Journal: Acta biochimica et biophysica Sinica

Article Title: METTL3-mediated m 6 A modification of pri-miRNA-31 promotes hypertrophic scar progression.

doi: 10.3724/abbs.2025033

Figure Lengend Snippet: Figure 4. miR-31-5p counteracts the effects of m6A methylation inhibitors on fibrosis and cell proliferation (A) miR-31-5p mimics partially reversed the m6A methylation inhibitor-mediated regulation of COL I/III and α-SMA in HSFBs. (B) Grayscale analysis of COL I/III and α-SMA levels. (C) miR-31-5p mimics partially attenuated the m6A methylation inhibitor-mediated regulation of cell proliferation in HSFBs. (D) Percentage of EdU-positive cells. (E) CCK-8 assay was used to measure the proliferative capacity of the cells from 24 to 96 h. (F) Percentage of EdU-positive cells. Groups: miR-31-5p-negative control (NC); miR-31-5p-NC+cycloleucine (CL, 40 μM) treatment; miR-31-5p-mimics; miR-31-5p-mimics+CL treat- ment. NS, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm.

Article Snippet: Cell treatment To inhibit m6A methylation, the cells were exposed to 40 μM cycloleucine (CL, HY-30008; MCE, Monmouth Junction, USA) for 48 h. Additionally, to inhibit METTL3, the cells were treated with 20 μM STM2457 (S9870; Selleck, Huston, USA) or a control (DMSO equivalent) for 24 h. Cell transfection Short hairpin RNAs (shRNAs) were subsequently cloned and inserted into the lentiviral vector pLKO.1 (Plasmid 10878; Addgene, Watertown, USA).

Techniques: Methylation, CCK-8 Assay, Negative Control

Journal: Acta biochimica et biophysica Sinica

Article Title: METTL3-mediated m 6 A modification of pri-miRNA-31 promotes hypertrophic scar progression.

doi: 10.3724/abbs.2025033

Figure Lengend Snippet: Figure 5. ZBTB20 is a downstream target gene of miR-31-5p (A) Heatmap of genes differentially expressed between the METTL3-knockdown groups and the control groups. (B) Volcano map of genes differentially expressed between the METTL3-knockdown groups and the control groups. (C) Venn diagram illustrating the overlap between the miRDB and TargetScan predictions and upregulated genes in METTL3-knockdown cells. ZBTB20, LITAF and SEPHS1 are downstream target genes of miR-31-5p. (D) Western blot analysis of ZBTB20, LITAF and SEPHS1 protein levels. (E) Grayscale analysis of ZBTB20, LITAF and SEPHS1. (F) The mRNA levels of ZBTB20, LITAF and SEPHS1 were detected by qRT-PCR. Groups: miR-31-5p-negative control (NC); miR-31-5p-NC+cycloleucine (CL, 40 μM) treatment; miR-31-5p-mimics; miR-31-5p-mimics+CL treat- ment. (G) The METTL3 inhibitor STM2457 can suppress the mRNA levels of ZBTB20 mediated by the miR-31-5p mimic. Groups: miR-31-5p- negative control (NC); miR-31-5p-NC+STM2457 treatment; miR-31-5p-mimics; miR-31-5p-mimics+STM2457 treatment. (H) Information on the mutated sequence of ZBTB20 used for Dual-Luciferase reporter gene assays. (I) Dual-luciferase reporter gene assays confirmed that ZBTB20 is a downstream target gene of miR-31-5p. (J) Knockdown of ZBTB20 increases the mRNA levels of COL I/III and α-SMA. ZBTB20-knockdown groups: si-ZBTB20-1# and si-ZBTB20-2#; control group: negative control (NC). NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell treatment To inhibit m6A methylation, the cells were exposed to 40 μM cycloleucine (CL, HY-30008; MCE, Monmouth Junction, USA) for 48 h. Additionally, to inhibit METTL3, the cells were treated with 20 μM STM2457 (S9870; Selleck, Huston, USA) or a control (DMSO equivalent) for 24 h. Cell transfection Short hairpin RNAs (shRNAs) were subsequently cloned and inserted into the lentiviral vector pLKO.1 (Plasmid 10878; Addgene, Watertown, USA).

Techniques: Knockdown, Control, Western Blot, Quantitative RT-PCR, Negative Control, Sequencing, Luciferase