ALKBH5 Expression Research Articles

The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

N6-Methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs. Although m6A is important in many biological processes, its roles in the placenta are unclear.Methods: Levels of global mRNA m6A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m6A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation.Results: We found that the level of global mRNA m6A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating CYR61 mRNA stability, and this RNA regulation is m6A dependent. Mechanistic analyses showed that decreased ALKBH5 in trophoblast increased the half-life of CYR61 mRNA and promoted steady-state CYR61 mRNA expression levels.Conclusions: We elucidated the functional roles of ALKBH5 and mRNA m6A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.

The perturbed expression of m6A in parthenogenetic mouse embryos.

Parthenogenetically activated oocytes cannot develop to term in mammals owing to abnormal epigenetic modifications. Methylation of the N6 position of adenosine (m6A) is a post-transcriptional epigenetic modification of RNA. To investigate the role of m6A methylation in parthenogenetic (PA) embryonic development, we analyzed METTL3, METTL14, FTO, ALKBH5, YTHDF2, IGF2BP1, and IGF2BP2 expression by quantitative real-time PCR. These genes were found dynamically expressed during the 2-cell, 4-cell, 8-cell, and blastocyst stages of the embryo. Compared to normally fertilized embryos, the expression of these genes was perturbed in PA embryos, especially at the 8-cell stage. Furthermore, immunofluorescence was used to detect m6A expression. The results demonstrated that m6A expression decreased in the 2-cell stage, whereas it increased in the 8-cell stage of PA embryos. Taken together, these results suggest that the expression of RNA methylation-related genes was perturbed, leading to abnormal m6A modification during early development in PA embryos.

ALKBH5 Functions As an Oncogene in Acute Myeloid Leukemia

N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) has been shown to play important roles in diverse cellular and pathological processes (Deng X, et al. Cell Res. 2018;28:507-517). ALKBH5, recently identified as a m6A demethylase, was reported to promote tumorigenesis and proliferation in glioblastoma stem-like cells (GSCs) (Zhang, S. et al. Cancer Cell. 2017;31:591-606) and breast cancer stem cells (BCSCs) (Zhang, C et al. PNAS. 2016;113: E2047-E2056). While ALKBH5 is well-recognized to function as an oncogene in solid tumors, it was reported that shallow/deep deletion of ALKBH5 is associated poor prognosis in patients with acute myeloid leukemia (AML), and is frequently co-existing with TP53 mutation (Kwok, C. T et al. J Hematol Oncol. 2017; 10(1): 39), implying that ALKBH5 may function as a tumor suppressor in AML. Thus, a systematic investigation of the definitive role of ALKBH5 in AML is warranted.To this end, we performed series of in vitro and in vivo experiments to determine the function of ALKBH5 in AML. For the in vitro experiments, we used three lentiviral shRNAs (shALKBH5-A, shALKBH5-D and shALKBH5-E) to deplete ALKBH5 expression in three human AML cell lines with different TP53 mutation status: NOMO-1 (TP53-mutant), MV4;11 (TP53-WT) and MA9.3 cells (TP53-WT). Somewhat surprisingly, ALKBH5 depletion significantly (p<0.05) inhibited AML cell proliferation/growth in all three AML cells lines, regardless of the status of TP53 mutation. We next conducted colony forming assays and found that ALKBH5 knockdown significantly (p<0.01) impaired the colony forming ability to 18% ~45% of the control group level in all three AML cell lines. We further showed that ALKBH5 depletion caused a significant increase in apoptosis (with a 1.5 ~ 4 fold increase; p<0.001) in all three AML cell lines, which is consistent with the previous report that knockout of ALKBH5 caused severe apoptosis of mouse testis cells (Zheng G et al. Mol Cell. 2013; 49:18-29). In contrast, ALKBH5 knockdown did not significantly affect cell cycles.To further confirm ALKBH5's role in AML development in vivo. We utilized Xenografted AML model as well as mouse bone marrow transplantation (BMT) model. Consistent with the in vitro results, we found that NSGS mice xeno-transplanted with MV4;11-ALKBH5-knockdown cells survived significantly longer than those with MV4;11 control cells (p<0.001). Moreover, we have also conducted mouse bone marrow transplantation (BMT) assays with MLL-AF9-transduced mouse bone marrow lineage negative (Lin-) progenitor cells collected from mice carrying Alkbh5 wild-type (Alkbh5+/+), or heterozygous (Alkbh5+/-) or homozygous (Alkbh5-/-) deletion. Consistent with the xeno-transplanted mouse model results, our BMT assays also showed that Alkbh5 depletion significantly inhibited leukemogenesis and prolonged survival in BMT recipient mice (median survival of ALKBH5wt/wt +MA9 vs. ALKBH5+/- +MA9 or ALKBH5-/- +MA9: 32 days vs. 64 days or 68 days; p<0.005).Taken together, our in vitro and in vivo functional studies data indicate ALKBH5 also functions as an oncogene in AML regardless of TP53 mutation status, similar to its role in solid tumors. We are currently conducting as series of studies to reveal the molecular mechanism(s) underlying the oncogenic role of ALKBH5 in AML. DisclosuresNo relevant conflicts of interest to declare.

N6-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression.

The mRNA modification N6-methyladenosine (m6A) is involved in many post-transcriptional regulatory processes including mRNA stability and translational efficiency. However, it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs), as compared with their primary cell predecessor. Furthermore, the m6A methyltransferase (METTL3) is decreased and the demethylase (ALKBH5) is increased in the immortalized and transformed cell lines, providing a possible mechanism for this basal change in m6A levels. Although the immortalized and transformed cells showed lower m6A levels than their primary parental cell line, overexpression of METTL3 and METTL14, or ALKBH5 knockdown to increase m6A levels in transformed cells increased proliferation and migration. Remarkably, these treatments had little effect on the immortalized cells. Together, these results suggest that m6A modification may be downregulated in immortalized cells as a brake against malignant progression. Finally, we found that m6A levels in the immortalized and transformed cells increased in response to hypoxia without corresponding changes in METTL3, METTL14 or ALKBH5 expression, suggesting a novel pathway for regulation of m6A levels under stress.