m(cid:0)A Demethylase ALKBH5 Promotes Tumor Cell Proliferation by Destabilizing IGF2BPs Target Genes and Worsens the Prognosis of Patients with Non-Small Cell Lung Cancer

Background: The modication of N 6 -methyladenosine (m 6 A) in RNA and its eraser ALKBH5, an m 6 A demethylase, play important roles across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-smallcell lung cancer (NSCLC) development remains to be completely elucidated. Methods: The current study investigated the involvement of ALKBH5 in NSCLC development using immunostaining of clinical NSCLC specimens as well as cancer-related cellular functions (cell proliferation, migration ability, cell cycle, and apoptosis) in ALKBH5-knockdown lung cancer cell lines. Moreover, a microarray was utilized to comprehensively analyze mRNA and m 6 A in ALKBH5-knockdown cells. m 6 A target genes were identied using the methylated RNA immunoprecipitation (MeRIP) assay with m 6 A antibody. Furthermore, mRNA stability and protein expression owing to m 6 A modication (the target genes) were examined. Results: Clinicopathological analysis revealed that increased ALKBH5 expression was an independent prognostic factor associated with unfavorable overall survival in NSCLC (hazard ratios, 1.468; 95% condence interval, 1.039–2.073). In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells as well as promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines BEAS2B and HEK293. Comprehensive analysis of microarray and MeRIP quantitative-polymerase chain reaction revealed that 3′ untranslated regions (3′ UTRs) of CDKN1A, TIMP3, E2F1, and CCNG2 mRNA were potential targets of ALKBH5. Depending on the lung cancer cell lines, increased expression of CDKN1A or TIMP3 and decreased cell proliferation were observed by ALKBH5 knockdown.These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline splicing the the heterogeneous nuclear ribonucleoprotein G (HNRNPG) alters RNA structures via RNA–protein interaction YTH domain family1 (YTHDF1), YTHDF3, METTL3, and eukaryotic initiation factor3 (eIF3) regulate translation eciency and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), YTHDF2, YTHDF3, and YTHDC2 alter mRNA stability Differences between experimental groups were assessed using Mann–Whitney U-test for continuous variables or Fisher’s exact test for categorical data. Data represent median (range) or number (n) (%). Prediction of mortality of patients with non–small-lung cancer. The univariate and multivariate Cox proportional hazards models were applied to generate the hazard ratios (HRs) of death. Multivariate analysis was adjusted by age, sex, smoking status, histology, stage, and ALKBH5. HR; hazard ratio, confdence interval, Ad; adenocarcinoma, Representative images of the wound-healing assay for HEK293 (d) and BEAS2B (f) cells. Wound areas relative to baseline at each time point were compared between ALKBH5 OE and NC HEK293 (e) and BEAS2B (g) cells (n = 3). Results are presented as mean ± SD. *P< 0.05indicates a signicant difference between the indicated groups. western blot analysis. Endogenous mRNA expression levels of cells or those in cells transfected with were analyzed via compared with

Over the past few years, several researchers investigating the role of m 6 A eraser in malignant tumors have revealed that m 6 A eraser proteins play a critical role in oncogenesis. A number of previous studies have demonstrated that ALKBH5 exerts a cancer-promoting effect in glioblastoma, osteosarcoma, colon cancer, ovarian cancer, esophageal squamous cell carcinoma, endometrial cancer, and renal cell carcinoma [23][24][25][26][27][28][29]. In contrast, ALKBH5 has been reported to play a tumor-suppressing effect in hepatocellular carcinoma and pancreatic cancer [30,31]. Several studies on lung cancer have shown that FTO plays a cancer-promoting role through m 6 A modi cation in lung squamous cell carcinoma and adenocarcinoma [32][33][34], whereas ALKBH5 inhibits NSCLC tumorigenesis by reducing YTHDFs-mediated YAP expression [35]. Conversely, ALKBH5 had also been found to promote NSCLC progression by regulating TIMP3 stability [36]. Thus, the precise role of ALKBH5 in NSCLC tumorigenesis across various conditions deserves further investigation.
Cell type and cell environment (e.g., during hypoxic conditions) as well as the m 6 A target gene and its recognition protein (reader) have been found to affect RNA metabolism caused by ALKBH5 perturbation [37,38]. Therefore, m 6 A-mediated gene expression regulated by ALKBH5 could result in various consequences in cancer cells depending on the surrounding environment and other factors. Several studies regarding m 6 A have focused on speci c genes in the speci c contexts, with their results showing that m 6 A is involved in the mechanisms through which these speci c genes are regulated. However, in actual human cancers, ALKBH5 catalyzes speci c m 6 A of numerous genes, which simultaneously alters several RNA and protein expressions through RNA recognition by reader proteins, consequently causing numerous interactions between them in vivo. As such, systematically clarifying the association between m 6 A modi cation and cancer development across each clinical and pathological setting is important.
Furthermore, elucidating the signi cance of m 6 A modi cation by ALKBH5 may facilitate the clinical usage of such molecules as therapeutic targets. Therefore, the current study aimed to examine the role of m 6 A demethylase in NSCLC focusing on ALKBH5 and determine its association with downstream targets, including "readers" and "target genes."

Immunohistochemistry
Resected NSCLC samples from Hamamatsu University School of Medicine and Seirei Mikatahara General Hospital were collected and named as the HUSM cohort. Tissue microarray (TMA) sections were analyzed using immunohistochemistry (IHC) as previously described [39]. Cores of insu cient quality or quantity were excluded from analysis. Antibodies for ALKBH5 (HPA007196, Atlas Antibodies, Stockholm, Sweden) and FTO (Ab124892, Abcam, Cambridge, UK) were diluted at 1:400, whereas those speci c for EGFR E746-A750 deletion (#2085, D6B6, Cell Signaling Technology [CST], Danvers, MA, USA) and EGFR L858R mutant (#3197, 43B2, CST) were diluted at 1:100, followed by incubation at room temperature for 0.5 h. Protein expression levels were then assessed using the H-score, which was calculated by multiplying the percentage of stained tumor area (0-100%) by the staining intensity (scored on a scale of 0-3) to yield a value ranging from 0 to 300.

Analysis Of Publicly Available Datasets
We used the lung cancer database in the Kaplan-Meier plotter (http://kmplot.com/analysis/index.php? p = service&cancer = lung) to analyze the association between prognosis and ALKBH5 and FTO mRNA expression in NSCLC cohorts. Data were downloaded on December 10, 2020. Kaplan-Meier curves for overall survival (OS) were generated and strati ed according to the median expression of each mRNA. To assess the mRNA expression of ALKBH5 and FTO, data from the Cancer Genome Atlas (TCGA) (NSCLC, Provisional) were downloaded from cBioPortal (http://www.cbioportal.org/) on November 11, 2019.
Expression data were obtained in the form of RNA-seq by Expectation Maximization (RSEM).

Immuno uorescence Analysis
Cells grown on coverslips were xed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% bovine serum albumin in PBS (−) at room temperature for 1 h, the cells were probed with primary antibodies against ALKBH5 (HPA007196, Atlas Antibodies) and then incubated with a Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (#A-11010, Thermo Fisher Scienti c, Waltham, MA, USA). Nuclei were stained with ProLong® Gold Antifade Reagent with DAPI (#8961, CST), after which the cells were imaged via uorescence microscopy using z-stack image reconstructions (BZ-9000; Keyence, Osaka, Japan).
More than two different sequences were used for one target gene to minimize off-target effects. Cells were cultured for 24 h before transfection, after which they were transfected with 15 nM of nal siRNA concentrations using Opti-MEM (31985070, Gibco, Dublin, Ireland) and Lipofectamine® 2000 (11668019, ThermoFisher). The cells were then used for further assays at 48-96 h after transfection. When no siRNA sample number was available, siRNA no. 1 (#1) and siRNA no. 3 (#3) were pooled for ALKBH5 unless otherwise speci ed. siIGF2BP1, siIGF2BP2, and siIGF2BP3 were pooled for all transfections.
Rna Isolation And Quantitative-polymerase Chain Reaction (Qpcr) Total RNA was extracted using the RNeasy Plus Mini Kit (#74136, QIAGEN, Hilden, Germany) according to the manufacturer's instructions, with the total RNA concentration calculated using Nanodrop (NanoDrop1000, Thermo Fisher Scienti c). cDNA was synthesized from 1 µg of total RNA using the ReverTra Ace qPCR RT Master Mix (FSQ-201, TOYOBO) according to the manufacturer's instructions. qPCR reactions were performed on a Step One Plus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scienti c) using the THUNDERBIRD qPCR Mix (QPS-201, TOYOBO, Osaka, Japan). The relative RNA expression levels were calculated using the ΔΔCt method, with the levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. All amplicons were con rmed as a single product using agarose gel visualization and/or melting curve analysis. The applied primer sequences are listed in Additional le 7: Table S1.

Protein Isolation And Western Blotting
Protein isolation and western blotting Total protein lysates were extracted from whole cells using 1× sodium dodecyl sulfate (SDS) sample buffer. The Pierce BCA Protein Assay Kit (Cat#23225, Thermo Fisher Scienti c) was used to determine the protein concentration. All proteins were separated using SDS-polyacrylamide gel electrophoresis and transferred to PVDF Blotting Membrane (P 0.45, A29532146, GE healthcare Life science, Chicago, IL, USA) using the Trans-Blot Turbo Cassette (Bio-Rad, Hercules, CA, USA). Blocking One (03953, Nacalai, Kyoto, Japan) or 5% skimmed milk were used for blocking. (1:1000 dilution, Ab8245; Abcam) were incubated for overnight at 4℃. Secondary antibodies for rabbit (1:20000 dilution, NA9340; GE healthcare Life science) or mouse (1:20000 dilution, NA9310; GE Healthcare Life Science) were incubated at room temperature with 1-5% skimmed milk for 1 h. Enhanced chemiluminescence (Pierce ECL Plus Substrate or West Atto Ultimate Sensitivity Substrate, Thermo Fisher Scienti c) was used to visualize the protein bands using ChemiDocTouch (Bio-Rad).

Cell Viability Assay
Cells were seeded into 96-well plates with 3000 cells per well after 48 h of knockdown or overexpression.
Cell proliferation was monitored using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. Thereafter, the cells were incubated with 10% CCK-8 for 1 h, followed by absorbance assessment at 450 nm in each well via spectrophotometry (Synergy HT, BioTek, Winooski,VT, USA) every 24 h.

Transwell Migration Assay
Cell migration was evaluated using a 24-well plate with cell culture inserts (353097, Falcon, Mexico City, Mexico) containing a lter with 8 µm-diameter pores. Brie y, after serum starvation for 24 h with 0.1% FBS-containing RPMI1640 medium, 1 × 10 5 cells resuspended in 500 µL of RPMI1640 medium (Gibco) were seeded into the upper chamber, after which RPMI1640 medium containing 10% FBS was placed in the lower compartment of the chamber. After incubation for 16 h, the upper surface of the membrane was wiped with a cotton-tipped applicator to remove non-migrating cells, whereas the migrating cells on the lower surface were xed with cold methanol and stained with 0.5% crystal violet. Migrating cells were automatically counted in three random microscopic elds using the Hybrid Cell Count software (BZ-Analyzer, Keyence, Osaka, Japan).

Wound-healing Assays
To assess cell migration, 2 × 10 5 cells were seeded into 6-well plates. Thereafter, cells were incubated in 5% CO 2 at 37℃ for 48 h and an additional 24 h with 0.1% FBS-containing RPMI1640 medium. A wound was scratched into the cells using a 200-µL plastic tip and washed with PBS (−). The cells were then incubated in RPMI1640 containing 10% FBS. The relative distance of the scratches was observed under an optical microscope (IX53, Olympus, Tokyo, Japan) at 3-6 time points after wounding and assessed using the Image J software.

Cell Cycle Assay And Apoptosis Assay
Cell Cycle Assay Solution Blue (C549, Dojindo) was used to measure the cell cycle according to the manufacturer's instructions. Brie y, treated cells were synchronized at the G1 phase through serum starvation with 0.1% FBS-containing medium for 48 h. At 24 h after the release of serum starvation, the treated cells were collected, washed with PBS (−), and incubated with 5 µL cell cycle assay solution for 15 min at 37℃. Thereafter, DNA content was determined based on staining intensity using a Gallios ow cytometer (Beckman Coulter, Miami, FL, USA). The Annexin V-FITC Apoptosis Detection Kit (15342-54 Nacalai) was used to detect apoptosis by measuring annexin V and propidium iodide (PI)-positive cells following the manufacturer's instructions. Brie y, cells were incubated for 96 h after siRNA transfection. To induce apoptosis, the cells were exposed to either 7.5 µM of ge tinib (078-06561, FUJIFILM) or 10 µM of cisplatin (P4394, Sigma-Aldrich) alone for 48 h after siRNA transfection. The treated cells were collected, washed with PBS (−), and incubated with 5 µL of annexin V-FITC solution and 5 µL of PI solution for 15 min. Thereafter, apoptotic cells were determined using a Gallios ow cytometer. Results were analyzed using the FlowJo software (Becton, Dickinson, Franklin Lakes, NJ, USA), after which the extent of apoptosis and cell cycle distribution were determined.

Rna Stability Assay
Cancer cells were incubated for 48 h after siRNA transfection. Cells were treated with actinomycin D at a nal concentration of 5 µg/mL. Total RNA was extracted at 0, 2, 4, and 6 h after adding actinomycin D.
The remaining CDKN1A and TIMP3 mRNA was measured through quantitative real-time PCR and normalized to RPL32 mRNA, which has a half-life of 25 h. Technically, 100 ng of polyA-enriched RNA was digested using 5.7 µL of acetic acid buffer and 3 µL of Nuclease P1 included in the 45-µL sample containing nuclease-free water at 37℃ for 30 min, followed by incubation with 6 µL of Tris Buffer and 0.3 µL of alkaline phosphatase at 37℃ for 30 min. After digestion, the sample was centrifuged at 14,000 g and 4℃ for 20 min using a Nanosep 3K Omega centrifugal device (Pall Corporation, Port Washington, NY, USA).
As an internal standard, N 6 -methyladenosine-d3 (m 6 A-d3; M275897, Toronto Research Chemicals, Toronto, Canada), which is a stable isotope of N 6 -methyladenosine labeled with three deuterium atoms on the N 6 -methyl group, was added to the nucleosides obtained via digestion of polyA-enriched RNA. These nucleosides were separated using an Acquity UPLC HSS T3 column ( 2 (Filgen, Aichi, Japan) was used for data normalization and subsequent processing. Differentially expressed mRNAs were identi ed using a set cutoff (fold change > 1.5 or < 0.67; P < 0.01). Gene set enrichment analysis (GSEA) was performed to examine the gene set regulated by ALKBH5 knockdown (http:/software.broadinstitute.org/gsea/omdex.jsp) [40]. For analysis, the false discovery rate (FDR) based on gene set permutation was used. Microarray data has been deposited in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) (accession number GSE165453).

Epitranscriptomic Microarray Analysis
Unfragmented total RNA was extracted from ALKBH5-knockdown or control PC9 cells at 96 h after transfection and quanti ed using the NanoDrop ND-1000. RNA samples were used for global m 6 A expression pro ling on an Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray (8 × 60 K; Arraystar), which includes 44,122 protein-coding mRNAs and 12,496 long non-coding RNAs. Microarray analyses were entrusted to Arraystar Inc. (Rockville, MD, USA). Sample preparation and microarray hybridization were performed based on Arraystar's standard protocols. Brie y, total RNAs were immunoprecipitated with an anti-m 6 A antibody (Synaptic Systems, 202003). The "immunoprecipitated (IP)" and "supernatant (Sup)" RNAs were labeled with Cy5 and Cy3, respectively, as cRNAs in separate reactions using the Arraystar Super RNA Labeling Kit. The cRNAs were combined and hybridized onto Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray (8 × 60 K, Arraystar). After washing the slides, the arrays were scanned in two-color channels using an Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Raw intensities of IP (Cy5-labeled) and Sup (Cy3-labeled) were normalized with an average of log2-scaled Spike-in RNA intensities. The "m 6 A methylation level" was calculated to determine the percentage of modi cation based on the IP (Cy5-labeled) and Sup (Cy3-labeled) normalized intensities. "m 6 A quantity" was calculated to determine the amount of m 6 A methylation based on the IP (Cy5-labeled) normalized intensities. Differentially m 6 A-methylated RNAs between both comparison groups were identi ed by ltering with a fold change of > 1.5 or < 0.67 (P < 0.01) through the unpaired t-test. Microarray data had been deposited in the GEO at the NCBI (accession number GSE165454).

Qpcr For Methylated Rna Immunoprecipitation (Merip) With Ma Antibody
ALKBH5-knockdown or control lung cancer cells were used for methylated RNA immunoprecipitation assay. The Magna MeRIP m 6 A kit (catalog no.17-10499, Millipore, Burlington, MA, USA) was used according to the manufacturer's protocol. Brie y, the polyA-enriched RNA was fragmented into 100-200 nucleotides incubated with RNA fragmentation buffer for 55 s (CS220011, Millipore). The size of polyAenriched RNA fragments was optimized using the Agilent 4200 TapeStation (Agilent technologies, Santa Clara, CA, USA). We used 0.5 µg of fragmented polyA-enriched RNA as input control and 5 µg of fragmented polyA-enriched RNA for m 6 A mRNA immunoprecipitation, followed by incubation with m 6 A antibody (MABE1006, Millipore)-or mouse IgG-conjugated Protein A/G Magnetic Beads in 500 µL 1× IP buffer supplemented with RNase inhibitors at 4℃ overnight. Methylated RNAs were immunoprecipitated with beads, eluted via competition with free m 6 A, and puri ed using the RNeasy kit (Qiagen). Moreover, modi cation of m 6 A toward particular genes was determined using qPCR analysis with speci c primers [primers for the positive control region (stop codon, EEF1A1+) or NC region (exon 5, EEF1A1−) of human EEF1A1 was included in the Magna MeRIP m6A kit]. To design primers for MeRIP qPCR, m 6 A sites of speci c genes were predicted using the sequence-based RNA adenosine methylation site predictor algorithm (http://www.cuilab.cn/sramp). We focused on the potential m 6 A sites in the 3′ UTRs near the stop codon and designed primers to ensure that the target sequence were present in these sites with a limited length of 120 nt. Self-designed primers for MeRIP qPCR are listed in Additional le 7: Table S1.

Statistical analysis
Discrete variables were expressed as numbers (percentages), whereas continuous variables were expressed as means ± standard deviations (SDs) unless otherwise speci ed. The Mann-Whitney U test was used to compare continuous individual samples, whereas Student's t-test was applied to compare continuous experimental data. Fisher's exact test for independence was used to compare categorical data between groups. The Wilcoxon matched-pairs signed-rank test was used to compare two corresponding groups. Spearman's correlation coe cient was used for correlation analysis. Kaplan-Meier curves with log-rank tests were used to analyze survival. Accordingly, OS was de ned as the duration from baseline to the date of death, whereas recurrence-free survival (RFS) was de ned as the duration from baseline to the recurrence date. Univariate and multivariate Cox proportional hazards models were applied to generate hazard ratios (HRs) for death while adjusting for other potential confounding factors. Cell proliferation and RNA stability assays were analyzed using two-way analysis of variance. Statistical analyses were performed using GraphPad Prism Version 8 (GraphPad Software, San Diego, CA, USA) and EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), with P values of < 0.05 indicating statistical signi cance.

Results
High ALKBH5 expression was associated with a worse prognosis in patients with NSCLC To investigate the impact of ALKBH5 and FTO in NSCLC, we examined the mRNA expression levels of ALKBH5 and FTO in non-cancerous lung tissues and NSCLC tissues using TCGA data. Accordingly, our results showed no signi cant difference in ALKBH5 mRNA expression between non-cancerous and cancerous tissues. By contrast, our ndings showed that NSCLC had a signi cantly lower FTO mRNA expression than non-cancerous tissues (Fig. 1a). We subsequently investigated the protein expression levels of ALKBH5 and FTO in non-cancerous lung alveolar tissue and corresponding NSCLC tissues using TMA of patient samples. Furthermore, our results showed that cancerous tissues had signi cantly higher H-scores for ALKBH5 and FTO than non-cancerous tissues (Fig. 1b). ALKBH5 and FTO expression were evaluated in immortalized bronchial epithelial cells (BEAS2B) and lung cancer cell lines. Consequently, qPCR analysis demonstrated that ALKBH5 mRNA expression was higher in lung cancer cell lines except LC-2/ad and RERF-LC-MS, whereas FTO mRNA expression was lower in lung cancer cell lines except HLC-1, ABC1, and PC3 (Fig. 1c). Western blot analysis demonstrated that ALKBH5 and FTO were endogenously expressed in all lung cancer cell lines except HLC-1 (Fig. 1d). IHC analysis showed that ALKBH5 and FTO were mainly localized in the nucleus of the cells (Fig. 1e). Furthermore, immuno uorescence analysis showed that ALKBH5 was localized in the nucleus of PC9 cells overexpressing ALKBH5 (Fig. 1f). We analyzed the clinical characteristics of 627 NSCLC cases used in IHC of TMA in the context of ALKBH5 or FTO expression in tumors of the HUSM cohort (  Figure S1a). Based on the median value, cases were divided into "high" and "low" expression groups, after which their association with clinical data as well as prognostic signi cance was examined. Lymph node metastasis, chemotherapy, and EGFR status signi cantly differed depending on ALKBH5 expression, whereas tumor status, lymph node metastasis, pathological stage, chemotherapy, and EGFR status signi cantly differed depending on FTO expression. Kaplan-Meier curves showed that patients with high ALKBH5 expression had signi cantly worse survival than those with low ALKBH5 expression (Fig. 1g: log-rank p = 0.0009 for OS; Additional le 1: Figure S1b: log-rank p = 0.0008 for RFS). Conversely, Kaplan-Meier curves showed no signi cant difference in survival between the low and high FTO expression groups (Fig. 1g: log-rank p = 0.20 for OS, Additional le 1: Figure S1c: log-rank p = 0.07 for RFS). Univariate analysis revealed high ALKBH5 expression as a predictor of unfavorable OS (HR, 1.675; 95% CI, 1.230-2.521). Moreover, multivariate analysis of age, sex, smoking status, histology, pathological stage, and ALKBH5 expression revealed that ALKBH5 expression was an independent prognostic factor associated with unfavorable OS (HR, 1.468; 95% CI, 1.039-2.073) ( Table 2). To validate the prognostic value of ALKBH5 and FTO in other cohorts of patients with NSCLC, the lung cancer database in the Kaplan-Meier plotter was used. Accordingly, Kaplan-Meier curves showed that patients with high ALKBH5 expression had a signi cantly worse survival than those with low ALKBH5 expression (Additional le 1: Figure S1d: log-rank p = 0.014 for OS).
In contrast, Kaplan-Meier curves showed that patients with high FTO expression had signi cantly favorable survival compared with those with low FTO expression (log-rank p < 0.0001 for OS) (Additional le 1: Figure S1e). These observations suggested that ALKBH5 played a critical role in the poor prognosis of patients with NSCLC.  (siFTO#1) and siRNA no. 3 (siFTO#3) were used in subsequent knockdown experiments (Fig. 2a, 2b, Additional le 2: Figure S2a). ALKBH5 knockdown signi cantly suppressed the proliferation of PC9 and A549 cells (Fig. 2c). By contrast, FTO knockdown showed no signi cant suppressive effects on the proliferation of PC9 and A549 cells (Fig. 2d). Thereafter, we assessed migration abilities in ALKBH5knockdown cells. Accordingly, the transwell migration assay showed no signi cant reduction in the migratory PC9 and A549 cells (Fig. 2e). Moreover, the wound-healing assay showed that ALKBH5 knockdown promoted no signi cant reduction in the migration ability of PC9 and A549 cells (Fig. 2f, 2g).
Together with the prognostic value of ALKBH5 in NSCLC, these observations suggested that ALKBH5 played a cancer-promoting role by regulating cell proliferation.
To subsequently examine the mechanism by which ALKBH5 knockdown suppressed cell proliferation, cell cycle and apoptosis analyses were performed using ow cytometry. Accordingly, ALKBH5 knockdown signi cantly increased the number of PC9 cells in the G1 phase and reduced the number of PC9 cells in the G2/M phase (Fig. 3a, 3b). Conversely, ALKBH5 knockdown promoted no signi cant differences, with a consistent result of two different sequences of siRNAs in the cell cycle of A549 (Fig. 3c, 3d). ALKBH5 knockdown increased the number of apoptotic PC9 cells (Fig. 3e, 3f). Furthermore, under drug-induced apoptosis via cisplatin and ge tinib administration, ALKBH5 knockdown increased the number of apoptotic PC9 cells (Fig. 3g, 3h). ALKBH5 knockdown also increased the number of apoptotic A549 cells (Fig. 3i, 3j). Moreover, ALKBH5 knockdown increased the number of apoptotic A549 cells with cisplatin ( Fig. 3k, 3l). Overall, the aforementioned data showed that ALKBH5 knockdown suppressed cell proliferation through G1 phase arrest and/or apoptosis induction in NSCLC cell lines.

Alkbh5 Overexpression Promoted Cell Proliferation In Immortalized Cells
To analyze whether ALKBH5 overexpression in immortalized cells promoted malignant changes in cell function, BEAS2B and HEK293 cells were infected with a doxycycline-inducible vector, pRetroX-TetOne puro-ALKBH5. ALKBH5 overexpression was con rmed in HEK293 and BEAS2B cells (Fig. 4a) and signi cantly enhanced HEK293 and BEAS2B cell proliferation ( Fig. 4b and 4c). In contrast, ALKBH5 overexpression showed no signi cant effects on the migration ability of HEK293 ( Fig. 4d and 4e) and BEAS2B cells (Fig. 4f and 4g). The aforementioned results provided further evidence that ALKBH5 played a cancer-promoting role by regulating cell proliferation.
ALKBH5 altered the abundance of m 6 A modi cation in polyA-enriched RNA To assess the amount of m 6 A in cells, a quantitative evaluation of m 6 A was performed via LC-MS/MS using polyA-enriched RNA extracted from cells with altered ALKBH5 gene expression (Fig. 5a). We investigated the technical variability that occurs when adenosine and N 6 -methyladenosine-d3 (m 6 A-d3) are used as internal standards. Although both adenosine (A) and m 6 A-d3 showed a strong positive correlation with m 6 A (r = 0.92 and r = 0.90), the measurement with m 6 A-d3 as the internal standard showed less technical variability than that with A as the internal standard (Additional le 3: Figure S3ac). Hence, we used m 6 A-d3 as the internal control for subsequent experiments. ALKBH5 knockdown increased m 6 A modi cation in PC9 and A549 cells (Fig. 5b, 5c), whereas ALKBH5 overexpression reduced m 6 A modi cation in a doxycycline concentration-dependent manner in PC9 cells (Fig. 5d, Additional le 3: S3D, S3E). Moreover, ALKBH5 overexpression reduced m 6 A modi cation regardless of the time that had elapsed after doxycycline addition (Fig. 5e). Furthermore, ALKBH5 overexpression reduced m 6 A modi cation in BEAS2B and HEK293 cells (Fig. 5f). The aforementioned results presented evidence suggesting that ALKBH5 alters the global m 6 A abundance in cells.

Alkbh5 Regulated The Expression Of Cell Proliferationrelated Genes
An expression microarray analysis was herein performed to investigate gene expression pro les in ALKBH5-knockdown PC9 cells with two different sequences of siRNA (ALKBH5#1 and ALKBH5#3). Differentially expressed genes (DEGs) were de ned as those with a fold change of > 1.5 or < 0.67 (P < 0.01). A total of 697 DEGs were detected for ALKBH5#1 comprising 392 upregulated and 305 downregulated genes (Fig. 6a), whereas 1394 DEGs were detected for ALKBH5#3 comprising 803 upregulated and 591 downregulated genes (Fig. 6b). Moreover, 82 upregulated genes (Additional le 8: Table S2) and 47 downregulated genes (Additional le 9: Table S3) overlapped between ALKBH5#1 and ALKBH5#3 (Fig. 6c). Except ALKBH5, genes associated with m 6 A modi cation described in a previous review [41] were not included in the overlapped DEGs (Additional le 10: Table S4). GSEA with the hallmark gene set revealed that the PC9 cells transfected with siALKBH5#1 and siALKBH5#3 had a more enriched expression of genes involved in cell cycle, such as MYC_TARGETS_V2, P53_PATHWAY, and G2/M_CHECKPOINT, than those transfected with siNC (Fig. 6d, 6e). We selected 10 DEGs associated with cell proliferation bibliographically and con rmed the upregulation of E2F1, GADD45A, TIMP3, and CDKN1A and downregulation of CASP14 and CCNG2 by qPCR (Fig. 6f). The aforementioned results revealed that ALKBH5 regulated the expression of genes associated with cell proliferation.
ALKBH5 altered the abundance of m6A in the 3′ UTR and regulated protein expression of target genes We performed m 6 A-speci c methylated RNA immunoprecipitation microarray analysis in PC9 cells on an Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray to comprehensively examine whether differentially regulated genes were associated with m 6 A modi cation using unfragmented total RNA. The median methylation level in unfragmented total RNA was 50.4% (6.8-94.5%) (Additional le 4: Figure   S4a). A positive correlation was observed between the methylation level in unfragmented total RNA and the RNA length of each transcript (r = 0.35) (Additional le 4: Figure S4b). Moreover, a negative correlation was noted between the rate at which ALKBH5 knockdown increased m 6 A modi cation (methylation level in siALKBH5 − methylation level in siNC) and methylation level at baseline (methylation level in siNC) (r = − 0.35) (Additional le 4: Figure S4c). The volcano plot showed that 1 RNA was hypermethylated by ALKBH5#1 knockdown (fold change > 1.5, P < 0.01) (Additional le 4: Figure S4d), whereas 28 RNAs were hypermethylated by ALKBH5#3 knockdown (fold change > 1.5; P < 0.01) (Additional le 4: Figure S4e). No hypermethylated genes overlapped between ALKBH5#1 and ALKBH5#3 knockdown with a fold change threshold of > 1.5 (P < 0.01) (Additional le 4: Figure S4f). GSEA showed no common hallmark gene set with an FDR q-value of < 0.25 for PC9 cells transfected with siALKBH5#1 and siALKBH5#3 compared with the control group (Additional le 4: Figure S4g).
Considering that the m6A levels of unfragmented RNAs regulated by ALKBH5 is affected by the baseline RNA length and endogenous m6A level, we performed methylated RNA immunoprecipitation (MeRIP) with m 6 A antibody using fragmented polyA-enriched RNA in ALKBH5-knockdown PC9 cells to investigate focal m 6 A alterations in the mRNA. The fragmentation conditions were optimized (Additional le 5 Figure S5a), and the m 6 A changes in the positive and NC were con rmed through qPCR using the primers included in the Magna MeRIP m 6 A Kit (Millipore) (Additional le 5: Figure S5b). To verify the accuracy of the MeRIP experiment, we selected MFAP5 out of the 11 genes that were hypermethylated (> 1.5 fold change and P < 0.05) in the human mRNA&lncRNA Epitranscriptomic Microarray (Additional le 11: Table S5) and analyzed the m 6 A target site via qPCR. Speci c primers were designed for the predicted m 6 A-harboring regions, and MeRIP qPCR con rmed that ALKBH5 knockdown increased m 6 A levels in the 3′ UTR of MFAP5 (Additional le 5: Figure S5c). Thereafter, MeRIP qPCR was performed in six DEGs veri ed using qPCR with ALKBH5-knockdown PC9 cells. Our results showed increased m 6 A levels in the 3′ UTRs of CDKN1A, TIMP3, E2F1, and CCNG2 in ALKBH5-knockdown PC9 cells (Fig. 7a, Additional le 5: Figure   S5d). The aforementioned results of the MeRIP qPCR suggest that ALKBH5 targeted the 3′ UTRs of m 6 A in these four transcripts (Fig. 7b).
Next, the protein expression levels of the potential target transcript of ALKBH5 were quanti ed by western blot analysis. Accordingly, our results showed that CDKN1A (p21) expression increased independent of p53 in ALKBH5-knockdown PC9 cells, whereas TIMP3 expression increased in ALKBH5-knockdown A549 cells (Fig. 7c).
IGF2BPs were required for ALKBH5 regulation of target mRNA expression via stabilization of mRNA and affected cell proliferation IGF2BP1, IGF2BP2, and IGF2BP3 (IGF2BPs) are well-known m 6 A-recognizing RNA-binding proteins and readers of m 6 A that have been known to stabilize mRNA. The expression of these proteins in a series of cell lines was analyzed by western blotting, and the results showed signi cant differences in their expression in lung cancer cells and immortalized bronchial epithelial cells according to the cell lines ( Fig. 8a).
Thereafter, ALKBH5 and IGF2BPs were knocked down with siRNA to investigate the association between ALKBH5 and IGF2BPs and the expression of CDKN1A or TIMP3 (Fig. S6A, S6B, S6C, S6D, 8b). Accordingly, ALKBH5 knockdown increased the mRNA expressions of CDKN1A in PC9 cells (Fig. 8c) and TIMP3 in A549 cells (Fig. 8d). In addition, the increased expression was offset by the knockdown of IGF2BPs. Actinomycin D assay showed that ALKBH5 knockdown stabilized CDKN1A mRNA in PC9 cells, and this stabilization was offset by the knockdown of IGF2BPs (Fig. 8e). ALKBH5 knockdown also stabilized TIMP3 mRNA in A549 cells, although not statistically signi cant, and this stabilization was decreased by IGF2BP3 knockdown (Fig. 8e). These results suggest that these alterations in mRNA expression were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs, and the decline of mRNAs were, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs.
Additionally, we evaluated the expression of ALKBH5 target mRNAs CDKN1A and TIMP3 in lung cancer using the TCGA dataset. Accordingly, cancerous tissues (high ALKBH5 expression) had lower CDKN1A and TIMP3 expression than non-cancerous tissue (low ALKBH5 expression) (Additional le 6: Figure S6e, Considering that the interaction between ALKBH5 and IGF2BPs was found to regulate the expression of genes associated with cell proliferation, cell proliferation assays were conducted using ALKBH5-and IGF2BPs-knockdown cells. Notably, ALKBH5 knockdown reduced cell proliferation in PC9 (Fig. 8g) and A549 cells (Fig. 8h). The reduction of cell proliferation was offset by IGF2BPs knockdown. These results support the hypothesis that IGF2BPs are required for ALKBH5 regulation of target mRNA expression and cell proliferation.

Discussion
The current study revealed that ALKBH5 promoted poor survival and cell proliferation in patients with NSCLC. Mechanistically, ALKBH5 knockdown had been found to increase the expression of CDKN1A (p21) and TIMP3 by altering mRNA stability in PC9 and A549 cells via m 6 A change. Moreover, these changes in mRNA stability were counteracted by IGF2BPs knockdown (particularly prominent in IGF2BP3). The aforementioned results suggest that the recognition of target transcripts by IGF2BPs stabilizes the mRNA of CDKN1A (p21) or TIMP3 and subsequently increases their expressions, thereby regulating cell proliferation, cell cycle, and apoptosis in lung cancer cell lines.
Over the last decade, considerable progression has been made on research regarding the molecular mechanism for m 6 A-mediated carcinogenesis of ALKBH5. Nevertheless, previous studies on ALKBH5 have shown con icting results regarding the carcinogenic mechanisms of ALKBH5 across several cancers [23][24][25][26][27][28][29][30][31]. Two previous studies have reported contradictory results regarding ALKBH5, suggesting that it acts as either an oncogenic factor or a tumor suppressor in NSCLC [35,36]. The current study concluded that ALKBH5 exerted cancer-promoting effects in NSCLC by suppressing CDKN1A (p21) or TIMP3. CDKN1A (p21) functions as a cell growth suppressor by inhibiting cell cycle progression. Multiple transcription factors, ubiquitin ligases, and protein kinases regulate the transcription, stability, and cellular localization of CDKN1A (p21) [42]. A previous study showed that ALKBH5 knockdown increased m 6 A modi cation and mRNA stability of CDKN1A, which subsequently increased p21 protein expression and acted as a tumor suppressor in esophageal cancer [27]. Similarly, our ndings showed that ALKBH5 knockdown in PC9 cells acted as a tumor suppressor by the upregulation of CDKN1A (p21) via m 6 A alteration. We also showed that p21 expression was p53-independent, indirectly reinforcing our nding that CDKN1A (p21) upregulation was an m 6 A-mediated response. Our results further indicated a novel mechanism wherein changes in CDKN1A expression via ALKBH5 knockdown were rescued by IGF2BPs knockdown, which supports our nding that alterations in CDKN1A (p21) expression were mediated by m 6 A.
The current study identi ed TIMP3 as another important target molecule downstream of ALKBH5. A previous study showed TIMP3 had several anticancer properties, including apoptosis induction and antiproliferative, antiangiogenic, and antimetastatic activities. The expression of TIMP3 is regulated by transcription factors and histone acetylation [43]. Several studies have shown that TIMP3 acts as a tumor suppressor in lung cancer [44]. Moreover, a previous report using A549 cell lines showed that ALKBH5 knockdown increases TIMP3 mRNA stability and TIMP3 expression via m 6 A modi cation [36].
Similarly, the current study also con rmed that ALKBH5 knockdown increased mRNA stability, which increased TIMP3 protein expression, and acted as a tumor suppressor in A549 cells. Furthermore, our experimental data for the rst time showed that the ALKBH5 knockdown-induced increase in TIMP3 was rescued by IGF2BPs, strongly suggesting that alterations in TIMP3 expression were mediated by m 6 A.
Previous studies have reported that IGF2BPs, which are known as m 6 A-recognizing RNA-binding proteins that stabilize m 6 A-containing RNA, have oncogenic properties. Studies on lung cancer have associated IGF2BPs with cancer progression and poor prognosis [45][46][47]. Notably, a previous report showed that ALKBH5-mediated m 6 A modi cation of LY6/PLAUR Domain Containing 1 (LYPD1) is recognized by IGF2BP1 and enhances the stability of LYPD1 mRNA in hepatocellular carcinoma [30]. Moreover, recent RNA-binding protein immunoprecipitation-sequencing analysis using HEK293T showed that the binding site of IGF2BPs is mainly distributed in the 3′ UTRs and that the target of IGF2BPs preferentially binds to the consensus sequence of UGGAC in the target mRNA [21]. These ndings support our experimental hypothesis that IGF2BPs recognize the m 6 A in the 3′ UTRs of CDKN1A or TIMP3 because UGGAC is present within three locations in the 3′ UTRs of CDKN1A and two locations in the 3′ UTRs of TIMP3. Our ndings also showed that the expression of IGF2BPs signi cantly differed with the lung cancer cell line, which can cause different fates of m 6 A-modi ed transcripts among cell lines.
FTO inhibitors have been shown to suppress the progression of acute myeloid leukemia and glioblastoma in vivo [48,49]. In contrast, the antitumor effects of ALKBH5 inhibitors, which enhanced the e cacy of cancer immunotherapy, have only been con rmed in melanomas [50]. Furthermore, several experimental facts have shown that ALKBH5 is associated with the malignant transformation of cancer [23][24][25][26][27][28][29], indicating that ALKBH5 inhibitors can be a target of tumor-agnostic therapy. However, it should be noted that ALKBH5 inhibitors may cause unexpected side effects in unknown target genes given that ALKBH5 inhibition alters the m6A modi cation of numerous transcripts and the expression of several genes.
Although the current study provided abundant evidence to conclude the remarkable role of the m 6 Aregulated ALKBH5 and IGF2BPs axis in NSCLC, several limitations warrant consideration. First, given that RNAs were not fragmented during our epitranscriptomic microarray, site-speci c changes in m 6 A modi cation could not be determined. Considering the correlation between RNA length and ratio of m 6 Amodi ed transcripts, the epitranscriptional micrarray analysis using unfragmented RNA does not allow the evaluation of multiple m6A modi cations occurring within a single transcript. As such, we conducted MeRIP q-PCR with fragmented RNAs to evaluate site-speci c differential m 6 A modi cation. Nevertheless, the epitranscriptomic microarray with unfragmented RNAs provided a holistic view of the degree of m 6 A modi cation for each transcript, establishing a landscape for m 6 A modi cation by ALKBH5 knockdown (Additional le 4: Figure S4a, S4b, S4c, S4d, S4e and S4g). Secondly, our epitranscriptomic microarray ndings showed that ALKBH5-knockdown reduced m6A methylation levels in approximately half of the transcripts. Although the detailed mechanism remains unclear, hypomethylation may occur when some of the m 6 A-rich transcripts bind to YTHDF2 and YTHDC2, reducing the stability of RNA containing m 6 A.
Consequently, the m 6 A-modi ed transcript then undergoes degradation. In other words, the target's transcript may also differ depending on the elapsed time after the perturbation of ALKBH5. However, the current study did not investigate the chronological alteration of the m 6 A abundance of each transcript following ALKBH5 knockdown. Third, as mentioned earlier, CDKN1A and TIMP3 are also regulated by transcription factors or miRNA, and we cannot deny the possibility that mechanisms other than m 6 A promoted changes in CDKN1A (p21) and TIMP3 expression. Nonetheless, the nding that IGF2BPs knockdown rescued the CDKN1A and TIMP3 expression supports our proposition that the changes in CDKN1A (p21) and TIMP3 expression were mediated by m 6 A.

Conclusions
The current study revealed that increased ALKBH5 expression was an independent unfavorable prognostic factor in NSCLC. Moreover, upregulation of ALKBH5 in NSCLC reduced m 6 A modi cations on the 3′ UTR of speci c genes. The loss of m 6 A decreased the opportunity for recognition by IGF2BPs and destabilized the target transcript, such as CDKN1A (p21) and TIMP3. Downregulation of CDKN1A (p21) and TIMP3 induced cell cycle alteration and inhibited apoptosis. Our results suggest that the ALKBH5-IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC. Our ndings provide a novel insight into the pathophysiological mechanisms of m 6 A epitranscriptomic modi cation in NSCLC (Fig. 9). Further in vivo studies are nonetheless required to determine whether ALKBH5 inhibitors can be incorporated in the treatment of NSCLC in the near future. High ALKBH5 expression was associated with a worse prognosis in patients with non-small cell lung cancer. (a) ALKBH5 and FTO mRNA levels were analyzed in the paired non-cancerous and NSCLC tissues using the TCGA database (n = 109 for each group). (b) ALKBH5 and FTO protein levels were assessed in the paired non-cancerous lung alveolar tissue and NSCLC tissues in the HUSM cohort via immunohistochemistry (IHC) using the H-score (n = 77 for each group). (c) Relative ALKBH5 and FTO mRNA expression levels were detected using qPCR in cell lines. Data were normalized to GAPDH and adjusted to the expression of BEAS2B cells (ALKBH5: n = 4, FTO: n = 6). (d) ALKBH5 and FTO protein expression levels were determined using western blot analysis in cell lines. (e) IHC staining for ALKBH5 and FTO were assessed using the TMA core of NSCLC tissues in the HUSM cohort. Staining intensity was categorized into 0 (absent), 1 (weak), 2 (moderate), or 3 (strong). (f) Immuno uorescence visualized subcellular localization in PC9 cells (×100). PC9 cells infected with pRetroX-TetOne puro-ALKBH5 were transduced by 100 ng/mL of doxycycline and used as ALKBH5 overexpression. PC9 cells without doxycycline were used as a negative control. (g) A Kaplan-Meier survival curve with log-rank test was utilized to analyze the overall survival of the HUSM cohort. Patients were strati ed into low (blue) or high expression groups (red) based on a cutoff determined by the median H-scores (n = 627). Results were presented as the median (a and b) or mean ± SD (c). ****P < 0.0001 indicates a signi cant difference between the indicated groups. Cell proliferation relative to baseline in PC9 and A549 cells transfected with siFTO (#1 and #3) or siNC were assessed using the CCK-8 assay (n = 3). (e) Migration ability of PC9 and A549 cells transfected with siALKBH5 (#1 and #3) or siNC were assessed usingtranswell migration assay. The bar charts indicate the number of migratory cells that passed through the chamber membrane (n = 3). (f, g) Migrationability of PC9 (f) and A549 (g) cells transfected with siALKBH5 (#1 and #3) or siNC was assessed using woundhealing assay (n = 3). Results were presented as mean ± SD. **P< 0.01, ***P< 0.001, ****P< 0.0001 indicates a signi cant difference between the indicated groups.   (OE), whereas those without DOX were designated as negative control (NC). Cell proliferation relative to baseline in ALKBH5 OE HEK293 and BEAS2B cells were assessed using the CCK-8 assay (n = 3). (d-g) The migration ability of HEK293 and BEAS2B cells was assessed via wound-healing assay.
Representative images of the wound-healing assay for HEK293 (d) and BEAS2B (f) cells. Wound areas relative to baseline at each time point were compared between ALKBH5 OE and NC HEK293 (e) and BEAS2B (g) cells (n = 3). Results are presented as mean ± SD. *P< 0.05indicates a signi cant difference between the indicated groups. TetOne puro-ALKBH5 vector (ALKBH5 OE) whose ALKBH5 overexpression was induced by 100 ng/mL DOX for 24, 48, 72, 96, 120 h was compared with those without DOX (NC) (n = 3). (f) m6A/m6A-d3 in HEK293 and BEAS2B (ALKBH5 OE) cells whose ALKBH5 overexpression was induced by 100 ng/mL DOX for 48 h were compared with those without DOX (NC) (n = 3). Results were presented as mean ± SD. *P< 0.05, **P < 0.01, ***P< 0.001, ****P< 0.0001 indicates a signi cant difference between the indicated groups by Student's t-test.   (middle two lanes), or those with siNC (right end indicating both siALKBH5 and siIGF2BPs were negative) were con rmed via western blot analysis. (c and d) Endogenous mRNA expression levels of CDKN1A in PC9 cells (c) or those of TIMP3 in A549 cells (d) transfected with siALKBH5 were analyzed via qPCR and compared with those in cells cotransfected with siALKBH5 and one of the siIGF2BPs. Gene expression was normalized to the GAPDH expression and was shown relative to the expression in siNC (n = 3). (d and e) The remaining RNA level of CDKN1A in PC9 cells (d) or of TIMP3 in A549 cells (e) after actinomysin D treatment for 0, 2, 4, and 6 h was determined using qPCR and normalized to the expression at 0 h. RNA decay rate in cells transfected with siALKBH5 and/or one of the siIGF2BPs and siNC were compared with the stability of CDKN1A and TIMPs (n = 3). (g and h) Cell proliferation relative to baseline in PC9 (e) and A549 (f) cells transfected with siALKBH5 was assessed via the CCK-8 assay and compared with that in cells cotransfected with siALKBH5 and one of the siIGF2BPs (n = 3 for each group).Results are presented as mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 indicates a signi cant difference between the indicated groups. Figure 9