Fig 2: miR-466f-3p-dependent AKT/GSK3β pathway was critical for radiation-induced EMT in MLE-12 cells. A The overlap of potential targets of miR-466f-3p predicted by Targetscan, miRmap, PITA, and miRanda databases. B KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for a panel of 367 overlapped targets of miR-466f-3p, and PI3K/AKT pathway was included among the top 10 enrichments. C, D Western blot analysis (C) and densitometric quantification (D) of p-AKT, p-GSK3β, and SNAIL in nonirradiated control and irradiated cells with or without AKT inhibitor (LY294002) at 1 h post-irradiation. E, F Western blot analysis (E) and densitometric quantification (F) of E-cadherin and Vimentin in irradiated cells with or without AKT inhibitor at 48 h post-irradiation. G-J Western blot analysis and densitometric quantification of p-AKT, p-GSK3β, and SNAIL (G, H), E-cadherin and Vimentin (I, J) in irradiated MLE-12 cells with scramble and miR-466f-3p mimic at 1 or 48 h post-irradiation. All the above western blot analysis used AKT, GSK3β, or GAPDH as the loading control. K Immunoflurescence staining for SNAIL (green) and DAPI (blue) in irradiated MLE-12 cells with scramble and miR-466f-3p mimic at 48 h post-irradiation. Data are presented as the mean ± SD. from three independent experiments, * p < 0.05

Fig 3: Ligustrazine inhibits hypoxia-induced PMC ECM deposition and EMT transition. (a) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α-SMA) in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. (b–g) qRT-PCR analysis of mRNA levels of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α-SMA) in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. Compared with the control, ∗P < 0.05 and ∗∗P < 0.01. (h–k) Immunofluorescence assay of mesothelial-related phenotypic biomarkers (E-cadherin, Cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail, α-SMA) in hypoxia-induced PMCs treated with 20 μM ligustrazine for 24 h (200x).

Fig 4: C-MET as a direct target of miR-466f-3p abrogated radiation-induced EMT via AKT/GSK3ß pathway in MLE-12 cells. A Seven candidates overlapped by AKT-pathway genes and potential targets predicted by Targetscan, miRmap, PITA, and miRanda databases. B Schematic illustration of the potential binding sites for miR-466f-3p in the 3’-UTRs of c-MET (upper). Dual-luciferase assay in H293T cells after co-transfected either wild-type (c-MET-wt) or mutant (c-MET-mut) vectors with miR-466f-3p mimic (lower). C Western blot and densitometric quantification for protein level of c-MET and GAPDH (for normalization) in irradiated MLE-12 cells with miR-466f-3p mimic and MLE-12 cells with miR-466f-3p inhibitor. D, E Western blot (D) and densitometric quantification (E) for protein level of c-MET, p-AKT, p-GSK3ß, SNAIL, E-cadherin and Vimentin in irradiated MLE-12 cells with or without siRNA against c-MET. AKT, GSK3ß, or GAPDH was used as the loading control. F Western blot and densitometric quantification for protein level of c-MET and GAPDH (for normalization) in c-MET expressing MLE-12 cells. G, H Western blot (G) and densitometric quantification (H) for protein level of p-AKT, p-GSK3ß, SNAIL, c-MET, E-cadherin and Vimentin in irradiated MLE-12 cells with or without mMSCs-Exo treatment and c-MET restoration at 48 h post-irradiation. AKT, GSK3ß, or GAPDH was used as the loading control. Data are presented as the mean ± SD. from three independent experiments, * p < 0.05

Fig 5: Ligustrazine suppresses hypoxia-induced PMC functions by activating PPARγ. (a, b) Western blot and qRT-PCR analysis of PPARγ levels in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. Compared with the control, ∗P < 0.05 and ∗∗P < 0.01. (c) Immunofluorescence analysis of PPARγ nuclear distribution in hypoxia-induced PMCs treated with 20 μM ligustrazine for 24 h (200x). (d) Flow cytometry analysis of PPARγ nuclear translocation in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h. (e) Western blot analysis of protein expression of profibrotic cytokines (VEGF and CTGF) in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h. (f, g) FDA staining assay of cell viability in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h (200x). Compared with the control, ∗P < 0.05 and ∗∗P < 0.01. Compared with the ligustrazine group, #P < 0.05 and ##P < 0.01. (h) Western blot analysis of protein expression of migration and adhesion-associated molecules (VCAM-1 and ICAM-1) in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h. (i) Western blot analysis of cytoskeleton protein (vinculin) expression in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h. (j) Western blot analysis of protein expression of FSP1 in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h. (k) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α-SMA) in hypoxia-induced PMCs treated with 20 μM ligustrazine and/or 1 μM GW9662 for 24 h.