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. 2023 Jan 6;12(1):1.
doi: 10.1186/s40164-022-00370-2.

ALKBH5 prevents hepatocellular carcinoma progression by post-transcriptional inhibition of PAQR4 in an m6A dependent manner

Weijian Wang #  1   2 , Qibo Huang #  1   2 , Zhibin Liao #  1   2   3 , Hongwei Zhang  1   2   3 , Yachong Liu  1   2 , Furong Liu  1   2 , Xiaoping Chen  1   2   3 , Bixiang Zhang  1   2   3 , Yan Chen  4   5   6 , Peng Zhu  7   8   9
Affiliations

ALKBH5 prevents hepatocellular carcinoma progression by post-transcriptional inhibition of PAQR4 in an m6A dependent manner

Weijian Wang et al. Exp Hematol Oncol. .

Abstract

Background: N6-methyladenosine (m6A) is a prevalent modification of mRNA and is known to play important roles in tumorigenesis in many types of cancer. The function of N6-methyladenosine (m6A) RNA methylation depends on a variety of methyltransferases and demethylases. AlkB homolog 5 (ALKBH5) is a demethylase, and its biological function has not been completely explored in HCC.

Results: ALKBH5 is downregulated and has antitumor effects in HCC cells. In addition, Progestin and AdipoQ Receptor 4 (PAQR4) was identified as a downstream target of ALKBH5 based on transcriptome sequencing and validation studies. We found that ALKBH5 decreases PAQR4 mRNA and protein expression in an N6-methyladenosine (m6A)-dependent manner. The study also showed that ALKBH5 changes PAQR4 expression via the m6A reader IGF2BP1. In both in vivo and in vitro experiments, PAQR4 showed a strong association with the development of HCC. Finally, we found that PAQR4 interacts with AKT and enhances PI3K/AKT pathway activation.

Conclusions: ALKBH5 inhibits HCC growth by downregulating PAQR4 expression in an m6A-dependent manner, therefore suppressing PI3K/AKT pathway activation.

Keywords: AKT; ALKBH5; HCC; Methylation; PAQR4.

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Conflict of interest statement

No potential competing interest were disclosed.

Figures

Fig. 1
Fig. 1
ALKBH5 is decreased in HCC and suppress HCC proliferation and invasion in vitro. A Protein level of ALKBH5 was detected by Western bolt assay in paired HCC samples (n = 60). CCK8 and EdU assays confirmed that overexpression of ALKBH5 inhibit proliferation of 97H and HLF (B, D and Additional file 1A), whereas knockdown of ALKBH5 promoted cell proliferation in LM3 (C, E and Additional file 1A). Transwell and wound healing assays showed that overexpression of ALKBH5 suppress the capability of migration and invasion in 97H and HLF (F, H and Additional file 1B, C). In contract, knockdown of ALKBH5 promoted migration and invasion in LM3 (G, I and Additional file 1B, C). GAPDH was used as internal controls in Western blotting analysis. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
ALKBH5 suppressed m6A modification of PAQR4 mRNA. A The heat map exhibited the changed genes of transcriptome sequencing after ALKBH5 overexpression. B, C RT–qPCR analysis showed the six altered target genes with ALKBH5 knockdown or ALKBH5 overexpression. D The result of m6A abundances in mRNA transcripts of ALKBH5 target genes was shown. E Western blot analysis showed the protein level of PAQR4 with overexpression of ALKBH5 and knockdown of ALKBH5 in HCC cells. F–H RT–qPCR analysis and Western blot verified both mRNA and protein expression of PAQR4 was decreased when co-transfected with ALKBH5-WT but not with ALKBH5-H204A. GAPDH was used as internal controls in Western blotting and RT–qPCR analysis. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
ALKBH5 downregulated PAQR4-mediated with IGF2BP1 in a m6A dependent manner. A Schematic representation exhibited totally 5 possible sites of m6A motifs in PAQR4 mRNA, and all of these positions were muted as described then constructed into psiCHECK-Vector plasmids to investigate the m6A roles on PAQR4 expression. PAQR4-WT was constructed into psiCHECK-Vector plasmids as control. B The psiCHECK PAQR4-MUT and psiCHECK PAQR4-WT plasmids were transfected into wild-type or ALKBH5-overexpression HLF cells and ALKBH5-knockdown LM3 cells for 24 h. Fluorescence intensity represented changes in PAQR4 transcriptional activity. C MeRIP-qPCR analysis indicated ALKBH5 overexpression abolish m6a modification on PAQR4 mRNA in HLF cells while ALKBH5 knockdown enriched m6a modification on PAQR4 mRNA. D Actinomycin D (ActD) assay showed overexpression of PAQR4 accelerate the degradation of mRNA whereas this process slowed down after ALKBH5 was knockdown. E PAQR4 expression was measured via RT–qPCR in two IGF2BP1 knockdown HCC cells. F The result of RIP-qPCR indicated that IGF2BP1 could bind to PAQR4 mRNA in HLF and LM3 cells. GAPDH was used as internal controls in qPCR analysis. G Representative immunohistochemical staining images of ALHBH5, IGF2BP1 and PAQR4 in human HCC tissues; scale bar: 200 μm. H Correlation analysis between ALHBH5 and PAQR4 in human HCC tissues. I Correlation analysis between IGF2BP1 and PAQR4 in human HCC tissues. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
PAQR4 promoted HCC cells proliferation in vivo and in vitro. CCK8 and EdU assays confirmed that overexpression of PAQR4 promote proliferation in Hep3B and HLF cells (A, C and E), whereas knockdown of ALKBH5 repressed cell proliferation in HLF and 97H cells (B, D and E). F Representative images of tumor xenograft models using PAQR4-overexpressing HLF cells (n = 5), HLF-Vector cells were used as control. (G and H) Tumor weight and volume of PAQR4-overexpressing and control group were shown. I The Immunohistochemistry (IHC) assays showed expression of ki-67 and PCNA in the tumor xenograft model. Scale bar, 20 μm (40 ×). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
PAQR4 promoted the migration and invasion capability of HCC cells in vitro and promoted HCC metastasis in vivo. Transwell and wound healing assays showed that overexpression of PAQR4 promote the capability of migration and invasion in Hep3B and HLF cells (A, C and Additional file 2A and B). Knockdown of PAQR4 suppress the capability of migration and invasion in HLF and 97H cells (B, D and Additional file 2A and B). Immunofluorescence and Western blot analysis EMT markers like E-ca, N-ca, Vimentin in Hep3B and HLF cells (E, F). Representative images of intrahepatic metastasis model using PAQR4-overexpressing HLF cells (n = 5), HLF-Vector cells were used as control (G). The numbers of metastasis nodules (H). The Immunohistochemistry (IHC) assays showed expression of EMT markers E-ca and N-ca on tumor sections. Scale bar, 20 μm (40 ×). i *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
PAQR4 promoted the development of HCC by activating AKT. A Silver staining indicated PAQR4 may interact with AKT. B Co-immunoprecipitation (Co-IP) assay identified the interaction between PAQR4 and AKT. C The colocalization of PAQR4 and AKT was observed by immunofluorescence in HLF and HepG2 cells. D Western blot analysis showed activation of AKT pathway with or without IGF-1 stimulation when PAQR4 was overexpressed. (E, F and Additional file 4A) CCK8 and EdU assays showed knockdown of AKT reduce proliferation caused by overexpression of PAQR4. (G, H and Additional file 4B, C) Transwell and wound healing assays showed that knockdown of AKT reduce increased migration and invasion capability caused by overexpression of PAQR4. GAPDH was used as internal controls in Western blotting analysis. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 7
Fig. 7
High expression of PAQR4 is associated with poor prognosis in HCC. A PAQR4 expression in the normal and HCC data were obtained from TCGA and the (B, C) Kaplan–Meier survival curves disease-free survival of patients with HCC from TCGA. D PAQR4 expression in human HCC data were obtained from 6 GEO datasets. E PAQR4 expression in DEN-induced mouse liver cancer. F Representative images of Immunohistochemistry (IHC) from human HCC samples and the expression of PAQR4 was compared between Adjacent tissue and tumor tissue according to the IHC scoring. G, H Kaplan–Meier survival curves and disease-free survival of patients with HCC from Tongji cohort. i Schematic representation of a model for the role of PAQR4 in HCC. *P < 0.05, **P < 0.01, ***P < 0.001
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