The RNA demethylase ALKBH5 promotes osteoblast differentiation by modulating Runx2 mRNA stability.

AlkB homolog 5 (ALKBH5) has been reported as a key m6A demethylase that is involved in development and diseases; however, the function of ALKBH5 in osteogenesis remains unknown. In this study, we report that ALKBH5 mRNA and protein expression were upregulated during osteoblast differentiation and that ALKBH5 knockdown suppressed osteoblast differentiation, mineralization, and the expression of osteogenic biomarkers. Conversely, ALKBH5 overexpression promoted osteogenesis. Moreover, the expression of wild-type ALKBH5, but not the m6A-modified active site mutant ALKBH5, could rescue ALKBH5 knockdown-induced osteogenesis inhibition. Furthermore, knockdown of ALKBH5 significantly impaired the mRNA stability of the transcription factor Runx2, which plays a key role in osteoblast differentiation. Taken together, our results suggest that ALKBH5 promotes osteogenesis through modulating Runx2 mRNA stability.

ALKBH5-Modified HMGB1-STING Activation Contributes to Radiation Induced Liver Disease via Innate Immune Response.

PURPOSE: Radiation therapy, which is vital for the treatment of primary liver cancer, comes with unavoidable liver injury, which limits its implementation. N6-methyladenosine (m6A) methylation is involved in many molecular functions. However, its role in radiation-induced liver diseases (RILD) remains unknown. Herein, we investigate the role of m6A methylation in RILD. METHODS AND MATERIALS: Methylated RNA-immunoprecipitation sequencing and RNA transcriptome sequencing were used to reveal the methylation pattern of human hepatic stellate cells (HSCs) exposed to irradiation. C3H/HeN mice and stimulator of interferon genes (STING)-deficient mice underwent x-ray irradiation of 24 Gy in 3 fractions. The m6A methylation of the high-mobility group box 1 (HMGB1) transcript was validated using methylated RNA immunoprecipitation, RNA immunoprecipitation, luciferase assays, and a messenger RNA decay assay. RESULTS: Human hepatic stellate cells showed significant differences in methylation patterns after 8 Gy of x-ray irradiation. Irradiation recruited AlkB homolog 5 (ALKBH5) to demethylate m6A residues in the 3' untranslated region of HMGB1, which resulted in the activation of STING-interferon regulatory factor 3 signaling. Changes in the transcription of the 3' untranslated region of HMGB1 occurred after the knockdown of ALKBH5, which were eliminated after m6A residue mutation. Strikingly, ALKBH5 deficiency or HMGB1 silencing both attenuated type I interferon production and decreased hepatocyte apoptosis. In vivo depletion of ALKBH5 abolished the upregulation of HMGB1-mediated STING signaling and decreased liver inflammation, which was consistent with STING-/- mice treated with irradiation. Notably, YTHDF2 (m6A reader protein) directly bound to HMGB1 m6A-modified sites and promoted its degradation. CONCLUSIONS: ALKBH5-dependent HMGB1 expression mediates STING-interferon regulatory factor 3 innate immune response in RILD.

A positive feedback loop between AlkB homolog 5 and miR-193a-3p promotes growth and metastasis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most frequent malignancies worldwide. miR-193a-3p acts as an oncogene or tumor suppressor in different cancers. However, the functional role and regulatory mechanism of miR-193a-3p in ESCC remain to be elucidated. Our results demonstrated that miR-193a-3p expression was significantly upregulated and associated with advanced TNM stage, recurrence, and poor prognosis in ESCC patients. miR-193-3p targeted ALKBH5 and suppressed its expression. ALKBH5 inhibited miR-193a-3p expression in turn. ALKBH5 affected the primary miR-193a-3p processing by negatively regulating its m6A modification. These findings suggested a positive feedback regulation between miR-193a-3p and ALKBH5 in ESCC cells. Moreover, the functional assays indicated that the miR-193-3p-ALKBH5 feedback loop promoted the proliferation, migration and invasion ability of ESCC cells in vitro, and facilitated tumor growth and metastasis in vivo. Collectively, our current study identified a novel positive feedback regulation between miR-193a-3p and ALKBH5 in ESCC, which may be helpful to gain insight into ESCC pathogenesis and provide novel therapeutic target for ESCC.

Principles of RNA methylation and their implications for biology and medicine.

RNA methylation is a post-transcriptional level of regulation. At present, more than 150 kinds of RNA modifications have been identified. They are widely distributed in messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), noncoding small RNA (sncRNA) and long-chain non-coding RNA (lncRNA). In recent years, with the discovery of RNA methylation related proteins and the development of high-throughput sequencing technology, the mystery of RNA methylation has been gradually revealed, and its biological function and application value have gradually emerged. In this review, a large number of research results of RNA methylation in recent years are collected. Through systematic summary and refinement, this review introduced RNA methylation modification-related proteins and RNA methylation sequencing technologies, as well as the biological functions of RNA methylation, expressions and applications of RNA methylation-related genes in physiological or pathological states such as cancer, immunity and virus infection, and discussed the potential therapeutic strategies.

N6-methyladenosine ALKBH5 promotes non-small cell lung cancer progress by regulating TIMP3 stability.

Mounting evidence demonstrates that N6-methyladenosine (m6A) play critical roles of m6A in the epigenetic regulation, especially for human cancer. The m6A modification is installed by methyltransferase and erased demethylases, leading to the significant modification for gene expression and cell fate. Here, we investigated the biological roles and mechanism of demethylase alkylation repair homolog protein 5 (ALKBH5) in the non-small cell lung cancer (NSCLC). Results revealed that ALKBH5 was ectopically up-regulated in the NSCLC tissue and cells, and closely correlated with the poor prognosis. Functionally, ALKBH5 promoted the proliferation and reduced apoptosis of NSCLC cells in vitro, and knockdown of ALKBH5 repressed the tumor growth in vivo. Mechanistically, RNA immunoprecipitation sequencing (RIP-Seq) revealed that ALKBH5 targeted the TIMP3. Moreover, ALKBH5 repressed TIMP3 mRNA stability and protein production. In conclusion, the present research confirmed the ALKBH5/TIMP3 pathway in the NSCLC oncogenesis progress, providing a novel insight for the epitranscriptome and potential therapeutic target for NSCLC.

ALKBH5-m6A-FOXM1 signaling axis promotes proliferation and invasion of lung adenocarcinoma cells under intermittent hypoxia.

Obstructive sleep apnea (OSA) is closely associated with cancer progression and cancer-related mortality. N6-methyladenosine (m6A) is involved in the process of intermittent hypoxia (IH) promoting tumor progression. However, it is unclear how m6A regulates the development of lung adenocarcinoma under IH. In this study, we found that ALKBH5 was elevated in lung adenocarcinoma cells and subcutaneous tumors in mice under IH, which was associated with decreased m6A levels in these cells and tissues. Next, we knocked out ALKBH5 in a human lung adenocarcinoma cell line under IH, and we found that the proliferation and invasion of these cells were significantly inhibited. Mechanistic analysis showed that under IH, knockout of ALKBH5 in lung adenocarcinoma cells upregulated the level of m6A in Forkhead box M1 (FOXM1) mRNA and decreased the translation efficiency of FOXM1 mRNA, resulting in downregulation of the FOXM1 protein. The FOXM1 protein is elevated in lung adenocarcinoma cells and subcutaneous tumor tissues of mice under IH. By knocking out FOXM1 in lung adenocarcinoma cells under IH, proliferation and invasion of these cells were inhibited, and overexpression of FOXM1 partially restored the inhibition of growth and invasion of lung adenocarcinoma cells due to ALKBH5 knockout. Collectively, our findings demonstrate that the m6A demethylase ALKBH5 affects the proliferation and invasion of lung adenocarcinoma cells under IH by downregulating m6A modification on FOXM1 mRNA and by promoting FOXM1 expression.

ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1.

N6-Methyladenosine (m6A) is the most common posttranscriptional modification of RNA and plays critical roles in cancer pathogenesis. However, the biological function of long noncoding RNA (lncRNA) methylation remains unclear. As a demethylase, ALKBH5 (alkylation repair homolog protein 5) is involved in mediating methylation reversal. The purpose of this study was to investigate lncRNA m6A modification and its role in gastric cancer (GC). Bioinformatics predicted interactions of ALKBH5 with lncRNAs. Five methods were employed to assess the function of nuclear paraspeckle assembly transcript 1 (NEAT1), including gene silencing, RT-PCR, separation of nuclear and cytoplasmic fractions, scrape motility assays, and transwell migration assays. Then, m6A RNA immunoprecipitation and immunofluorescence were used to detect methylated NEAT1 in GC cells. Rescue assays were performed to define the relationship between NEAT1 and ALKBH5. NEAT1 is a potential binding lncRNA of ALKBH5. NEAT1 was overexpressed in GC cells and tissue. Additional experiments confirmed that knockdown of NEAT1 significantly repressed invasion and metastasis of GC cells. ALKBH5 affected the m6A level of NEAT1. The binding of ALKBH5 and NEAT1 influences the expression of EZH2 (a subunit of the polycomb repressive complex) and thus affects GC invasion and metastasis. Our findings indicate a novel mechanism by which ALKBH5 promotes GC invasion and metastasis by demethylating the lncRNA NEAT1. They may be potential therapeutic targets for GC.

Potential link between m6A modification and systemic lupus erythematosus.

The field of m6A modification and epitranscriptomics has recently attracted much attention. More methods allowing for precise m6A site profiling and location are developed and crucial players of m6A modification machinery are increasingly identified. Although some challenges remain, m6A modification is found to modulate almost all aspects of RNA metabolism, such as splicing, stability, structure, translation, and export. Thus, m6A modification adds a new layer of post-transcriptional gene expression regulation, and it is implicated in T cell response to HIV infection, type I interferon production, and T cell differentiation and homeostasis. Moreover, evidence supporting its involvement in various human diseases including cancers is accumulating. Given the role of m6A modification in gene expression regulation and immune response, it invites the speculation that m6A modification may justify the pathogenesis of systemic lupus erythematosus (SLE) and take part in the initiation and progression of SLE. In this review, we introduce the widespread existence of m6A modification and briefly discuss components of m6A modification machinery in mammals. We mainly summarize the studies reporting the mechanisms of m6A modification in gene expression regulation through modulating pre-mRNA splicing, mRNA stability, RNA structure, translation, and pri-miRNA processing. Biological functions related to immune response of m6A modification and the implication of m6A modification in cancers are highlighted. In the end, we surmise the potential link between m6A modification and SLE.

Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.

N(6)-methyladenosine (m(6)A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m(6)A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of AlkB homolog 5 (ALKBH5), an m(6)A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m(6)A residue in the 3'-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3'-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.