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Review
. 2023 Nov 28;24(1):300.
doi: 10.1186/s12931-023-02606-z.

METTL3-mediated m6A RNA methylation induces the differentiation of lung resident mesenchymal stem cells into myofibroblasts via the miR-21/PTEN pathway

Yi Lu #  1 , Zeyu Liu #  1 , Yunjiao Zhang  1 , Xiuhua Wu  1 , Wei Bian  1 , Shan Shan  2 , Danrong Yang  3 , Tao Ren  4
Affiliations
Review

METTL3-mediated m6A RNA methylation induces the differentiation of lung resident mesenchymal stem cells into myofibroblasts via the miR-21/PTEN pathway

Yi Lu et al. Respir Res. .

Abstract

Background: The accumulation of myofibroblasts is the key pathological feature of pulmonary fibrosis (PF). Aberrant differentiation of lung-resident mesenchymal stem cells (LR-MSCs) has been identified as a critical source of myofibroblasts, but the molecular mechanisms underlying this process remain largely unknown. In recent years, N6-methyladenosine (m6A) RNA modification has been implicated in fibrosis development across diverse organs; however, its specific role in promoting the differentiation of LR-MSCs into myofibroblasts in PF is not well defined.

Methods: In this study, we examined the levels of m6A RNA methylation and the expression of its regulatory enzymes in both TGF-β1-treated LR-MSCs and fibrotic mouse lung tissues. The downstream target genes of m6A and their related pathways were identified according to a literature review, bioinformatic analysis and experimental verification. We also assessed the expression levels of myofibroblast markers in treated LR-MSCs and confirmed the involvement of the above-described pathway in the aberrant differentiation direction of LR-MSCs under TGF-β1 stimulation by overexpressing or knocking down key genes within the pathway.

Results: Our results revealed that METTL3-mediated m6A RNA methylation was significantly upregulated in both TGF-β1-treated LR-MSCs and fibrotic mouse lung tissues. This process directly led to the aberrant differentiation of LR-MSCs into myofibroblasts by targeting the miR-21/PTEN pathway. Moreover, inhibition of METTL3 or miR-21 and overexpression of PTEN could rescue this abnormal differentiation.

Conclusion: Our study demonstrated that m6A RNA methylation induced aberrant LR-MSC differentiation into myofibroblasts via the METTL3/miR-21/PTEN signaling pathway. We indicated a novel mechanism to promote PF progression. Targeting METTL3-mediated m6A RNA methylation and its downstream targets may present innovative therapeutic approaches for the prevention and treatment of PF.

Keywords: Lung resident mesenchymal stem cells (LR-MSCs); M6A methylation; METTL3/miR-21/PTEN pathway; Myofibroblast; Pulmonary fibrosis (PF).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
LR-MSCs differentiate into myofibroblasts in response to TGF-β1. A Morphologies of mouse LR-MSCs under a standard light microscope. Scale bar: 200 μm. B Flow cytometric analysis of surface markers (CD44, CD73, CD90, CD34, CD45, CD14 and CD19R) on LR-MSCs. C–E Adipogenic (oil red O staining), osteogenic (alizarin red staining) and chondrogenic (alcian blue staining) differentiation potential of LR-MSCs. F Cartilage nodules formed by LR-MSCs during chondrogenic differentiation. G–I Relative changes in the mRNA and protein levels of the indicated markers related to myofibroblast differentiation (α-SMA, type I collagen, and vimentin) in LR-MSCs at baseline and after TGF-β1 stimulation, as assessed by qRT‒PCR, WB assays and immunofluorescence analysis. β-Actin was used as a reference gene. Scale bars = 20 μm, **P < 0.01
Fig. 2
Fig. 2
TGF-β1-induced differentiation of LR-MSCs into myofibroblasts is dependent on METTL3-mediated m6A RNA methylation. A Relative m6A levels (arbitrary units) of LR-MSCs at baseline and after TGF-β1 stimulation. B, C The mRNA and protein expression levels of METTL3, METTL14, WTAP, FTO, and ALKBH5 in LR-MSCs at baseline and after TGF-β1 stimulation by qPCR and WB assays. D Relative m6A levels (arbitrary units) of LR-MSCs in the following four cases: control group, METTL3 silencing group, TGF-β1-treated group, and METTL3 silencing group treated with TGF-β1. E, G The mRNA and protein expression levels of myofibroblast markers (α-SMA, type I collagen, and vimentin) in LR-MSCs treated as described above. β-Actin was used as a reference gene. Scale bars = 20 μm, **P < 0.01
Fig. 3
Fig. 3
METTL3-mediated m6A RNA methylation is involved in TGF-β1–induced pulmonary fibrosis in the murine lung. A, B The m6A levels (arbitrary units) in mouse fibrotic lungs relative to normal lungs. C, D The mRNA and protein expression levels of METTL3 in mouse fibrotic lungs relative to normal lungs by qRT‒PCR and WB assays. E HE staining of lung tissues from mice exposed to bleomycin with or without METTL3 silencing. F The α-SMA, type I collagen, and Vimentin expression levels in lung tissues from mice exposed to bleomycin with or without METTL3 silencing. β-Actin was used as a reference gene. G, H The m6A levels and METTL3 levels in primary LR-MSCs isolated from mouse fibrotic lungs and normal lungs. *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
METTL3-mediated m6A RNA methylation promotes pri-miR-21 processing by DGCR8. A Prediction score distribution of the potential m6A modification sites along the pri-miR-21 sequence. B RIP-qPCR analysis of pri-miR-21 enrichment by METTL3, m6A and DGCR8 in LR-MSCs with or without METTL3 silencing. (C-D) qRT‒PCR analysis of pri-miR-21 (C) and miR-21 (D) expression levels in the METTL3 overexpression group or METTL3 silencing group compared to the control. E, F qRT‒PCR analysis of pri-miR-21 (E) and miR-21 (F) expression levels in the following four cases: control group, METTL3 silencing group, TGF-β1-treated group, and METTL3 silencing group treated with TGF-β1. U6 and β-Actin were used as reference genes. **P < 0.01
Fig. 5
Fig. 5
METTL3 is involved in TGF-β1-induced differentiation of LR-MSCs into myofibroblasts by targeting miR-21. A, B The mRNA and protein expression levels of myofibroblast markers (α-SMA, type I collagen, and Vimentin) in LR-MSCs with or without TGF-β1 treatment and miR-21 inhibitor. C, D The mRNA and protein expression levels of myofibroblast markers (α-SMA, type I collagen, and Vimentin) in LR-MSCs with or without METTL3 silencing, combined with or without treatment with TGF-β1 and miR-21 inhibitor. β-Actin was used as a reference gene. **P < 0.01
Fig. 6
Fig. 6
miR-21 inhibits PTEN expression in LR-MSCs. A qRT‒PCR analysis of miR-21 expression with the addition of miR-21 inhibitor or mimics in LR-MSCs. B, C qRT‒PCR and WB analysis of PTEN expression with the addition of miR-21 inhibitor or mimics in LR-MSCs. D Construction of the luciferase reporter system. E Dual-luciferase reporter assay to validate the direct binding between miR-21 and the PTEN 3′-UTR. F RIP-qPCR analysis of miR-21 and its interacting 3′ UTR of PTEN enrichment by Ago2. G, H The mRNA and protein expression levels of PTEN in LR-MSCs with or without TGF-β1 treatment and miR-21 inhibitor. β-Actin was used as a reference gene. **P < 0.01
Fig. 7
Fig. 7
miR-21 affects the TGF-β1-induced differentiation of LR-MSCs into myofibroblasts by regulating PTEN expression. A–C The mRNA and protein expression levels of myofibroblast markers (α-SMA, type I collagen, and Vimentin) in LR-MSCs with or without PTEN silencing, combined with or without treatment with TGF-β1 and miR-21 inhibitor. β-Actin was used as a reference gene. Scale bars = 20 μm, **P < 0.01
Fig. 8
Fig. 8
TGF-β1 induces the differentiation of LR-MSCs into myofibroblasts by modulating the METTL3/miR-21/PTEN pathway. A, B The mRNA and protein expression levels of PTEN in LR-MSCs with or without METTL3 silencing, combined with or without treatment with TGF-β1 and miR-21 mimics. C–E The mRNA and protein expression levels of myofibroblast markers (α-SMA, type I collagen, and vimentin) in LR-MSCs with or without METTL3 silencing, PTEN overexpression, and treatment with TGF-β1 and miR-21 mimics. β-Actin was used as a reference gene. Scale bars = 20 μm, **P < 0.01
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