The YTHDC1 reader protein recognizes and regulates the lncRNA MEG3 following its METTL3-mediated m6A methylation: a novel mechanism early during radiation-induced liver injury

Abstract

While apoptotic cell death is known to be central to the pathogenesis of radiation-induced liver injury (RILI), the mechanistic basis for this apoptotic activity remains poorly understood. N⁶-methyladenosine (m⁶A) modifications are the most common form of reversible methylation observed on lncRNAs in eukaryotic cells, with their presence leading to pronounced changes in the activity of a range of biological processes. The degree to which m⁶A modification plays a role in the induction of apoptotic cell death in response to ionizing radiation (IR) in the context of RILI remains to be established. Here, IR-induced apoptosis was found to significantly decrease the levels of m⁶A present, with a pronounced decrease in the expression of methyltransferase-like 3 (METTL3) at 2 d post radiation in vitro. From a mechanistic perspective, a methylated RNA immunoprecipitation assay found that lncRNA MEG3 was a major METTL3 target. The expression of MEG3 was upregulated via METTL3-mediated m⁶A in a process that was dependent on YTHDC1, ultimately reversing the miR-20b-mediated inhibition of BNIP2 expression. Together, these findings demonstrate that the responsivity of METTL3 activity to IR plays a role in IR-induced apoptotic cell death, leading to the reverse of miR-20b-mediated BNIP2 inhibition through the YTHDC1-dependent m⁶A modification of MEG3, suggesting that this process may play a central role in RILI incidence.

Fig. 1. The impact of irradiation on METTL3, YTHDC1, MEG3, miR-20b, and BNIP2 levels in BNL CL2 cells.

A The METTL3 protein level (**p < 0.01) at different time points within 24 h after radiation in BNL CL2 cells. B The YTHDC1 protein level (**p < 0.01) at different time points within 24 h after radiation in BNL CL2 cells. C The MEG3 level (**p < 0.01) at different time points within 24 h after radiation in BNL CL2 cells. D The miR-20b level (**p < 0.01) at different time points within 24 h after radiation in BNL CL2 cells. The BNIP2 mRNA (E) and protein (F) levels (**p < 0.01) at different time points within 24 h after radiation in BNL CL2 cells. A–F n = 3. A–F were followed by one-way ANOVA.

Fig. 2. YTHDC1 impacts BNL CL2 cell viability and apoptotic induction following radiation-induced injury.

A Western blotting revealed the downregulation of YTHDC1 in BNL CL2 cells on day 2 post irradiation (**P < 0.01). B Flow cytometry was used to examine the impact of YTHDC1 on BNL CL2 cell apoptosis on day 2 post irradiation (**P < 0.01). C. A CCK-8 assay was used to examine the impact of YTHDC1 on BNL CL2 cell viability with or without irradiation (**P < 0.01). D. The methylation level of MEG3 in BNL CL2 cells a at 2 d after 6 Gy radiation was detected by MeRIP assay (**P < 0.01). A, B, D, n = 3; C, n = 5. A, B were followed by one-way ANOVA. C, D were performed by two-tailed unpaired T-test.

Fig. 3. MEG3 influences BNL CL2 cell viability and apoptosis following radiation-induced injury.

A Flow cytometry was used to assess the effects of MEG3 on BNL CL2 cell apoptosis on day 2 post irradiation (**P < 0.01). B. MEG3 was significantly upregulated on day 2 post irradiation (**P < 0.01). C A CCK-8 assay was used to assess the impact of MEG3 on BNL CL2 cell viability (**P < 0.01). D A CCK-8 assay was used to assess the impact of MEG3 on BNL CL2 cell viability on day 2 post irradiation (**P < 0.01). E Hoechst staining was used to examine the impact of MEG3 on BNL CL2 cell apoptosis on day 2 post irradiation. A, B n = 3; C, D n = 5; scale bar: 20 µm. A, B were followed by one-way ANOVA. C, D were performed by two-tailed unpaired T-test.

Fig. 4. The m⁶A methylation of MEG3 mediated by METTL3 plays a role in radiation-induced hepatocyte injury.

A Predictive analysis of MEG3-m⁶A methylation sites. B Predictive analysis of putative binding sites between MEG3 and YTHDC1. C Through the combination of results from (A, B), YTHDC1-dependent methylation sites were identified. D Confirmation of the successful knockdown of METTL3 in BNL CL2 cells (**P < 0.01). qPCR was used to detect levels of MEG3 in cells following METTL3 knockdown without (E) or with (F) 6 Gy irradiation (**P < 0.01). MeRIP was used to detect MEG3 methylation levels in BNL CL2 cells in which METTL3 was knocked down without (G) or with (H) 6 Gy irradiation (**P < 0.01). I MEG3 methylation levels were detected via MeRIP assay in BNL CL2 cells at 2 h following 6 Gy irradiation (**P < 0.01). J Overview of the MeRIP experimental approach. D–I n = 3. D–I were performed by two-tailed unpaired T-test.

Fig. 5. METTL3 controls MEG3 levels in a YTDC1-dependent fashion in the context of RILI.

A Confirmation of the establishment of YTHDC1-knockdown BNL CL2 cells (**P < 0.01). B MEG3 expression in BNL CL2 cells was analyzed via qPCR in YTHDC1 knockdown cells (**P < 0.01). C. MEG3 expression levels were analyzed via qPCR following YTHDC1 knockdown and irradiation in BNL CL2 cells (**P < 0.01). D, E RIP assays were used to pull down MEG3 with anti-YTHDC1 in BNL CL2 cells following METTL3 knockdown and irradiation (**P < 0.01). F MEG3 levels were assessed in cells in which METTL3 had been knocked down and co-transfected with TYHDC1 with or without irradiation (**P < 0.01). G MEG3 levels were detected in NC or YTHDC1 shRNA-treated cells by qPCR following actinomycin D (1 µg/ml) treatment for the indicated periods (**P < 0.01). H MEG3 and YTHDC1 target binding sites. I YTHDC1 can recognize METTL3-mediated MEG3-m⁶A modification. J RIP experimental overview. A–G, n = 3. A–G were performed by two-tailed unpaired T-test.

Fig. 6. MEG3 regulates radiation-induced injury in BNL CL2 cells via regulating the miR-20b-mediated control of BNIP2 expression.

A A dual-luciferase reporter assay was used to probe the impact of miR-20b on wild-type or mutant MEG3 reporter activity (**P < 0.01; NS no significance); The effects of irradiation (6 Gy) on miR-20b levels were assessed following the overexpression (B) or knockdown (C) of MEG3 (**P < 0.01). The impact of overexpressing MEG3 on BNIP2 mRNA and protein levels without (D) or with (E) irradiation (**P < 0.01). The impact of knocking down MEG3 on BNIP2 mRNA levels without (F) or with (G) irradiation. H The impact of knocking down MEG3 on BNIP2 protein levels in the presence or absence of irradiation (**P < 0.01, NS, no significance); MEG3 is capable of reversing miR-20b-mediated BNIP2 downregulation in the absence (I) or presence (J) of irradiation (**P < 0.01). B–J n = 3. B–G were performed by two-tailed unpaired T-test. H–J were followed by one-way ANOVA.

Fig. 7. miR-20b protects against radiation-induced injury in BNL CL2 cells through targeting BNIP2.

A A dual-luciferase reporter assay was used to probe the impact of miR-20b on the activity of wild-type or mutant BNIP2 (**P < 0.01; NS, no significance). The impact of overexpressing miR-20b on BNIP2 mRNA and protein levels within BNL CL2 cells without (B) or with (C) irradiation. (**P < 0.01). D The impact of knocking down miR-20b on the mRNA and protein levels of BNIP2 in BNL CL2 cells that were or were not irradiated (**P < 0.01, NS no significance). E A CCK-8 assay was used to assess the effects of miR-20b inhibition on BNL CL2 cell viability following miR-20b inhibition and irradiation (**P < 0.01). F Hoechst staining was used to assess the impact of miR-20b inhibition on BNL CL2 cell apoptosis following irradiation. B–D, n = 3; A, E, n = 5; scale bar: 20 µm. B–E were performed by two-tailed unpaired T-test. D was also followed by one-way ANOVA.

Fig. 8. YTHDC1 plays a role in RILI in mice through the identification of m⁶A-modified MEG3.

A Schematic overview of the preparation of a YTHDC1-knockdown mouse model. B Hepatic MEG3 methylation modification levels were measured following YTHDC1 knockdown in mice without or with radiation (**P < 0.01). C The impact of knocking down YTHDC1 on hepatic YTHDC1 protein levels was assessed in mice that were or were not irradiated (**P < 0.01) D The impact of silencing YTHDC1 on the hepatic protein levels of METTL3 was assessed in mice that were or were not irradiated (**P < 0.01); E. The impact of silencing YTHDC1 on the hepatic levels of miR-20b was assessed in mice that were or were not irradiated (**P < 0.01); F The impact of silencing YTHDC1 on the hepatic MEG3 levels was assessed in mice that were or were not irradiated (**P < 0.01); G The effects of silencing YTHDC1 on BNIP2 protein in the liver of mice that were or were not irradiated (**P < 0.01); H Pathological changes in murine liver tissue were assessed via H&E staining following the knockdown of YTHDC1 and irradiation-induced damage. B–G n = 8; scale bar: 20 µm. B was performed by two-tailed unpaired T-test. C–G were followed by one-way ANOVA.

Fig. 9

YTHDC1-dependent METTL3-mediated m⁶A modification of MEG3 early during RILI and consequent regulation of the miR-20b-mediated inhibition of BNIP2.