Hypoxia blocks ferroptosis of hepatocellular carcinoma via suppression of METTL14 triggered YTHDF2-dependent silencing of SLC7A11

Abstract

Residue hepatocellular carcinoma (HCC) cells enduring hypoxic environment triggered by interventional embolization obtain more malignant potential with little clarified mechanism. The N⁶ -methyladenosine (m⁶ A) biological activity plays essential roles in diverse physiological processes. However, its role under hypoxic condition remains largely unexplored. RT-qPCR and Western blot were used to evaluate METTL14 expression in hypoxic HCC cells. MDA assay and electronic microscopy photography were used to evaluate ferroptosis. The correlation between SLC7A11 and METTL14 was conducted by bioinformatical analysis. Flow cytometry was used to verify the effect of SLC7A11 on ROS production. Cell counting kit-8 assay was performed to detect cells proliferation ability. Hypoxia triggered suppression of METTL14 in a HIF-1α-dependent manner potently abrogated ferroptosis of HCC cells. Mechanistic investigation identified SLC7A11 was a direct target of METTL14. Both in vitro and in vivo assay demonstrated that METTL14 induced m⁶ A modification at 5'UTR of SLC7A11 mRNA, which in turn underwent degradation relied on the YTHDF2-dependent pathway. Importantly, ectopic expression of SLC7A11 strongly blocked METTL14-induced tumour-suppressive effect in hypoxic HCC. Our investigations lay the emphasis on the hypoxia-regulated ferroptosis in HCC cells and identify the HIF-1α /METTL14/YTHDF2/SLC7A11 axis as a potential therapeutic target for the HCC interventional embolization treatment.

Keywords: METTL14; SLC7A11; ferroptosis; hepatocellular carcinoma; hypoxia.

FIGURE 1

The levels of METTL14, ROS and lipid peroxidation were limited by HIF‐1α in hypoxia condition. (A) Hypoxia downregulated METTL14 in Huh7 and HCCLM3 cells. (B&C). Effect of HIF‐1α knockdown on hypoxia‐induced METTL14 suppression. (D&E) Effect of HIF‐1α knockdown on hypoxia‐induced ROS accumulation detected by flowcytometry (D) and the quantification was also shown (E). F. Effect of HIF‐1α knockdown on hypoxia‐induced MDA accumulation. (G) The morphological change of mitochondria was detected by electronic microscopy. The white arrows referred to typical mitochondria. ‘NS’, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 2

METTL14 negatively regulates SLC7A11 expression in HCC. (A–C) Bioinformatics analysis from ‘Guichard Liver’ and ‘Guichard Liver 2’ database analysed by Oncomine (https://www.oncomine.org/resource/main.html) and TCGA database analysed by OncoLnc (http://www.oncolnc.org/) with the keywords ‘METTL14’, ‘Hepatocellular Carcinoma vs. Normal Analysis’. (D–F) Bioinformatics analysis from ‘Wurmbach Liver’, ‘Roessler Liver’ and ‘Roessler Liver 2’ database analysed by Oncomine (https://www.oncomine.org/resource/main.html) with the keywords ‘SLC7A11’, ‘Hepatocellular Carcinoma vs. Normal Analysis’. (G) The protein expression pattern of METTL14 among seven HCC cell lines (Huh7, HepG2, 7721, HCCLM3, MHCC97H, PLC/PRF/5 and Bel‐7402), comparing to normal liver cell line L02 detected by Western blot (low panel). The relative METTL14 protein level was quantified as showed in up panel. (H&I) The mRNA and protein levels of SLC7A11 among seven HCC cell lines (Huh7, HepG2, SMMC‐7721, HCCLM3, MHCC97H, PLC/PRF/5 and Bel‐7402), comparing to normal liver cell line L02 detected by qPCR and Western blot, separately. (J&K) The effect of METTL14 on SLC7A11 expression in Huh7 and HCCLM3 cells under hypoxia. The SLC7A11 mRNA and protein levels were detected by RT‐qPCR and Western blot, respectively. ‘NS’, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 3

METTL14 triggers m6A methylation of SLC7A11 mRNA at 5’UTR and the anti‐tumour effect of METTL14 is dependent on its methylation activity in HCC. (A) Schematic diagram of SLC7A11 mRNA and the predicted ‘m⁶A’ sites at 5‘UTR are highlighted in red. Base A in the middle of ‘DRACH’ was replaced by T to make the mutant plasmid for luciferase reporter assay. (B&C) The effect of wide‐type METTL14 and METTL14‐R298P mutant on SLC7A11 expression in HCCLM3 cells. The mRNA and protein level of SLC7A11 was detected by qPCR and Western blot, separately. (D) DOT BLOT showed the total m⁶A level HCCLM3 that stably expressed wide‐type METTL14 and METTL14‐R298P mutant. (E) Schematic diagram of the luciferase reporter of SLC7A11. (F) Relative activity of the WT or MUT luciferase reporters based on pGL3‐basic plasmid in METTL14 transfected HCCLM3 cells was determined (normalized to vector control groups). (G) MeRIP analysis followed by RT‐qPCR was applied to assess the m⁶A modification of SLC7A11 in HCCLM3 expressed wide‐type METTL14 or METTL14‐R298P mutant. The enrichment of m⁶A in each group was calculated by m⁶A‐IP/input and IgG‐IP/input. (H) The effect of wide‐type METTL14 and METTL14‐R298P mutant on HCC tumour growth. Nude mice were subcutaneously injected with HCCLM3 cells that stably expressed METTL14, METTL14‐R298P or control vector. Tumour growth was calculated twice every week. (I) Tumour growth curve of stable wide‐type METTL14 or METTL14‐R298P mutant overexpressing HCCLM3 cells (or negative control) in the xenograft model was presented. (J) The expression pattern of COX2, SLC7A11 and METTL14 in the xenograft detected by Western blot. (K) The correlation between METTL14 and COX2 in the xenograft. (L) The correlation between METTL14 and SLC7A11 in the xenograft. M. H&E stained section of three kinds of xenografts. (N) The expression pattern of COX2, SLC7A11 and METTL14 in the xenograft detected by immunohistochemistry. ‘NS’, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 4

m6A methylated 5’UTR regulates mRNA degradation of SLC7A11. (A&B) The mRNA decay rate was determined in Huh7 and HCCLM3 cells after treatment with Actinomycin D (normalized to 0h). (C) RT‐qPCR and Western blot showed the impact of silencing YTHDF2 on SLC7A11 mRNA and protein levels in Huh7 and HCCLM3. (D) Relative activity of the WT or MUT luciferase reporters in Huh7 and HCCLM3 cells transfected with siYTHDF2 was determined (normalized to vector control groups). ‘NS’, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 5

Knockdown of SLC7A11 stimulates ferroptosis and exhibits an anti‐tumour effect in HCC. A&B. ROS detection was performed by flow cytometry according to the manufacturers’ instruction (A) and quantified (B). The Huh7 and HCCLM3 cells were treated with or without 10mM concentrations of NAC for 36 h. (C&D) The protein and mRNA levels of EMT related E‐cadherin, N‐cadherin and Vimentin were detected by Western blot and qPCR in Huh7 and HCCLM3 cells, respectively. Cells were treated with or without 10 mM concentrations of NAC for 36 h. (E) The morphological alterations of mitochondria treated with/without NAC or shSLC7A11 in HCCLM3 cells were detected by electronic microscopy. The white arrows referred to typical mitochondria. (F) The effect of SLC7A11 knockdown on the tumour growth. G. Tumour volume was monitored during the time course of 5 weeks. (H) H&E stained section of three kinds of xenografts described above. (I) The expression pattern of SLC7A11 and COX2 in the xenograft detected by immunohistochemistry. ‘NS’, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 6

Ectopic expression of SLC7A11 abrogates METTL14‐induced tumour‐suppressive effect under hypoxia in HCC. A&B. Western blot showed the expression pattern of METTL14 and SLC7A11 in Huh7 and HCCLM3 cells. Cells stably expressed wide‐type METTL14 or R298P mutant were overexpressed (transfected) with SLC7A11. C‐F. Wound healing showed the migration rate of Huh7 and HCCLM3 cells transfected with or without SLC7A11 plus METTL14‐control, METTL14 overexpression or METTL14‐R298P under hypoxic condition, respectively. G&H. CCK‐8 assay illustrated the cell viability of Huh7 and HCCLM3 cells transfected with or without SLC7A11 plus METTL14‐control, METTL14 overexpression or METTL14‐R298P under hypoxia, respectively. ‘NS’, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 7

Model of METTL14‐induced YTHDF2/SLC7A11/ROS axis mediated ferroptosis under hypoxia triggered by embolization in HCC cells