ALKBH5-mediated m 6 A modification of CDC6 mRNA impairs gastric cancer tumorigenesis and has prognostic significance

doi:10.21203/rs.3.rs-3236157/v1

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ALKBH5-mediated m 6 A modification of CDC6 mRNA impairs gastric cancer tumorigenesis and has prognostic significance

https://doi.org/10.21203/rs.3.rs-3236157/v1

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Background: N6-methyladenosine (m⁶A) RNA methylation and its associated demethylase ALKBH5 participate in pathogenesis and progression of multiple malignancies. The present study aimed to reveal the biological function and clinical significance of ALKBH5 in gastric cancer (GC).

Method: The prognostic significance of ALKBH5 expression was assessed by using tissue microarray, immunohistochemical analyses and TCGA dataset in human GC. The functional role of ALKBH5 in GC tumorigenesis were explored in vitro and in vivo. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq and MeRIP-PCR were utilized to identify the target genes of ALKBH5. RNA pull-down and RIP assays were conducted to validate the binding of CDC6 with ALKBH5 and YTHDF1.

Results: ALKBH5 was markedly downregulated in GC tissue samples and reduced expression of ALKBH5 correlated with poor prognosis. Multivariate Cox regression analysis unveiled that ALKBH5 expression was an independent prognostic factor for overall survival in human patients with GC. Furthermore, ALKBH5 overexpression suppressed GC cell proliferation and cycle progression in vitro and in vivo, whereas knockdown of ALKBH5 harbored the opposite effects. Mechanistically, ALKBH5 abolished m⁶A modification of CDC6 mRNA, and the m⁶A reader YTHDF1 could directly recognize and bind to the m⁶A site on CDC6 mRNA. CDC6 acted as an oncogenic factor and had poor prognosis in GC.

Conclusions: Our findings demonstrate that ALKBH5-mediated m⁶A modification of CDC6 mRNA impairs GC tumorigenesis and has prognostic significance in patients with GC.

m6A

ALKBH5

CDC6

growth

gastric cancer

Gastric cancer (GC) is one of the most common gastrointestinal malignancies and possesses the fifth incidence and the third cancer-related mortality worldwide [1]. Despite the tremendous advances in prevention of gastric carcinogenesis, such as Helicobacter pylori eradication and lifestyle alterations [2], the advanced patients with GC still display discouraging prognosis ascribed to the tumor invasiveness and metastasis [3]. Therefore, comprehensive observation of the pathogenic mechanisms underlying GC progression is essential for the early diagnosis and therapy.

Epigenetic dysregulations including m⁶A RNA methylation act emerging roles in modification of target genes involved in cancer progression [4, 5]. m⁶A methylation modifications can be dynamically regulated by methyltransferases such as METTL3/14/WTAP/KIAA1429 (“writers”), demethylases FTO and ALKBH5 (“erasers”) and recognition proteins such as YTHDF1-3 and IGF2BP1 (“readers”) [6]. The deregulated METTL3/14 [7-9], FTO [10] and YTHDF1 [11] are linked to the prognosis and progression of GC [12].

Alkylation repair homolog protein 5 (ALKBH5) as an m⁶A demethylase is provided with diverse functions in cancer. On the one hand, accumulating data show that ALKBH5 is downregulated in non-small cell lung cancer (NSCLC) [13], pancreatic cancer (PC) [14, 15], hepatocellular carcinoma (HCC) [16], indicates favorable prognosis [14-16] and suppresses tumorigenesis and metastasis [13-16]. On the other hand, ALKBH5 is upregulated in ovarian cancer (OC) [17], HCC [18], NSCLC [19], renal cell carcinoma (RCC) [20], multiple myeloma (MM) [21], harbors poor prognosis [18-21] and promotes carcinogenesis [17-21].

ALKBH5 has been reported to accelerate bile acid-induced gastric carcinogenesis [22]. However, comprehensive understanding of the function and molecular mechanism of ALKBH5 is required for GC pathogenesis. In the present study, we found that downregulation of ALKBH5 as an independent prognostic factor was associated with poor survival in patients with GC, and ALKBH5-mediated m⁶A modification of CDC6 mRNA impaired GC tumorigenesis, offering a promising therapeutic target for GC.

Tissue microarray (TMA) and immunohistochemistry (IHC)

Human microarray slides (TMA1 and TMA2) of GC were purchased from Shanghai Outdo Biotech Co. Ltd. (Shanghai, China). TMA1 consisted of 90 pairs of tumor and adjacent normal tissues and the clinicopathological information from TMA1 could be provided in Supplementary Table S1. The samples were incubated with anti-ALKBH5 (1:200 dilution in PBS, ab195377, Abcam, USA ). The protein levels of ALKBH5 in GC tissues were detected using IHC as described previously [11].

Immunofluorescence (IF)

TMA2 contained 4 normal, 28 pairs of tumor and adjacent normal tissue and the clinicopathological information from TMA2 could be provided in Supplementary Table S2. The slides were immersed in EDTA antigen repair buffer (pH 8.0) and heated for antigen repair. After being washed, the slide was blocked with 3% BSA and incubated with anti-ALKBH5 (1:200 dilution in PBS) overnight at 4℃. Then, the slide was incubated with fluorescent secondary antibody (1:100, Alexa Fluor 488, Abcam), counterstained with DAPI (Solaibio, China), and sealed for observing. The sections were scanned using PANNORAMIC MIDI (3DHISTECH, Hungary) and analyzed by DensitoQuant 1.15.4 of QuantCenter (3DHISTECH, Hungary). The representative images of TMA2 were captured using a Nikon confocal microscope (A1HD25, Nikon).

Bioinformatic analysis

The mRNA expression levels of ALKBH5 and clinical information of GC patients in TCGA-STAD dataset were downloaded from TCGA database (https://portal.gdc.cancer.gov/). The expression levels of target genes of ALKBH5 in GC and normal tissues were analyzed using GEPIA database (http://gepia.cancer-pku.cn/index.html).

Cell culture

Human gastric epithelial cell line GES-1 and GC cell lines (MKN-28, HGC-27, BGC-823, SGC-7901 and AGS) were obtained from Digestive Disease Laboratory of Shanghai Sixth People’s Hospital and cultured in RPMI 1640 medium with 10% fetal bovine serum (Gibco, USA), 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 ◦C and 5% CO2.

Transfection of lentivirus, siRNA and plasmids

The construction and synthesis of overexpression (OE), knockdown (KD) and corresponding control lentiviruses used in this experiment were accomplished by Hanheng Biotechnology Co,, Ltd (Shanghai, China). The sequences of ALKBH5 shRNA were presented in the Supplementary Table S4. The cells were seeded into six-well plates and infected with lentivirus as the manufacturer’s instructions. OE plasmids and siRNA against CDC6 were obtained from Hanheng Biotechnology, and the sequence of siRNA against CDC6 was listed in the Supplementary Table S5. The transfection was conducted using Lipo3000 (Invitrogen, USA) based on the manufacturer’s instructions.

RNA extraction and quantitative real-time PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA ) following manufacturer’s instructions. 1000 ng of total RNA were used for cDNA synthesis on the ABI VeritiTM 96-well Thermal Cycler (Thermo Fisher Scientific, USA) by the HiScript II QRT SuperMix for quantitative PCR (Vazyme Biotech Co. Ltd., Nanjing, China). The primer sequences were presented in Supplementary Table S3. The mRNA expression levels of target genes were calculated using a 2^-^△△^Ct method and normalized to β-actin.

Western blotting

Cells or human GC tissues were lysed using RIPA buffer (Beyotime, Shanghai, China). Equal amount of total proteins were separated on 10% SDS-PAGE gels (EpiZyme, Shanghai, China) and transferred to PVDF membranes (0.45 µm, Millipore,USA). The membranes were blocked in 5% milk-TBST for 1 h at room temperature and incubated with the primary antibodies (1:500 or 1:1000 dilution) including anti-ALKBH5 (ABE547, Merck Millipore, Germany), anti-YTHDF1 (ab220162, Abcam), anti-CDC6 (ab109315, Abcam), anti-FEN1 (DF6309, Affinity), anti-CDK4 (DF6102, Affinity), anti-CDKN1C (AF6819, Affinity), anti-CDC45 (DF6779, Affinity), anti-MCM7 (DF6272, Affinity), anti-E2F1 (DF6797, Affinity), anti-β-actin (T0022, Affinity), and anti-GAPDH (Affinity Biosciences, USA). Then, membranes were incubated with species-specific secondary antibodies (1:3000, Beyotime, China).

Cell counting Kit-8 (CCK8) and EdU assay

Cell viability was monitored by a CCK8 assay kit (Dojindo corp, Japan) as previously described [11]. Cell proliferation assays were detected using a Cell-Light EdU Apollo567 in vitro Kit (RiboBio, China) following the manufacturer’s instruction. EdU-positive cell rates were calculated by Image J software (NIH, USA).

Colony formation assay

GC cells during the logarithmic phase were seeded in the 6-well plate with 1000 per well and cultured for 7 days. After being washed with PBS, the cells were fixed by 4% paraformaldehyde. Subsequently, cells were washed by PBS twice and then stained with 0.1% crystal violet solution. The colonies were imaged and countered.

Cell cycle assay

Cell cycle assays were conducted using Cell Cycle DNA Content Quantitation Assay (Solarbio, China). Briefly, cell samples were collected and fixed in cold 70% ethanol overnight at -20℃. Subsequently, cells were washed by cold PBS twice and added RNaseA for incubating at 37°C for 30 minutes. Then, cells were dyed with PI in the dark for 30 min at room temperature. Then cell cycle tests were performed by CytoFLEX flow cytometer (Beckman Coulter, United States). Modfit LT software was used for the analysis.

M⁶A dot blot

The complete mRNAs were denatured at 65℃ for 5 min and placed on the ice for 5 min. mRNAs (500ng, 250ng or 125ng) were spotted onto positively charged nylon (GE Healthcare, USA), and then conducted as previously reported [23].

RNA sequencing (RNA-seq) analysis

Total RNAs from ALKBH5 OE and Vector transfected MKN-28 cells were collected for poly(A) mRNA purification by NEB Next Poly(A) mRNA Magnetic Isolation Module. The next generation sequencing (NGS) was conducted by Aksomics Biotech (Shanghai, China). Differential expression genes (DEGs) were obtained using “DESeq” package in R (|log2FC| ≥ 0.585), and used for KEGG and GO analysis mainly with “clusterProfiler”, “ggplot2”, and “enrichplot” packages. Additionally, DEGs related to cell proliferation were acquired from intersection between all DEGs and gene sets of Cell cycle (c2.cp.kegg.v7.5.1.symbols.gmt, https://www.gsea-msigdb.org/gsea/index. jsp) .

Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-qPCR

The complete mRNA was obtained from total RNA using Seq-StarTM poly(A) mRNA Isolation Kit (Arraystar, USA). The mRNA was fragmentated and incubated with m⁶A antibody (ABE572, Merck Millipore, Germany). The MeRIP m⁶A Kit (Merck Millipore) was used for the immunoprecipitation. Then, the m⁶A enrichment of mRNA was analyzed by NGS (conducted by Aksomics Biotech) and qPCR.

RNA immunoprecipitation (RIP) assay

RIP assay was performed using an RNA Immunoprecipitation Kit (Geneseed Biotech, China) based on the manufacturer’s instructions. Briefly, MKN-28 cells were collected from T75 flasks at about 75% confluence and lysed on the ice. 3μg ALKBH5 antibody (ABE547, Merck Millipore, Germany), YTHDF1 (ab220162, Abcam) and control rabbit IgG were incubated with Dynabeads (Thermo Fisher Scientific) for conjugation. After 2 times washes, 400μL cell lysis supernatant and 350μL buffer A were added for antigen capture. After washes, beads were resuspended by Buffer E and filtrated by DR Coulumns for RNA extraction. Finally, co-immunoprecipitated and input RNAs were obtained and used for qPCR analysis.

RNA pull-down assay

To confirm the interaction of ALKBH5 or YTHDF1 protein with CDC6 mRNA in GC cells, RNA pull-down assay was performed using biotin-labeled CDC6 probe (RiboBio, China) (Supplementary Table S6). The enriched proteins were identified by Western blotting analysis.

Animal experiment

A subcutaneous xenograft tumor model in nude mice was used to assess tumor growth in vivo. Six-week old BALB/c nude mice were purchased from Shanghai Slake Laboratory Animal Center (Shanghai, China). MKN-28 cells (5×10⁶) stably transfected with ALKBH5 OE or Vector lentiviruses and HGC-27 cells with sh-ALKBH5 or Vector lentiviruses were injected subcutaneously into the BALB/c mice, respectively. Tumor volumes (length × width²)/2 were examined every 3 d. All mice were sacrificed after 4 weeks. Tumor volume and weight were calculated and tumor tissues were paraffin-embedded for IHC analysis of positive Ki-67 (ab16667, Abcam, USA) levels.

Statistical analysis

Statistical tests were conducted using GraphPad Prism (version 8.0, USA) and R software (version 4.0.2). Quantitative data were presented as mean and standard deviation (SD). Unpaired, paired t-test or Chi-square test was performed between GC and normal groups. One-way ANOVA was conducted for comparisons among three or more groups. Correlations were analyzed using Pearson’s correlation. Survival analysis was carried out using Kaplan-Meier curves via “survival” and “survminer” packages in R. .Univariate and multivariate Cox regression analyses were performed using R “survival” package. P < 0.05 was considered statistically significant.

Downregulation of ALKBH5 correlates with poor prognosis in patients with GC.

We examined the protein expression levels of ALKBH5 in GC by using the TMA1 and IHC analyses, which showed that ALKBH5 protein levels were dramatically increased in non-matched (n = 87) and matched GC tissue samples (n = 82) as compared with the adjacent normal tissues (Figure 1A, B). The similar results were confirmed in 28 pairs of GC tissue samples by using IF and Laser Confocal microscopy (Figure 1C, D) as well as in 10 pairs of GC tissue samples by Western blotting (Figure 1E). In addition, we found that ALKBH5 protein levels were correlated with TNM stage rather than pathological stage, and with the TNM grading level increasing, the protein expression of ALKBH5 decreased in GC from the TMA1 cohort (Figure 1F). We also found that deregulation of ALKBH5 displayed no association with Tumor Mutation Burden (TMB) but associated with Microsatellite Instability (MSI) in Stomach Adenocarcinoma (STAD) (Supplementary Figure S1). Kaplan-Meier analysis was used to assess the prognosis of ALKBH5 in patients with GC and indicated that the patients with ALKBH5 downregulation harbored the poorer overall survival as compared with those with ALKBH5 upregulation in both TMA1 and TCGA-STAD cohorts (Figure 1G). Univariate (Figure 1H) and multivariate cox analyses (Figure I) unraveled that ALKBH5 expression was an independent prognostic marker for overall survival of patients with GC.

ALKBH5 inhibits GC cell growth in vitro

To estimate the function of ALKBH5 in GC, we constructed stable ALKBH5-overexpressing GC cell lines (MKN-28 and SGC-7901) and ALKBH5-knockdwon cell lines (AGS and HGC-27) (Figure 2A, Supplementary Figure S2A-C). The elevated expression of ALKBH5 repressed cell viability, DNA synthesis and clonogenic ability in MKN-28 and SGC-7901 cells, while knockdown of ALKBH5 promoted these effects in AGS cells (Figure 2B, C and D). Flow cytometry showed that ALKBH5 upregulation led to the decreased cell number in S phase but increased cell number in G1 phase in MKN-28 and SGC-7901 cells (Figure 2E).

RNA-seq reveals the implication of ALKBH5 in regulating GC proliferation and cycle progression

The RNA-seq was used to screen the differentially-expressed genes (DEGs) between ALKBH5-overexpression (OE) and control transfected MKN-28 cells. According to the p < 0.05 and | fold change (FC) | > 0.585, Volcano Maps showed that 90 downregulation genes and 76 upregulation genes were identified in ALKBH5-overexpressiong MKN-28 cells as compared with the control group, and the distribution for these DEGs could be displayed on human chromosomes (Figure 3A). KEGG analysis indicated that the downregulated genes caused by ALKBH5-OE were predominantly enriched in cell cycle (Figure 3B). GO analysis unveiled that these downregulated genes were associated with DNA replication and cell proliferation (Figure 3C). We then seized the intersection of Cell cycle and DNA replication-related genes (n = 147) and DEGs from the KEGG dataset (n = 159), and screened out 6 target genes of ALKBH5 which participated in regulating cell cycle and cell proliferation (Figure 3D). Bubble chart displayed that ALKBH5 OE reduced the mRNA levels of CDK4, CDC45, CDC6, FEN1, MCM7, E2F1 and increased those of CDKN1C compared to the control group in MKN-28 cells (Figure 3E), and these results were further validated in the protein levels by Western blotting (Figure 3F). In addition, we analyzed the expression levels of these 7 genes in GC tissue samples by using the TCGA (Figure 3G) and GEPIA datasets (Figure 3H), which exhibited that CDK4, CDC45, CDC6, FEN1 and E2F1 were upregulated but CDKN1C was downregulated in GC tissue samples compared to the normal tissues.

CDC6 is identified as an m⁶A target of ALKBH5 in GC cells

To investigate the effects of ALKBH5 on the total m⁶A levels in GC cells, we conducted the m⁶A dot blot, which demonstrated that ALKBH5 OE reduced the total m⁶A levels in MKN-28 and SGC-7901 cells, whereas ALKBH5 knockdown elevated the m⁶A levels in AGS and HGC-27 cells (Figure 4A). MeRIP-seq unveiled that m⁶A peaks of 2724 transcripts displayed decreased abundance while those of 1001 transcripts exhibited increased abundance (p < 0.05) (Figure 4B). The m⁶A peaks were uniquely acquired with the CUGUAA(G)UC motifs, mainly enriched in 3' untranslated region (UTR) region (Figure 4C, D). KEGG analysis showed that the decreased mRNAs in m⁶A peaks were enriched in PI3K/AKT and mTOR signaling pathways (Figure 4E). GO analysis uncovered that these decreased mRNAs in m⁶A peaks were associated with cellular protein modification process (Supplementary Figure S3). Intriguingly, 2 transcripts were overlapped in the MeRIP-seq and RNA-seq data and they were from two different genes ZNF5 and CDC6 (Figure 4F). Further analysis verified that ALKBH5 OE decreased the mRNA and protein levels of CDC6 rather than ZNF5, while ALKBH5 knockdown promoted CDC6 expression in GC cells (Figure 4G, H).

Furthermore, analysis of CDC6 mRNA with SCRAMP program forecasted the potential m⁶A sites on 3' UTR region (Figure 5A), which was consistent with our results indicating m⁶A peaks were located on CDC6 mRNA in ALKBH5 and control transfected MKN-28 cells, especially on the 3' UTR region as demonstrated by the MeRIP-seq ﬁndings (Figure 5B). We observed the correlation of CDC6 with the m⁶A family members in GC tissue samples and found that CDC6 harbored a negative correlation with ALKBH5 expression but a positive correlation with YTHDF1 expression in GC (Figure 5C). MeRIP-PCR showed that ALKBH5 OE markedly reduced the m⁶A levels of CDC6 mRNA as compared with the IgG group (Figure 5D). RIP-qPCR of CDC6 mRNA was used to confirm the interaction between ALKBH5 or YTHDF1 and CDC6 mRNA, and uncovered that CDC6 mRNA was significantly enriched in ALKBH5 or YTHDF1-bound mRNAs in MKN-28 cells (Figure 5E). To verify the binding of CDC6 mRNA with YTHDF1 or ALKBH5 protein, we conducted a RNA pull-down assay by using a biotinylated CDC6 probe and found that YTHDF1 or ALKBH5 protein was abundantly pulled down by the biotin-coupled CDC6 probe as compared with the IgG group in MKN-28 cells (Figure 5F, G).

CDC6 inhibition abolishes ALKBH5 knockdown-induced cell proliferation and indicates clinical prognosis in GC

TCGA cohort demonstrated that CDC6 not only harbored a negative correlation with ALKBH5 expression but a positive correlation with cell cycle-related markers such as CDK2, CDK4, CyclinE1/2 and CylinD1 (Supplementary Figure S4). We further assessed the functional link of CDC6 with ALKBH5 in GC cells. The dramatically increased expression of CDC6 in CDC6 OE-transfected MKN-28 cells was determined by RT-qPCR analysis (Figure 6A). The significantly decreased expression of CDC6 in si-CDC6-transfected HGC-27 cells pretreated with Vector or sh-ALKBH5 lentiviruses was validated by RT-qPCR analysis (Figure 6B). Function assays unveiled that CDC6 OE accelerated the cell colony formation (Figure 6C) and DNA synthesis (Figure 6D) in MKN-28 cells. Likewise, CDC6 downregulation suppressed the colony formation and DNA synthesis, and abolished ALKBH5 knockdown-induced tumor-promoting effects (Figure 6E, F). Online Kaplan-Meier analysis was used to estimate the clinical prognosis of CDC6 in patients with GC and revealed that the patients with CDC6 upregulation possessed the poorer overall survival as compared with those with CDC6 downregulation in both ALL GEO and GSE15459 cohorts (Figure 6G).

ALKBH5 represses GC tumorigenesis in vivo

We established tumour xenograft models to confirm the roles of ALKBH5 in vivo, and the results indicated that ALKBH5 OE suppressed the tumour growth (Figure 7A) whereas knockdown of ALKBH5 accelerated tumour growth (Figure 7B), as reflected by the tumour size and weight compared to those of tumours derived from the control cells (Figure 7A, B). IHC analysis demonstrated that Ki-67, a biomarker of cell proliferation, was remarkably dwindled in the ALKBH5-overexpressiong tumour tissues (Figure 7C), but upregulated in ALKBH5-knockdown tumour tissues compared to that in the control group (Figure 7D).

Dysregulated m⁶A methylation gradually becomes a pivotal hallmark of cancer. Increasing evidence reveals that m⁶A modification is involved in GC pathogenesis and progression [7-12]. It has been reported that ALKBH5 as an m⁶A demethylase is downregulated and associated with encouraging prognosis in multiple solid malignancies including GC [13-16, 24]. In coinciding with these reports, we found that ALKBH5 expression was decreased in GC tissue samples and ALKBH5 downregulation as an independent prognostic marker was associated with poor clinical prognosis in patients with GC. Nevertheless, several opposite reports indicate the upregulation of ALKBH5 in malignant tumors [17-21]. The contradictory results for ALKBH5 expression in these cancers may be related to ALKBH5-fomred positive-feedback loop [18], DNA hypermethylation modification [25] and ALKBH5-specific tissue distribution in cancer.

ALKBH5 has been shown to act as a tumor suppressor [13-16] or an oncogene [17-21] in cancer. ALKBH5 promotes GC invasion and metastasis by decreasing lncRNA NEAT1 m⁶A methylation [26] but suppresses GC by decreasing PKMYT1 m⁶A modification [24]. In the present study, we used the RNA-seq to reveal that ALKBH5 was involved in the regulation of cell proliferation and cycle progression in GC. In consistence with the previous reports [13-16, 24], our functional assays demonstrated that ectopic expression of ALKBH5 impaired cell proliferation, DNA synthesis, and cycle progression in vitro and in vivo, whereas reduced expression of ALKBH5 promoted these effects, suggesting that ALKBH5 might be an anti-oncogenic factor in GC.

Mechanistically, ALKBH5 acts by m⁶A-dependent modification of mRNA stability, miRNA biogenesis and lncRNA expression in cancers. ALKBH5 inhibits tumor progression by m⁶A modification of YAP mRNA in NSCLC [13], PER1 mRNA in PC [14] and LYPD1 mRNA in HCC [16] but promotes tumorigenesis by m⁶A modification of HBx mRNA in HCC [18], TIMP3 mRNA in NSCLC [19] and AURKB mRNA in RCC [20]. In addition, ALKBH5 impedes esophageal cancer by m⁶A demethylation of pri-miR-194-2 [27] and promotes pulmonary fibrosis through m⁶A demethylation of pri-miR-320a-3p [28]. Moreover, ALKBH5 acts by m⁶A demethylation of lncRNA PVT1 in osteosarcoma [29], KCNQ1OT1 in laryngeal squamous cell carcinoma (LSCC) [30] and KCNK15-AS1 in PC [31]. We herein found that, ALKBH5 decreased CDC6 mRNA and protein levels by m⁶A methylation in an m⁶A-YTHDF1-dependent manner.

Previous studies showed that CDC6 upregulation indicates poor prognosis in epithelial ovarian cancer [32] and enhances cell proliferation and cancer radio-resistance [33] and CDC6 downregulation suppresses osteosarcoma tumorigenesis [34]. We herein found that elevated CDC6 expression possessed a negative correlation with ALKBH5 and indicated unfavorable survival in patients with GC, and CDC6 inhibition abolished ALKBH5 knockdown-induced tumor-promoting effects. These findings suggested that, ALKBH5 downregulation in GC led to the increased m⁶A modifications, which promoted the expression of CDC6 in an m⁶A-dependent manner, contributing to GC tumorigenesis (Figure 8).

In conclusion, our findings demonstrated that downregulation of ALKBH5 was associated with poor prognosis in patients with GC, and ALKBH5-mediated m⁶A demethylation modification of CDC6 mRNA impaired tumorigenesis and cycle progression of GC cells, providing a promising therapeutic target for patients with GC.

m6A：N6-methyladenosine； METTL3/14: methyltransferase 3/14; ALKBH5: alkB homolog 5, RNA demethylase; CDC6: cell division cycle 6; YTHDF1/2/3: YTH N6-methyladenosine RNA binding protein 1/2/3; IGF2BP1: insulin like growth factor 2 mRNA binding protein 1; WTAP: WT1 associated protein; FEN1: flap structure-specific endonuclease 1; CDK4: cyclin dependent kinase 4; CDKN1C: cyclin dependent kinase inhibitor 1C; MCM7: minichromosome maintenance complex component 7; E2F1: E2F transcription factor 1; RIP: RNA immunoprecipitation, TCGA: The Cancer Genome Atlas, TMA: Tissue microarray; IHC: Immunohistochemistry; IF: Immunofluorescence; OE: overexpression ; KD: knockdown; MeRIP: Methylated RNA immunoprecipitation; RIP: RNA immunoprecipitation; PKMYT1: protein kinase, membrane associated tyrosine/threonine 1; TIMP3: TIMP metallopeptidase inhibitor 3; PVT1: Pvt1 oncogene; GC: Gastric cancer, STAD: stomach adenocarcinoma; NSCLC: non-small cell lung cancer; PC: pancreatic cancer; HCC: Hepatocellular carcinoma; OC: ovarian cancer; RCC: renal cell carcinoma; MM: multiple myeloma; TMA: Tissue microarray, FBS: Fetal bovine serum, IOD: Immunofluorescence accumulation optical density, RT-qPCR: real-time quantitative PCR, TNM: Tumor-Node-Metastasis.

Acknowledgements

Thanks for the technical support of Aksomics Biotechnology Co., Ltd.

Authors’ contributions

JSZ and JZ designed this study and JZ drafted the manuscript. RL and XYC performed the experiments and contributed equally to the article. YY, YLY and ZC collected the data and HF conducted the statistical analysis. All authors read and approved the final manuscript.

Funding

Our work was supported by the grants from Natural Science Foundation of Shanghai (No. 21ZR1448700). China Postdoctoral Science Foundation (No. 2022M722141)and National Natural Science Foundation of China (No. 82074161).

Availability of data and materials

All data generated or analysed during this study are included in this published article and its additional files.

Declarations

Ethics approval and consent to participate

The present study was approved by the Hospital’s Protection of Human Subjects Committee.

Consent for publication

Consent for publication has been obtained from the patients.

Competing interests

The authors declare that they have no competing interests.

Author details

Department of Gastroenterology, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China.

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ALKBH5-mediated m 6 A modification of CDC6 mRNA impairs gastric cancer tumorigenesis and has prognostic significance

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