ALYREF (Aly/REF export factor): A potential biomarker for predicting cancer occurrence and therapeutic efficacy.

5-Methylcytosine (m5C) methylation is present in almost all types of RNA as an essential epigenetic modification. It is dynamically modulated by its associated enzymes, including m5C methyltransferases (NSUN, DNMT and TRDMT family members), demethylases (TET family and ALKBH1) and binding proteins (YTHDF2, ALYREF and YBX1). Among them, aberrant expression of the RNA-binding protein ALYREF can facilitate a variety of malignant phenotypes such as maintenance of proliferation, malignant heterogeneity, metastasis, and drug resistance to cell death through different regulatory mechanisms, including pre-mRNA processing, mRNA stability, and nuclear-cytoplasmic shuttling. The induction of these cellular processes by ALYREF results in treatment resistance and poor outcomes for patients. However, there are currently few reports of clinical applications or drug trials related to ALYREF. In addition, the looming observations on the role of ALYREF in the mechanisms of carcinogenesis and disease prognosis have triggered considerable interest, but critical evidence is not available. For example, animal experiments and ALYREF small molecule inhibitor trials. In this review, we, therefore, revisit the literature on ALYREF and highlight its importance as a prognostic biomarker for early prevention and as a therapeutic target.

A DNA adenine demethylase impairs PRC2-mediated repression of genes marked by a specific chromatin signature.

BACKGROUND: The Fe (II)- and α-ketoglutarate-dependent AlkB family dioxygenases are implicated in nucleotide demethylation. AlkB homolog1 (ALKBH1) is shown to demethylate DNA adenine methylation (6mA) preferentially from single-stranded or unpaired DNA, while its demethylase activity and function in the chromatin context are unclear. RESULTS: Here, we find that loss-of-function of the rice ALKBH1 gene leads to increased 6mA in the R-loop regions of the genome but has a limited effect on the overall 6mA level. However, in the context of mixed tissues, rather than on individual loci, the ALKBH1 mutation or overexpression mainly affects the expression of genes with a specific combination of chromatin modifications in the body region marked with H3K4me3 and H3K27me3 but depleted of DNA CG methylation. In the similar context of mixed tissues, further analysis reveals that the ALKBH1 protein preferentially binds to genes marked by the chromatin signature and has a function to maintain a high H3K4me3/H3K27me3 ratio by impairing the binding of Polycomb repressive complex 2 (PRC2) to the targets, which is required for both the basal and stress-induced expression of the genes. CONCLUSION: Our findings unravel a function of ALKBH1 to control the balance between the antagonistic histone methylations for gene activity and provide insight into the regulatory mechanism of PRC2-mediated H3K27me3 deposition within the gene body region.

The N6-methyladenine DNA demethylase ALKBH1 promotes gastric carcinogenesis by disrupting NRF1 binding capacity.

DNA N6-methyladenine (6mA) is an epigenetic modification that regulates various biological processes. Here, we show that gastric cancer (GC) cells and tumors display a marked reduction in 6mA levels compared with normal gastric tissues and cells. 6mA is abundant in the surrounding transcription start sites and occurs at consensus motifs. Among the 6mA regulators, ALKBH1, a demethylase, is significantly overexpressed in GC tissues compared with adjacent normal tissues. Moreover, high ALKBH1 expression is associated with poor survival of patients with GC. ALKBH1 knockout in mice impairs chemically induced gastric carcinogenesis. Mechanistically, ALKBH1 mediates DNA 6mA demethylation to repress gene expression. In particular, the 6mA sites are enriched in NRF1 binding sequences and targeted for demethylation by ALKBH1. ALKBH1-induced 6mA demethylation inhibits NRF1-driven transcription of downstream targets, including multiple genes involved in the AMP-activated protein kinase (AMPK) signaling pathway. Accordingly, ALKBH1 suppresses AMPK signaling, causing a metabolic shift toward the Warburg effect, which facilitates tumorigenesis.

DNA 6mA demethylase ALKBH1 regulates DDX18 expression to promote proliferation of human head and neck squamous cell carcinoma.

PURPOSE: Human head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Currently, surgical resection plus a combination of chemotherapy and radiotherapy is the standard treatment for HNSCC, and the 5-year survival rate of patients with HNSCC remains very low because of the higher incidence of metastasis with consequent recurrence. Here, we aimed to investigate the potential role of DNA N6-methyladenine (6mA) demethylase ALKBH1 in tumor cell proliferation in HNSCC. METHODS: The expression of ALKBH1 in 10 pairs of HNSCC/normal tissues and 3 HNSCC cell lines were measured by qRT‒PCR and western blotting. Colony formation, flow cytometry, patient-derived HNSCC organoid assays were used to assess the role of ALKBH1 in HNSCC cell proliferation in cell lines and human HNSCC patients. MeDIP-seq, RNA sequencing, Dot blotting and western blotting were used to evaluate the regulatory effect of ALKBH1 on the expression of DEAD-box RNA helicase DDX18. A dual-luciferase reporter assay was used to assess the putative effect of DNA 6mA levels on DDX18 transcription. RESULTS: ALKBH1 was highly expressed in HNSCC cells and patient tissues. Functional experiments revealed that ALKBH1 knockdown in SCC9, SCC25, and CAL27 cells inhibited their proliferation in vitro. Using patient-derived HNSCC organoid assay, we found that knockdown of ALKBH1 inhibited the proliferation and colony formation of HNSCC patients-derived organoids. Moreover, we found that ALKBH1 can enhance DDX18 expression by erasing DNA 6mA level and regulating its promoter activity. ALKBH1 deficiency blocked tumor cell proliferation by inhibiting DDX18 expression. Exogenous overexpression of DDX18 rescued the cell proliferation arrest caused by ALKBH1 knockdown. CONCLUSION: Our data reveal the important role of ALKBH1 in regulating proliferation of HNSCC.

ALKBH1 contributes to renal cell carcinoma progression by reducing N6-methyladenine of GPR137.

BACKGROUND: Renal cell carcinoma (RCC) accounts for approximately 4% of all adult malignancies with high mortality worldwide. Although conventional chemotherapy and radiotherapy treatment has been applied for RCC in clinic, the mortality rate of patients is increasing each year, and patients with metastatic RCC are still suffering from poor prognosis. Thus, further investigation of the molecular mechanisms responsible for the development and progression of RCC is of particular importance. METHODS: Total of 10 pairs of RCC tissues and adjacent nontumor tissues were collected for examination of ALKBH1 and GPR137 expression. The correlations between ALKBH1 and GPR137 expression in RCC patient were assessed by GEPIA online tool and were analyzed using auto best cutoff. The human RCC cell lines Caki-1, 786-O, ACHN, Osrc2, A498, and 769-P, were used for mechanistic investigation. RESULTS: Here, we report that the expression of AlkB homologue 1 (ALKBH1) is upregulated in RCC tissues, which is correlated with G-protein-coupled receptor 137 (GPR137) expression. The elevated expression of ALKBH1 is associated with RCC cell malignant characteristics, including cell proliferation and movement (migration and invasion). Mechanistic investigation further reveals that ALKBH1 reduces m6 A levels of GPR137 mRNA in RCC cells, which upregulates GPR137 mRNA levels, resulting in the increased GPR137 protein expression subsequently and the enhanced RCC cell biological actions consequently. In contrast, the suppression of GPR137 effectively alleviates the ALKBH1-induced malignancies of RCC cells. CONCLUSION: Our results indicate that ALKBH1-GPR137 axis might be used as a potential therapeutic target in RCC, contributing to finding new prognostic biomarkers for RCC at an early stage.

ALKBH1-mediated m1 A demethylation of METTL3 mRNA promotes the metastasis of colorectal cancer by downregulating SMAD7 expression.

Colorectal cancer (CRC) is one of the most common malignancies, and the main cause of death from CRC is tumor metastasis. m1 A RNA modification plays critical role in many biological processes. However, the role of m1 A modification in CRC remains unclear. Here, we find that the m1 A demethylase alkB homolog 1, histone H2A dioxygenase (ALKBH1) is overexpressed in CRC and is associated with metastasis and poor prognosis. Upregulation of ALKBH1 expression promotes CRC metastasis in vitro and in vivo. Mechanistically, knockdown of ALKBH1 results in a decrease in methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3) expression, probably due to m1 A modification of METTL3 mRNA, followed by m6 A demethylation of SMAD family member 7 (SMAD7) mRNA. In addition, downregulation of SMAD7 establishes an aggressive phenotype. More importantly, the cell migration and invasion defects caused by ALKBH1 depletion or METTL3 depletion are significantly reversed by SMAD7 silencing. Considering these results collectively, we propose that ALKBH1 promotes CRC metastasis by destabilizing SMAD7 through METTL3.

Selected ALKBH dioxygenases are overexpressed in salivary gland tumours.

Salivary gland tumours (SGTs) are a heterogeneous group of benign tumours of various origins and pathologies, showing a number of DNA modifications. Previously, in malignant head and neck cancer (HNSCC), we found overexpression of ALKBH proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate and Fe(II) dependent dioxygenase. Moreover, we proved the connection of some of these dioxygenases with cancer development. Here, we studied the expression of five of these ALKBH dioxygenases: 1, 3, 4, 5, and FTO in benign SGTs. Using Western blot analysis, we found overexpression of three proteins: ALKBH1, 4, and FTO in SGT as compared to the surrounding, unaffected tissue. ALKBH4 was overexpressed in 76% of patient samples, whereas ALKBH1 and FTO in 65% of the samples. These results differ from those obtained in HNSCC, where FTO overexpression has been observed in 90% of patient samples. We also investigated the relationships between ALKBHs' expression levels in normal and SGT tissues and identified two correlated pairs: ALKBH1-ALKBH3 and ALKBH1-ALKBH5. Additionally, in tumour tissue ALKBHs: ALKBH1, ALKBH3, ALKBH4, and ALKBH5 levels were correlated with each other. Together, these findings show that the ALKBH proteins exhibit pro cancerogenic action in SGT, even though the levels ALKBHs are generally lower in benign SGT than in malignant HNSCC. We suggest that the overexpression of the ALKBHs, especially FTO, may be used as a cancer marker and for its grading.

Tyrosyl-DNA phosphodiesterase 1 excises the 3'-DNA-ALKBH1 cross-link and its application for 3'-DNA-ALKBH1 cross-link characterization by LC-MS/MS.

The apurinic/apyrimidinic (abasic, AP) site is one of the most abundant DNA lesions. Previous studies by others demonstrated that human AlkB homologue 1 (ALKBH1) catalyzes the DNA strand incision at an AP site, resulting in suicidal cross-linking of the enzyme to the 3'-DNA end. Prior site-directed mutagenesis experiments had reported that Cys129 of ALKBH1 is the predominant nucleophile that conjugates to the C3' position of the incised AP site, 3'-phospho-α,ß-unsaturated aldehyde (3'-PUA), to form a 3'-PUA-ALKBH1 cross-link. However, direct evidence to support this mechanism was lacking. The 3'-PUA-ALKBH1 cross-link is so far the only adduct that has been found to form via a Michael addition reaction between a protein and 3'-PUA. It is unclear whether and how this type of cross-link is repaired. In this study, we first demonstrated that the 3'-PUA-ALKBH1 cross-link is fairly stable under physiological temperature and pH as only ~10% of the adduct decomposed after a 3-day incubation. Using a gel-based assay with an aldehyde-reacting probe, we demonstrated that the 3'-PUA-ALKBH1 cross-link has a free aldehyde group that is in line with the Michael addition mechanism. Moreover, we found that the 3'-PUA-ALKBH1 cross-link can be excised by human tyrosyl-DNA phosphodiesterase 1 (TDP1) and the removal efficiency is significantly enhanced if the adduct is pre-digested by trypsin. Notably, we employed TDP1 as a molecular tool to homogeneously release the cross-linked peptides from DNA to facilitate liquid chromatography tandem mass spectrometry analysis, and demonstrated that Cys129 and Cys371 of ALKBH1 cross-link to 3'-PUA.

The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells.

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N6-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.

Characterization of the prognostic and diagnostic values of ALKBH family members in non-small cell lung cancer.

BACKGROUND: AlkB homolog (ALKBH) family genes have been known to play a crucial role in the development of several types of cancers. Nevertheless, the prognostic and diagnostic values of ALKBH family members have not been systematically investigated in non-small cell lung cancer (NSCLC). METHODS: The mRNA expression, genomic mutations, and biological functions of ALKBH family genes in NSCLC were evaluated using ONCOMINE, UALCAN, Kaplan-Meier Plotter, cBioPortal, Metascape, and SurvivalMeth. ALKBH2 expression and associated clinicopathological features were analyzed in LUAD tissues using immunohistochemistry (IHC). RESULTS: The mRNA levels of ALKBH1/2/4/6 were significantly upregulated in NSCLC patients, while those of ALKBH7 and FTO were downregulated. Also, a higher expression of ALKBH2/4/6 correlated with poor overall survival (OS), first-progression survival (FPS), and post-progression survival (PPS). ALKBH3/8 and FTO were upregulated and related to better OS, FPS, and PPS. ALKBH2/4/6 and FTO showed a higher diagnostic value in differentiating NSCLC patients from healthy individuals. Furthermore, the ALKBH family mutation rate was as high as 57% in lung adenocarcinoma (LUAD) patients and mutations in ALKBHs were related to poor OS. ALKBH family genes were also involved in universal DNA methylation in NSCLC. Finally, it was confirmed using tissues microarray that ALKBH2 shows a high expression and has a great diagnosis value in LUAD. CONCLUSIONS: Firstly, our results provided prognostic and diagnostic values of ALKBH family genes in NSCLC at both the DNA and RNA levels. Secondly, ALKBH2 is a potential novel diagnostic biomarker for LUAD.

DNA N6-methyladenine involvement and regulation of hepatocellular carcinoma development.

DNA N6-methyladenine (6 mA) is a new type of DNA methylation identified in various eukaryotic cells. However, its alteration and genomic distribution features in hepatocellular carcinoma (HCC) remain elusive. In this study, we found that N6AMT1 overexpression increased HCC cell viability, suppressed apoptosis, and enhanced migration and invasion, whereas ALKBH1 overexpression induced the opposite effects. Further, 23,779 gain-of-6 mA regions and 11,240 loss-of-6 mA regions were differentially identified in HCC tissues. The differential gain and loss of 6 mA regions were considerably enriched in intergenic regions. Moreover, 7% of the differential 6 mA modifications were associated with tumors, with 60 associated with oncogenes and 57 with tumor suppressor genes (TSGs), and 17 were common to oncogenes and TSGs. The candidate genes affected by 6 mA were filtered by gene ontology (GO) and RNA-seq. Using quantitative polymerase chain reaction (qPCR), BCL2 and PARTICL were found to be correlated with DNA 6 mA in certain HCC processes.

DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling.

DNA N6-adenine methylation (6mA), as a novel adenine modification existing in eukaryotes, shows essential functions in embryogenesis and mitochondrial transcriptions. ALKBH1 is a demethylase of 6mA and plays critical roles in osteogenesis, tumorigenesis, and adaptation to stress. However, the integrated biological functions of ALKBH1 still require further exploration. Here, we demonstrate that knockdown of ALKBH1 inhibits adipogenic differentiation in both human mesenchymal stem cells (hMSCs) and 3T3-L1 preadipocytes, while overexpression of ALKBH1 leads to increased adipogenesis. Using a combination of RNA-seq and N6-mA-DNA-IP-seq analyses, we identify hypoxia-inducible factor-1 (HIF-1) signaling as a crucial downstream target of ALKBH1 activity. Depletion of ALKBH1 leads to hypermethylation of both HIF-1α and its downstream target GYS1. Simultaneous overexpression of HIF-1α and GYS1 restores the adipogenic commitment of ALKBH1-deficient cells. Taken together, our data indicate that ALKBH1 is indispensable for adipogenic differentiation, revealing a novel epigenetic mechanism that regulates adipogenesis.

Demethyltransferase AlkBH1 substrate diversity and relationship to human diseases.

AlkBH1 is a member of the AlkB superfamily which are kinds of Fe (II) and α-ketoglutarate (α-KG)-dependent dioxygenases. At present, only demethyltransferases FTO and AlkBH5 have relatively clear substrate studies among these members, the types and mechanisms of substrates catalysis of other members are not clear, especially the demethyltransferase AlkBH1. AlkBH1, as a demethylase, has important functions of reversing DNA methylation and repairing DNA damage. And it has become a promising target for the treatment of many cancers, the regulation of neurological and genetic related diseases. Many scholars have made important discoveries in the diversity of AlkBH1 substrates, but there is no comprehensive summary, which affects the design inhibitor target of AlkBH1. Herein, We are absorbed in the latest progress in the study of AlkBH1 substrate diversity and its relationship with human diseases. Besides, we also discuss future research directions and suggest other studies to reveal the specific catalytic effect of AlkBH1 on cancer substrates.

ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease.

Vascular calcification (VC) predicts cardiovascular morbidity and mortality in chronic kidney disease (CKD). To date, the underlying mechanisms remain unclear. We detected leukocyte DNA N6-methyladenine (6mA) levels in patients with CKD with or without aortic arch calcification. We used arteries from CKD mice infected with vascular smooth muscle cell-targeted (VSMC-targeted) adeno-associated virus encoding alkB homolog 1 (Alkbh1) gene or Alkbh1 shRNA to evaluate features of calcification. We identified that leukocyte 6mA levels were significantly reduced as the severity of VC increased in patients with CKD. Decreased 6mA demethylation resulted from the upregulation of ALKBH1. Here, ALKBH1 overexpression aggravated whereas its depletion blunted VC progression and osteogenic reprogramming in vivo and in vitro. Mechanistically, ALKBH1-demethylated DNA 6mA modification could facilitate the binding of octamer-binding transcription factor 4 (Oct4) to bone morphogenetic protein 2 (BMP2) promoter and activate BMP2 transcription. This resulted in osteogenic reprogramming of VSMCs and subsequent VC progression. Either BMP2 or Oct4 depletion alleviated the procalcifying effects of ALKBH1. This suggests that targeting ALKBH1 might be a therapeutic method to reduce the burden of VC in CKD.

Genetic characteristics and prognostic implications of m1A regulators in pancreatic cancer.

Studies have identified the methylation of N1 adenosine (m1A), an RNA modification, playing an important role in the progression of the tumorigenesis. The present study aimed to analyze the genetic characteristics and prognostic value of m1A regulators in pancreatic cancer. In the present study, data on gene mutations, single-nucleotide variants (SNVs), and copy number variation (CNV) were obtained from 363 patients with pancreatic cancer in the Cancer Genome Atlas (TCGA) database, and survival analysis was performed using the logarithmic rank test and Cox regression model. The chi-squared test was used to examine the relationship between the changes in m1A regulatory factors and clinicopathological characteristics. And we used ICGC database to verify the reliability of prognostic markers. The results show that changes in m1A-regulating genes are related to clinical stage and that the expression of some m1A-regulating genes is positively correlated with CNV. In addition, the low expression of the 'eraser' gene ALKBH1 is related to the poor prognosis of patients with pancreatic cancer, and its expression level has important clinical significance for patients with pancreatic adenocarcinoma (PAAD). Mechanistically, ALKBH1 may participate in the occurrence and development of pancreatic cancer through mTOR and ErbB signaling pathway. The expression of m1A-regulating genes can be used as a prognostic marker for pancreatic cancer. These findings provide valuable clues for us to understand the epigenetics of m1A in pancreatic cancer.

N6-methyladenine demethylase ALKBH1 inhibits the differentiation of skeletal muscle.

DNA N6-methyladenine (N6-mA) was recently recognized as a new epigenetic modification in mammalian genome, and ALKBH1 was discovered as its demethylase. Knock-out mice studies revealed that ALKBH1 was indispensable for normal embryonic development. However, the function of ALKBH1 in myogenesis is largely unknown. In this study, we found that N6-mA showed a steady increase, going along with a strong decrease of ALKBH1 during skeletal muscle development. Our results also showed that ALKBH1 enhanced proliferation and inhibited differentiation of C2C12 cells. Genome-wide transcriptome analysis and reporter assays further revealed that ALKBH1 accomplished the differentiation inhibiting function by regulating a core set of genes and multiple signaling pathways, including increasing chemokine (C-X-C motif) ligand 14 (CXCL14) and activating ERK signaling. Taken together, our results demonstrated that ALKBH1 is critical for the myogenic differentiation of C2C12 cells, and suggested that N6-mA might be a new epigenetic mechanism for the regulation of myogenesis.

ALKBH1 promotes lung cancer by regulating m6A RNA demethylation.

Lung cancer has surpassed breast cancer as the leading cause of cancer death in females in developed countries and the leading cause of cancer death in males. Despite extensive research on lung cancer, the pathogenesis of lung cancer is not fully understood. ALKBH1 is a 2-oxoglutarate and Fe (II)-dependent dioxygenase responsible for the demethylation of 6-methyladenine (m6A) in RNA and is essential to multiple cellular processes in human. Numerous recent studies suggest that ALKBH1 plays a role in tumorigenesis and tumor progression, but the role of ALKBH1 in lung cancer is largely unknown. In this study, we demonstrated that the expression levels of ALKBH1 in lung cancer tissues and cells were up regulated. The invasion and migration abilities of lung cancer cells were significantly suppressed in vitro upon the silencing of ALKBH1 while they were significantly promoted upon its overexpression. We next characterized the enzyme biochemically by analyzing the contribution of essential residues Y184, H231, D233, H287, R338, and R344 to its m6A demethylation activity. Lastly, our 3.1-Å crystal structure of mouse ALKBH1 revealed that the N-terminal domain of the protein forms close contacted with the core catalytic domain and might be responsible for the recognition of nucleic acid substrates. In summary, our combined cellular, biochemical, and structural results provide insight into the potential ALKBH1-based drug design for cancer therapies.

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation.

tiRNAs are small non-coding RNAs produced when tRNA is cleaved under stress. tRNA methylation modifications has emerged in recent years as important regulators for tRNA structural stability and sensitivity to cleavage and tiRNA generation during stress, however, the specificity and higher regulation of such a process is not fully understood. Alkbh1 is a m1A demethylase that leads to destabilization of tRNA and enhanced tRNA cleavage. We examined the impact of Alkbh1 targeting via gene knockdown or overexpression on B35 rat neuroblastoma cell line fate following stresses and on tRNA cleavage. We show that Alkbh1 impact on cell fate and tRNA cleavage is a stress specific process that is impacted by the demethylating capacity of the cellular stress in question. We also show that not all tRNAs are cleaved equally following Alkbh1 manipulation and stress, and that Alkbh1 KD fails to rescue tRNAs from cleavage following demethylating stresses. These findings shed a light on the specificity and higher regulation of tRNA cleavage and should act as a guide for future work exploring the utility of Alkbh1 as a therapeutic target for cancers or ischaemic insult.

MiRNA-339-5p suppresses the malignant development of gastric cancer via targeting ALKBH1.

OBJECTIVE: To uncover the role of microRNA-339-5p (miRNA-339-5p) in the development of gastric cancer (GC) and its possible molecular mechanism. METHODS: Differential expressions of miRNA-339-5p in GC and adjacent normal tissues were detected. The relationship between miRNA-339-5p level and clinical features in GC patients was analyzed. Proliferative and migratory changes in BGC-823 and SGC-7901 cells overexpressing miRNA-339-5p were examined. Finally, luciferase assay and rescue experiments were conducted to explore the regulatory mechanism of miRNA-339-5p in its downstream gene ALKBH1, and their interaction in the development of GC. RESULTS: MiRNA-339-5p was downregulated in GC tissues. Lowly expressed miRNA-339-5p was unfavorable to prognosis in GC because of high rates of lymphatic metastasis and distant metastasis. Overexpression of miRNA-339-5p markedly reduced proliferative and migratory abilities in GC cells. ALKBH1 was identified to be the downstream gene of miRNA-339-5p. In GC tissues, ALKBH1 was upregulated and negatively correlated to miRNA-339-5p level. Overexpression of ALKBH1 was able to reverse the inhibitory effects of overexpressed miRNA-339-5p on proliferative and migratory abilities in GC. CONCLUSIONS: Lowly expressed miRNA-339-5p is closely related to metastasis and poor prognosis in GC patients. MiRNA-339-5p suppresses the malignant development of GC by negatively regulating ALKBH1.