Fig 2: Exosomal miR-30b-5p promoted angiogenesis through suppressing GJA1. (A-B) Rescue experiments, the expression of GJA1 in RNA (A) and protein (B) level was detected after co-incubation of hypoxic BxPC-3 derived exosomes and miR-30b-5p inhibitor. (C-D) Rescue experiments, the tube formation and endothelial cell migration were tested after co-transfection of miR-30b-5p mimic and overexpression vector of GJA1. (E) The relative expression of total miR-30b-5p in plasma between PDAC and healthy subjects. (F) The correlation for expression of total miR-30b-5p between peripheral blood plasma and portal vein plasma. (G) ROC plot showing the diagnostic value of total miR-30b-5p in plasma to distinguish PDAC and healthy subjects. (H) The relative expression of exosomal miR-30b-5p in plasma between PDAC and healthy subjects. (I) The correlation for expression of exosomal miR-30b-5p between peripheral blood plasma and portal vein plasma. (J) ROC plot showing the diagnostic value of exosomal miR-30b-5p in plasma to distinguish PDAC and healthy subjects.

Fig 3: The mechanism of miR-130a aggravating mitochondrial dysfunction in myocardial IRI.In myocardial IRI, miR-130a is up-regulated and the increase of miR-130a can aggravate mitochondrial dysfunction by suppressing mitochondrial function and mitochondrial quality control system, which are represented by the cell viability, ATP production, MMP, and mitochondrial fusion decrease, and the cell apoptosis and ROS content increase. MiR-130a appears to be critically involved in FUNDC1-mediated mitophagy by down-regulating the mitochondria-related gene GJA1. In addition, the reduction of GJA1 might suppress the binding of FUNDC1 to LC3 by reducing FUNDC1 dephosphorylation, and further suppress mitophagy. Therefore, the depression of the FUNDC1-mediated mitophagy contributes to the accumulation of damaged mitochondria and the promotion of cellular apoptosis.

Fig 4: GJA1 was required for miR-130a to aggravate mitochondrial dysfunction in myocardial HRI.A The levels of GJA1, FUNDC1, PARKIN, BNIP3, and LC3 were measured by Western blot and quantified. n = 3 in each group. B The mRNA level of GJA1 expression was detected by qRT-PCR. n = 3 in each group. C The co-localization experiments were performed to observe mitophagy activity. Cells were stained with Mito-tracker (green) and Lyso-tracker (red). Scale bars, 50 μm and 10 μm in magnified. D The quantitative analysis of the co-localization of lysosome and mitochondria was assessed by the Pearson’s correlation coefficient. Data were expressed as mean ± SD; Statistical significance by two-tailed Student’s t-test, *P < 0.05, **P < 0.01 vs. Ctrl NC; #P < 0.05, ##P < 0.01 vs. HR NC; $$P < 0.01 vs. Ctrl miR-130a KD; &&P < 0.01 vs. HR miR-130a KD.

Fig 5: GJA1 protected myocardial cells by decreasing cell apoptosis, enhancing cell viability & ATP production rate & oxidative phosphorylation, protecting mitochondrial membrane potential & activating mitophagy.A, B Apoptosis in H9c2 cells was measured by Flow Cytometry under normoxia or HR conditions with GJA1 OE. n = 3 in each group. C Cell viability was determined by using a cell counting kit-8 under normoxia or HR conditions with GJA1 OE and absorbance was measured at 450 nm. n = 16 in each group. Relative cell activity (%) = [(As-Ab)/(Ac-Ab)] ×100%. As: absorbance of the experimental group; Ab: absorbance of the blank group; Ac: absorbance of the Ctrl NC group. APC: allophycocyanin; FITC: Fluorescein Isothiocyanate. D Total ATP production rates were measured by Agilent Seahorse analyzer under normoxia or HR conditions. The ECAR and OCR were measured under basal conditions. By obtaining these data under basal conditions and after serial addition of Rotenone/antimycin A, total cellular ATP Production Rates could be measured in real-time. n = 10 in each group. The results of seahorse tests were normalized to the value per 5000 cells. E Mitochondrial respiration was measured including ATP production, maximal respiration, Proton leak, spare respiratory capacity, and basal respiration by Agilent Seahorse analyzer under normoxia or HR conditions. n = 10 in each group. The results of seahorse tests were normalized to the value per 5000 cells. FCCP: Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone. F–G MMP was observed with JC-1 staining. Nuclei were stained with DAPI. The cells were observed by laser confocal microscopy. Fluorescence intensity was measured by ImageJ software. Scale bars, 50 μm and 10 μm in magnified. n = 3 in each group. H The co-localization experiments to observe mitophagy activity. Cells were stained with mito-tracker (green) and lyso-tracker (red). Scale bars, 50 μm and 10 μm in magnified. I The quantitative analysis of the co-localization of lysosome and mitochondria was assessed by the Pearson’s correlation coefficient. J The levels of FUNDC1-mediated mitophagy-associated proteins were assessed by Western blot. n = 3 in each group. Data were expressed as mean ± SD; Statistical significance by two-tailed Student’s t-test, *P < 0.05, **P < 0.01 vs. Ctrl NC; #P < 0.05, ##P < 0.01 vs. HR NC.