRNPA2B1KD) cells (Fig. 1c, d, Additional file 1: Fig. S1i). Flow cytom-etry and WB analyses illustrated that knockdown of HNRNPA2B1 promoted cellular apoptosis (Fig.  1e, f ). The nude mouse xenograft model by subcutaneous injec-tion of HNRNPA2B1OE cells showed overexpression of

HNRNPA2B1 accelerating tumor growth in vivo (Fig. 1g and Additional file 1: Fig. S1j, k, l). These data indicated that HNRNPA2B1 promoted MM cellular growth in vitro and in vivo.

Next, m6A IP and RNA-seq analyses (MeRIP-Seq) found that ILF3 was downregulated (P < 0.05) of both m6A and transcription in HNRNPA2B1KD cells com-pared with controls (Fig. 1h & Additional file 1: Fig. S2). The m6A consensus sequence (RRACH) motif [11] is

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Fig. 1 HNRNPA2B1 promotes MM proliferation and increases ILF3 protein expression through recognizing m6A modification and maintaining stabilization of ILF3 mRNA. a HNRNPA2B1 mRNA levels were significantly increased in MM samples. The signal level of HNRNPA2B1 was shown on the y-axis. Patients were designated as healthy donors with normal bone marrow plasma cells (NP, n = 22), monoclonal gammopathy of undetermined significance (MGUS, n = 44), or multiple myeloma (MM, n = 351), which were sorted on the x-axis. b Elevated HNRNPA2B1 mRNA was associated with poor overall survival (OS) in MM patients from the APEX patient cohort. c Overexpression of HNRNPA2B1 was confirmed by Western blotting after lentivirus infection in MM cells. d Overexpression of HNRNPA2B1 promoted cell proliferation in ARP1 and H929 cells. e Flow cytometry showed that HNRNPA2B1 inhibited MM cells apoptosis. f Expression of HNRNPA2B1 and apoptosis-related proteins in HNRNPA2B1KD MM cells. g Tumor tissues were dissected from NOD-SCID mice injected with wild-type and HNRNPA2B1OE cells. h Hot map of 44 differentially expressed genes (P < 0.05) in both m6A and transcription in ARP1 HNRNPA2B1KD cells compared with controls. i HORMER motif analysis of m6A peaks in ARP1 and H929 cells. "Target" showed the percentage of peaks containing the identified consensus motif, "Background" presented the percentage of genome background regions that contain the identified motif. j IGV tracks depicted the position of m6A modification of ILF3 gene. k HNRNPA2B1 gene knockdown affected expression of ILF3 mRNA in MM cells. l RIP-qPCR assay was used to determine the interaction between HNRNPA2B1 and ILF3 mRNA in ARP1 cells. m The expression of ILF3 RNA was detected in ARP1 cells upon treating with cyclolencine in the concentration of 0 mM, 50 mM and 100 mM. n RT-qPCR following the addition of Actinomycin D (5 μg/mL) was performed to detect ILF3 mRNA stability. k HNRNPA2B1 (red) localization was examined by confocal microscopy. (i) Nuclei were stained with DAPI. Scale bar, 5 μm. (ii) Immunolocalization of HNRNPA2B1 in MM cells. (iii) Immunolocalization of ILF3. (iv) Merger of images of i, ii and iii, with the colocalized regions shown in orange. (*P < 0.05, **P < 0.01, ***P < 0.001)

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shown in Fig. 1i, in which the m6A modification of ILF3 was enriched in 3′-noncoding regions (Fig.  1j). Consist-ently, ILF3 expression was reduced in HNRNPA2B1KD cells (Fig. 1k, Additional file 1: Fig. S3a) while increased in HNRNPA2B1OE cells (Additional file 1: Fig. S3b, c). RNA immunoprecipitation (RIP)-qPCR assay revealed that ILF3 mRNA was enriched in the precipitates of HNRN-PA2B1 antibody and silencing of HNRNPA2B1 decreased the abundance of the ILF3 transcript binding to HNRN-PA2B1 (Fig.  1l). To further verify the role of m6A in regulation of ILF3 expression, methylation inhibitor cyclolencine was used that induced remarkable reduc-tion of ILF3 in MM cells (Fig.  1m, Fig. S3d, e). There-fore, we can conclude that HNRNPA2B1 may stabilize ILF3 mRNA to play an important role. As expected, the stability of ILF3 was decreased in HNRNPA2B1KD cells (Fig. 1n, Additional file 1: Fig. S3f ). The biological effects of HNRNPA2B1 might be related to its nucleocytoplas-mic localization, as HNRNPA2B1 was distributed in both nuclear and cytoplasmic along with increased cytoplas-mic localization of ILF3 (Fig.  1o, Additional file  1: Fig. S3g). The above results illustrated that HNRNPA2B1-induced expression of ILF3 was due to the enhanced sta-bility of ILF3 mRNA transcripts upon recognition and bound of the m6A sites to HNRNPA2B1.

Notably, ILF3 expression was significantly elevated in plasma cells from MM patients (Fig.  2a) and associated with poor survival (Fig.  2b, Additional file  1: Fig. S4a, b). We established the ILF3 knockdown (ILF3KD) cells (Fig. 2c), which displayed decreased cell growth rate after induction compared to the non-induced cells (Fig.  2d). Apoptosis assay showed that ILF3 inhibited MM cel-lular apoptosis (Additional file 1: Fig. S4c, d, e). In addi-tion, MTT result demonstrated that ILF3 knockdown by siRNA could reverse the cellular proliferation induced by increased HNRNPA2B1 suggesting that ILF3 is one of the most important m6A/HNRNPA2B1 targets in MM (Additional file 1: Fig. S4f, g).

Further RNA immunoprecipitation-sequencing (RIP-seq) analysis indicated that MAPK pathway was enriched (Fig. 2e) and AKT3 mRNA was significantly enriched by anti-ILF3 antibody in ARP1 cells. The ILF3 binding sites represented by peaks were enriched in AKT3 mRNA transcripts (Fig.  2f ). In agreement with above results, decreased AKT3 was observed in ILF3KD cells (Fig.  2g, h) and RIP-qPCR using anti-ILF3 antibody confirmed a significantly reduced affinity of ILF3 to AKT3 mRNA in ARP1 ILF3KD cells compared to control (Fig.  2i). RNA decay assay showed a relatively lower stability of AKT3 transcripts in ILF3KD cells (Fig.  2j, Additional file  1: Fig S5a) correspondingly. These data suggested that ILF3 promoted MM progression through stabilization of AKT3 transcripts.

Finally, we verified the effect of HNRNPA2B1 on AKT3. The expression of AKT3 was decreased in HNRNPA2B1KD cells (Fig.  2k, l), whereas the elevated expression of AKT3 was observed in HNRNPA2B1OE cells (Additional file  1: Fig. S5b, c). While AKT3 was interfered with siRNA (Fig. 2m), the cellular prolifera-tion induced by HNRNPA2B1 was attenuated (Fig. 2n). Intriguingly, immunohistochemistry correlation analy-sis showed that HNRNPA2B1, ILF3 and AKT3 were highly increased in MM patients with statistical-corre-lated expression trend significantly compared to nor-mal controls (Fig. 2o).

In summary, we demonstrate the m6A-dependent effect of HNRNPA2B1 on regulating AKT signaling pathway and the correlation between HNRNPA2B1 and MM cell growth. It is disclosed that the HNRN-PA2B1/m6A/ILF3/AKT3 axis plays a key role in MM progression.

Abbreviationsm6A: N6-methyladenosine; HNRNPA2B1: Heterogeneous nuclear ribonu-cleoprotein A2B1; MM: Multiple myeloma; MeRIP-Seq: M6A IP seqing; ILF3: Interleukin enhancer-binding factor 3; RIP: RNA immunoprecipitation; AKT3: AKT serine/threonine kinase 3; ActD: Actinomycin D.

Supplementary InformationThe online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13045- 021- 01066-6.

Additional file 1. HNRNPA2B1 is a high-risk MM marker and promotes MM progression via enhancing ILF3-mediated expression of AKT3 in vitro and in vivo.

Additional file 2. Detailed materials and methods.

Additional file 3.

AcknowledgementsWe thank Dr. Jinjun Qian for providing technical support.

Authors’ contributionsCG and YY designed the project, integrated the data and revised the manu-script; FJ, XT, CT, ZH, MK, CW, JZ and SG performed experiments, analyzed the data and drafted the manuscript; AJ, SJ, MB and FZ supervised this project and the manuscript; FZ offered part of the MM cohorts’ database. All authors have read and approved the final version of the manuscript.

FundingThis work was supported by National Key R&D Program of China (No. 2020YFA0509400) and National Natural Science Foundation of China 81970196 and 81670200 (to CG & YY); A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine); Innovation Team of Six Talent Peaks Project in Jiangsu Province (TD-SWYY-015); Natural Science Foundation of Jiangsu Province BK20200097 (to CG).

Availability of data and materialsAll supporting data are included in the manuscript and supplemental files. Additional data are available upon reasonable request to the corresponding author.

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Declarations

Ethics approval and consent to participateThis study was approved by the Animal Ethics Committee of Nanjing Univer-sity of Chinese Medicine (Ethics Registration No. 201905A003) in China.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1 The Third Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210001, China. 2 School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023,

China. 3 Department of Hematology, Jagiellonian University Medical College, Cracow, Poland. 4 Division of Hematology and Oncology, Medical College of Wisconsin, Milwaukee, USA. 5 Department of Hematology, School of Medi-cine, Ankara University, Ankara, Turkey. 6 Internal Medicine, University of Iowa, Iowa City, USA. 7 Myeloma Center, University of Arkansas, Little Rock, USA.

Received: 24 December 2020 Accepted: 23 March 2021

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Fig. 2 HNRNPA2B1 facilitates MM progression via enhancing ILF3-mediated expression of AKT3 in vitro. a ILF3 mRNA levels were significantly increased in MM samples. The signal level of ILF3 was shown on the y-axis. Patients were designated as healthy donors with normal bone marrow plasma cells (NP, n = 22), monoclonal gammopathy of undetermined significance (MGUS, n = 44), or multiple myeloma (MM, n = 351), which were sorted on the x-axis. b Increased ILF3 mRNA was associated with poor overall survival (OS) in MM patients from the APEX patient cohort. c The shRNA-mediated ILF3 repression was confirmed by Western blotting after lentivirus infection in ARP1 and H929 cells. d Effect of ILF3 knockdown on cell proliferation in MM cells. e The top 20 enriched KEGG pathways of the RIP-seq were presented as scatter plot. f Visualization of RIP-seq signal surrounding the AKT3 locus. g and h AKT3 expression under ILF3 silence was detected by RT-qPCR (g) and Western blotting (h). i RIP-qPCR assay was used to determine the interaction between ILF3 and AKT3 mRNA. j RT-qPCR following the addition of ActD (5 μg/mL) was used to detect AKT3 mRNA stability in ARP1 cells. k HNRNPA2B1 knockdown affected the expression of ILF3, AKT3 at mRNA level in ARP1 and H929 cells. l HNRNPA2B1 knockdown affected the expression of ILF3, AKT3 at protein level in ARP1 and H929 cells. m Protein expression was examined after HNRNPA2B1OE cells treated with AKT3 small interfering RNA. n MTT assay indicated that targeting AKT3 by siRNA could reverse MM cell proliferation induced by HNRNPA2B1 overexpression. o Immunohistochemistry staining of HNRNPA2B1, ILF3 and AKT3 in primary MM samples (n = 12) and normal control (NP) (n = 12). (*P < 0.05, **P < 0.01, ***P < 0.001)

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