Research ArticleExperimental Studies

ADAR2 Regulates Malignant Behaviour of Mesothelioma Cells Independent of RNA-editing Activity

KEN-ICHIRO SAKATA, KOJIRO MAEDA, NOZOMI SAKURAI, SHANSHANG LIANG, SEITARO NAKAZAWA, KAZUYOSHI YANAGIHARA, TAKANORI KUBO, HIRONORI YOSHIYAMA, YOSHIMASA KITAGAWA, JUN-ICHI HAMADA and HISASHI IIZASA
Anticancer Research March 2020, 40 (3) 1307-1314; DOI: https://doi.org/10.21873/anticanres.14072

Abstract

Background/Aim: Malignant pleural mesothelioma (MPM) is an intractable cancer, and causes of its malignant transformation are not well known. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme that converts adenosine into inosine in double-stranded RNAs potentially involved in malignant development. Materials and Methods: To examine the role of ADAR1 and ADAR2 in MPM, small interfering RNAs (siRNAs) against ADAR1 or ADAR2 were used. Results: Transfection of siRNA against ADAR2 suppressed proliferation, motility, and invasiveness of MPM cells expressing both ADAR1 and ADAR2; however, siRNA against ADAR1 did not affect these cellular activities. Overexpression of ADAR2, that was incapable of binding to RNA, suppressed growth, motility, and invasion of MPM cells. However, overexpression of ADAR2 that had no enzyme activity did not alter the malignant properties of MPM cells. Conclusion: Enhancement of the malignant characteristics of cultured MPM cells via ADAR2 was independent of RNA-editing activity.

Malignant pleural mesothelioma (MPM) originates from mesothelial cells in the parietal or pleural pleura following asbestos exposure. MPM manifests few early clinical symptoms; therefore, total excision is often impossible at the time of diagnosis because the tumour has already invaded into the chest wall, mediastinum, and diaphragm. There are no standard treatments that extend survival (1). To fight against MPM, it is essential to better understand the biological characteristics of MPM cells and identify factors associated with their malignant progression. Hypoxia, the Hippo/yes-associated protein (YAP) pathway, and BRCA1 associated ptotein-1 (BAP1) mutations promote malignant progression of MPM cells; however, molecular mechanisms underlying this are not well understood (2, 3). Therefore, other factors associated with the malignancy of MPM cells should be identified and underlying molecular mechanisms elucidated.

Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme with double-stranded RNA-binding and adenosine deaminase domains. It converts adenosine to inosine in double-stranded RNA (4). Inosine is recognized as guanine during translation. RNA editing effectively modifies the translation of codons without an associated mutation in the genomic DNA, resulting in corresponding modifications in the structure of the protein synthesized. RNA-editing enzymes have been implicated in both cancer promotion and suppression. Antizyme induction leads to degradation of the cell cycle regulatory protein cyclin D1, and antizyme inhibitor 1 (AZIN1) can suppress antizyme activity. In liver cancer cells, AZIN1 mRNA editing by ADAR1 suppresses antizyme-enhanced cyclin D1 expression (5). Moreover, RNA editing worsens renal cancer prognosis (6). Meanwhile, ADAR2 suppresses glioma growth (7). However, effects of ADAR1 and ADAR2 on the tumour biology of MPM remain to be investigated. Therefore, in the present study, we examined the effects of ADAR2 on the proliferation, motility, and invasiveness of MPM cells in vitro and explored the underlying mechanism.

Materials and Methods

Cell culture. TCC-MESO-1, TCC-MESO-2, TCC-MESO-3, HMM1, and HMM3 cell lines were established from primary and metastatic tumours of MPM patients, as previously described (8, 9). NCI-H226 human mesothelioma cells were obtained from the National Institutes of Health, National Cancer Institute (Rockville, MD, USA). The cells were cultured in DMEM/Ham's F-12 medium (Wako Pure Chemical, Osaka, Japan) supplemented with 10% inactivated foetal bovine serum (FBS, Cambrex, East Rutherford, NJ, USA) at 37°C and 5% CO2.

RNA extraction and quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Total RNA was extracted using RNAiso Plus (TAKARA Bio, Shiga, Japan). After DNase treatment (DNase I Amp Grade, Invitrogen, Carlsbad, CA, USA), single-stranded cDNA was synthesized from 1 μg RNA and oligo dT using SuperScript III (Invitrogen), following the manufacturer's instructions. The following primers were used: 5’-GATGCCTTT GCAGAACACCA-3’ (FW) and 5’-TGCCTTCTGATGCTGAGAACC-3’ (RV) for human ADAR1; 5’-GTCTGTGGAGGTGAATGGCC-3’ (FW) and 5’-CCGTGTTGACAGACAGGGTC-3’ (RV) for human ADAR2; and 5’-TTGCCGACAGGATGCAGAA-3’ (FW) and 5’-GCCGAT CCACACGGAGTACT-3’ (RV) for human ACTB (β-actin). qRT-PCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN, Valencia, CA, USA) on the StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). ACTB was used as the internal standard; results were analyzed using the ΔΔCt method.

RNA-editing efficiency. RNA-editing efficiency was analyzed by direct sequencing of PCR products (10). PCR products included human BIRC4, as an ADAR1 substrate, and human COPA, as an ADAR2 substrate. BIRC4-specific primers were 5’-AGGGATAGCAAGGGACAATTGTATC-3’ (FW) and 5’-TGGGTGGTCAGATTCTACTTT GAATC-3’ (RV) (11). COPA-specific primers were 5’-CTGAGTGCCTCCGATGATCAGA-3’ (FW) and 5’-AACTGGGCTGCCTTCCACC-3’ (RV) (12). PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and sequence primers (human COPA FW, human BIRC4 FW) following the kit manufacturer's instructions. Nucleotide sequences were analyzed using the 3130 Genetic Analyzer (Life technologies, Carlsbad, CA, USA). RNA-editing efficiency was calculated on the basis of a histogram of sequences using the following formula: [(edited RNA / edited RNA + unedited RNA) ×100 (%)].

Construction of ADAR2 and dominant negative ADAR2 expression vectors. A plasmid containing human ADAR2 cDNA (MHS1010-9206176) was obtained from Open Biosystems (Huntsville, AL, USA). PCR of the protein-coding region of human ADAR2 cDNA was performed using KOD Plus-Neo (TOYOBO, Osaka, Japan); the cloning primers were 5’-CACCATGGATATAGAAGATGAAGAAAACATGA-3’ (FW) and 5’-TTATCAGGGCGTGAGTGAGAA-3’ (RV). PCR products were incorporated into pCMV-3xFLAG, p3XFLAG-CMV-7.1, and pCMV-hADAR2 (Sigma, St Louis, MO, USA). Replacing glutamic acid at the position 375 in the deaminase domain of ADAR2-T375A with alanine resulted in the formation of a dimer without RNA-editing activity (13). Using pCMV-hADAR2 as a template, the PCR product was amplified using KOD Plus-Neo with the primers 5’-ATGACTGCCATGCAGGAGCAATAATA TC-3’ (FW) and 5’-TTAATGCAAGGCCCACGTCACTC-3’ (RV), and digested with Dpn I (TaKaRa Bio). The remaining PCR product was ligated with T4 DNA ligase (Wako Pure Chemical) and T4PNK (TaKaRa Bio). ADAR2 EAA completely lacking the RNA-binding ability was provided by Dr. Kazuko Nishikura (Wistar Institute) (14).

Transfection. Small interfering RNAs (siRNAs) were designed by BLOCK-iT RNAi Designer (Invitrogen). The primer sequences were as follows: 5’-CCAGUGAGAGGGAGCUCUG UGTT-3’ (sense) and 5’-CACAGAGCUCCCUCUCACUGGTT-3’ (antisense) for ADARp110; 5’-GAGAGGAGGAGCAUAGUUCTT-3’ (sense) and 5’-GAACUAUGCUCCUCCUCUCTT-3’ (antisense) for ADARp150; and 5’-GGAAGAAUCCCGUGAUGAUTT-3’ (sense) and 5’-AUC AUCGGGAUUCUUCCT-3’ (antisense) for ADAR2. AllStars Negative Control siRNA (QIAGEN) was used as a negative control in RNAi experiments. For transfection, 1×106 tripsinized cells were suspended in 100 μl Opti-MEM (Invitrogen) and incubated with siRNA at a final concentration of 4 μM. Gene transfer was performed by electroporation with CUY21 Pro-Vitro-S (NEPAGENE, Ichikawa, Japan). The transfected cells were transferred onto DMEM/Ham's F-12 nutrient mixture containing 10% FBS and cultured at 37°C and 5% CO2 for 48 h before RNA isolation.

Western blotting. Total protein was extracted from cells with RIPA buffer [25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS with protease inhibitor (cOmplete Mini EDTA-free, Sigma)]. The extracted proteins were separated by electrophoresis, transferred onto polyvinylidene difluoride membranes (Immobilon P, Millipore, Burlington, MA, USA), and blocked with TBS-T [10 mM Tris–HCl (pH 7.5)/100 mM NaCl/0.1% Tween 20 containing 5% skim milk] for 1 h at room temperature. Membranes were incubated with primary antibodies including anti-ADAR1 (11.8.1) diluted in Can Get Signal Solution 1 (TOYOBO), anti-ADAR2 (1.3.1) (kindly provided by Dr. Kazuko Nishikura), and anti-β-actin (MAB1501, Millipore), at room temperature for 1 h. Membranes were incubated with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG F (ab')2 (GE Healthcare Bioscience, Pittsburgh, PA, USA) secondary antibody diluted with Can Get Signal Solution 2 (TOYOBO) at room temperature for 45 min. Protein expression was detected with an Immobilon Western HRP substrate (Millipore) and visualized with an ImageQuant LAS4000 mini biomolecular imager (GE Healthcare Bioscience).

Figure 1.
Figure 1.

ADAR expression in MPM cells with or without siRNA transfection. (A) ADAR1 and ADAR2 mRNA expression in MPM cells. White column: ADAR1; black column: ADAR2. mRNA expression was normalized against β-actin expression. Expression in TCC-MESO-1 cells was set at 1. (B) Effects of siRNA on ADAR1 mRNA expression. ADAR1 mRNA expression level was normalized against β-actin expression; expression with no siRNA was set at 1. Protein extracts from siRNA-treated cells were incubated with antibodies against ADAR1 or β-actin. Control: control siRNA; p110: ADAR1p110; p150: ADAR1p150. (C) Effect of siRNA on ADAR2 mRNA expression. ADAR2 mRNA expression was normalized against β-actin expression; expression with no siRNA was set at 1. Protein extracts from siRNA-treated cells were incubated with antibodies against ADAR2 or β-actin. (D) ADAR1 and ADAR2 RNA-editing efficiency in siRNA-treated samples. White column: BIRC4 mainly edited by ADAR1; black column: COPA edited by ADAR2. RNA-editing efficiency was determined by sequence histogram analysis. All experiments were performed in triplicate; RNA-editing values are reported as means. No siRNA: Transfection without siRNA; Control: Control siRNA; p110: ADAR1p110 siRNA; p150: ADAR1p150 siRNA. *p<0.05.

Cell proliferation assay. Cell proliferation was assayed as described by Kueng et al. (15). Cells were seeded at a density of 3×105/well in 6-well plates and cultured for 48 h at 37°C in a 5% CO2/95% air atmosphere. Cells were washed with phosphate-buffered saline containing 1 mM Ca2+ and 0.5 mM Mg2+ and fixed in 10% formalin. After washing with distilled water, cells were dried and stained with 0.2% crystal violet and 100 mM pH 9.4 CAPS buffer for 30 min. After staining, cells were washed with distilled water and dried. Cells were then suspended in 5 ml 10% acetic acid, and 100 μl aliquots were transferred to 96-well plates. Absorbance was measured at 595 nm using a model 680 micro plate reader (Bio-Rad, Hercules, CA, USA).

Phagokinetic track assay. Cell motility was measured as described by Alberecht-Buehler (16). A solution containing 10.6 mM Na2CO3 and 1.27 mM HAuCl4 was heated with a gas burner, and 0.01% formaldehyde was added immediately after boiling to prepare a colloidal gold solution. A 22×22 mm2 cover glass (Matsunami, Tokyo, Japan) coated with 1% bovine serum albumin (Sigma) was placed in the culture dish. The colloidal gold solution was dropped onto it and allowed to stand for 45 min. After washing twice with DMEM/Ham's F-12, 1×103 cells were cultured onto DMEM/Ham's F-12 supplemented with 2% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. After culture at 37°C in 5% CO2 incubator for 24 h, the cells were observed under an inverted microscope. Migrating cells visible at 5× magnification in five randomly selected fields were counted, and the area of trajectory was measured with Photoshop (Adobe systems, San Jose, CA, USA).

Cell invasion assay. Cell invasion assay was performed using Transwell chambers (Costar, Cambridge, MA, USA) with 8-μm pore membranes using the same method described in our previous report (17), with some modifications. Type I collagen solution (Cellmatrix type I-A, Nitta Gelatin, Yao, Japan), 10-fold concentrated DMEM (Wako Pure Chemical), neutralizing buffer (NaHCO3/HEPES), and FBS were mixed at a ratio of 8:1:1:0.5, respectively. The mixed collagen solution was added to the wells of a 24-well plate, and a Transwell insert was placed on each well. The plates were placed in a CO2 incubator for 30 min to create a gel. A suspension of 2×104 TCC-MESO-1 cells in 5% FBS DMEM was added to the upper chamber, and cells were cultured for 24 h at 37°C in a 5% CO2 incubator. After culture, the upper chamber was removed and the number of cells infiltrating into the gel in the lower chamber was counted in three fields in each well at ×200 magnification under an inverted microscope.

Statistical analysis. Statistical analyses were performed with JMP9 (SAS Institute, Cary, NC, USA). Tukey–Kramer HSD test was performed with a significance level of p<0.05.

Results

QRT–PCR confirmed that all cell lines (TCC-MESO-1, TCC-MESO-2, TCC-MESO3, HMM1, HMM3, and H226) expressed both ADAR1 and ADAR2 (Figure 1A). Differences in ADAR1 and ADAR2 expression were compared between TCC-MESO-1 cells transfected with siRNAs against the two genes and control cells (Figure 1B). Cells transfected with ADAR1p110-specific siRNA showed suppressed ADAR1 mRNA expression by 50% compared with non-transfected control cells, and cells transfected with ADAR1p150-specific siRNA showed suppressed ADAR1 mRNA expression by 70% compared with non-transfected control cells. Cells transfected with both siRNAs showed suppressed ADAR1 mRNA expression by 80%. ADAR1 protein expression in cells transfected with siRNA against ADAR1 was consistent with its mRNA expression. TCC-MESO-1 cells transfected with ADAR2-specific siRNA showed suppressed ADAR2 mRNA expression by 53% compared with control cells (Figure 1C). Cells transfected with ADAR2-specific siRNA showed ADAR2 protein expression consistent with its mRNA expression. Reduced RNA-editing efficiencies of BIRC4 (ADAR1) and COPA (ADAR2) in cells transfected with specific siRNAs were consistent with changes observed in ADAR1 and ADAR2 mRNA expression (Figure 1D).

Cell proliferation was inhibited by decreased ADAR2 expression but not by decreased ADAR1 expression (Figure 2A). In vitro invasion of TCC-MESO-1 cells into type I collagen gels was inhibited by transfection with siRNA against ADAR2 but not by transfection with ADAR1-specific siRNA (Figure 2B). In phagokinetic track assay, the motility of cells transfected by ADAR2-specific siRNA was significantly reduced. siRNAs targeting ADAR1 did not alter cell motility (Figure 2C).

Involvement of the T375A and EAA domains of the ADAR2 protein in the proliferation, motility, and invasion of MPM cells is depicted in Figure 3. Wild-type (WT) or a dominant negative ADAR2 mutant was transfected into TCC-MESO-1 cells (Figure 3A). The T375A-ADAR2 mutant, harbouring a point mutation in the deaminase domain, could bind to but not edit RNA (9). The EAA-ADAR2 mutant could not bind to RNA (17). The expression of WT and mutant ADAR2 proteins in the transfected cells was confirmed by western blotting. Cells expressing WT ADAR2 showed higher COPA mRNA editing efficiency than control cells. COPA mRNA editing was absent in TCC-MESO-1 cells transfected with either T375A-ADAR2 or EAA-ADAR2 (Figure 3B). Enforced expression of either WT or ADAR2-T375A did not affect the proliferation of TCC-MESO-1 cells. Enforced expression of ADAR2-EAA significantly reduced cell proliferation (by 36%) (Figure 3C). Motility and invasion of cells expressing ADAR2-EAA were significantly decreased compared with those of control cells; these properties remained unaffected in cells expressing WT or ADAR2-T375A (Figure 3D and 3E).

Discussion

Suppression of ADAR1 expression did not alter the malignant properties of TCC-MESO-1 cells, although suppression of ADAR2 expression decreased MPM cell proliferation, motility, and invasion (Figures 2 and 3). Effects of ADAR1 and ADAR2 expression in HMM3 cells were similar (data not shown). ADAR2 was previously reported as a tumour suppressor gene in glioma (5); however, RNA editing of SLC22A3 by ADAR2 promoted tumour malignancy in oesophageal cancer (18). ADAR2 consists of functional RNA-binding and deaminase domains. Cell proliferation, motility, and invasion of MPM cells were reduced by expression of ADAR2 without RNA-binding ability (Figure 3). Overexpression of WT ADAR2 did not alter these malignant characteristics (Figure 3). Whether ADAR2-specific substrates other than COPA are present in MPM cells remains unclear, although endogenous ADAR2 expression may be sufficient to promote tumour malignancy.

Figure 2.
Figure 2.

Effect of ADAR siRNA on MPM malignancy index. (A) Cell proliferation in ADAR siRNA-transfected TCC-MESO-1MPM cells. Crystal violet staining was measured at an absorbance of 595 nm (n=3). (B) Invasion of ADAR siRNA-transfected TCC-MESO-1 cells (n=5). (C) Motility of ADAR siRNA-transfected TCC-MESO-1 cells. Left: cover slip coated with colloidal gold particles after cell movement. Right: motility assay after removal of the colloidal gold and digitizing (n=9). No siRNA: Transfection without siRNA; Control: control siRNA; p110: ADAR1p110 siRNA; p150: ADAR1p150 siRNA; ADAR2: ADAR2 siRNA. *p<0.05.

Functions of ADAR2 other than RNA editing are not well understood. ADAR2 competitively inhibited, poly(A) specific ribonuclease (PARN) and human antigen R (HuR) which mediate mRNA degradation, and this function stabilized nuclear non-coding RNA (19). Similarly, ADAR1 also antagonized staufen1 and suppressed degradation of mRNA with a double-stranded structure at the 3’ UTR (20). Meanwhile, changes in miRNA synthesis independent of RNA editing have been reported in ADAR2-deficient mice, although molecular mechanisms underlying this phenomenon remain unclear (21). MPM is one of the cancers with poor prognosis, and factors involved in tumor malignancy such as BAP1, Hippo pathway related genes and miR-126 have been identified (22, 23). Interestingly, up-regulated ADAR2 expression in mesothelioma tissues compared with that in normal lung tissues was recently found to be correlated to decreased survival, which is consistent with the in vitro results of the present study (24).

In the present study, ADAR2 promoted growth, motility, and invasion of MPM cells, and this ADAR2-dependent malignant progression was independent of its RNA-editing activity. Therefore, investigating ADAR2 as a novel therapeutic target for MPM may be promising.

Figure 3.
Figure 3.

Effect of dominant negative ADAR2 overexpression on MPM malignancy index. (A) WT and dominant mutant ADAR2 expression. Mutant expression plasmids were introduced into TCC-MESO-1 cells, and protein expression was assayed using western blots, with β-actin as a loading control. (B) ADAR2 RNA-editing efficiency in TCC-MESO-1 cells expressing WT or overexpressing mutant ADAR2. Black column: COPA edited by ADAR2. RNA-editing efficiency was determined by sequence histogram analysis (n=3); RNA-editing frequencies are reported as means. (C) Proliferation of TCC-MESO-1 cells expressing WT and overexpressing mutant ADAR2. Crystal violet staining was measured at an absorbance of 595 nm. (D) Invasion of WT ADAR2 and mutated ADAR2 transfected TCC-MESO-1 cells (n=5). (E) Motility of TCC-MESO-1 cells expressing WT and overexpressing mutant ADAR2. Left: glass cover slip coated with colloidal gold particles after cell movement. Right: motility assay following removal of the colloidal gold and digitizing (n=9). *p<0.05. WT: Wild-type ADAR2; T375A: ADAR2 T375A RNA-editing activity mutant; EAA: ADAR2 EAA RNA-binding activity mutant.

Acknowledgements

The study was supported by the Ministry of Education, Culture, Sports, Science and Technology's Scientific Research Fund [26460465(H.I.), 26460466 (J.H.), 19K21361(K.S.)]. The figures were created by Ms. Sayuri Hamada.

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Authors' Contributions

    K.S. and K.M. mainly performed the experiments and analyzed data. J.H., H.I. H.Y., and K.Y. drafted the manuscript, N.S., L.S., and S.N. performed the experiments. K.Y. and T.K. analyzed data. J.H. and H.I. designed and supervised the research and complied the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest regarding this study.

  • Received December 14, 2019.
  • Revision received January 30, 2020.
  • Accepted February 3, 2020.

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ADAR2 Regulates Malignant Behaviour of Mesothelioma Cells Independent of RNA-editing Activity
KEN-ICHIRO SAKATA, KOJIRO MAEDA, NOZOMI SAKURAI, SHANSHANG LIANG, SEITARO NAKAZAWA, KAZUYOSHI YANAGIHARA, TAKANORI KUBO, HIRONORI YOSHIYAMA, YOSHIMASA KITAGAWA, JUN-ICHI HAMADA, HISASHI IIZASA
Anticancer Research Mar 2020, 40 (3) 1307-1314; DOI: 10.21873/anticanres.14072
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