Supplementary Fig. 1
Expression of miR-34a in transfected H9c2 cells. miRNA expression in H9c2 cells was measured by real-time polymerase chain reaction using TaqMan probes after treatment. miR-34a expression of H9c2 cells transfected with negative control (NC), miR-34a inhibitor, or miR-34a mimic. All data are expressed as mean±standard deviation. aP<0.05 vs. NC, bP<0.05 vs. miR-34a inhibitor.
dmj-44-173-s002.pdf
Supplementary Fig. 3
Proposed regulatory mechanisms of cardiac microRNAs in cardiomyocytes. Granulocyte-colony stimulating factor (G-CSF) treatment inhibits miR-34a expression, which increases B-cell lymphoma 2 (Bcl-2) protein expression, there by blocking cardiomyocyte apoptosis. DM, diabetic cardiomyopathy model.
dmj-44-173-s004.pdf
Fig. 1Experimental protocol. Normal rats (n=8) were fed a standard diet and diabetic rats (n=15) were fed with a high-fat diet for 7 weeks. At 13 weeks of age, the diabetic rats were intraperitoneally injected with streptozotocin (STZ; 30 mg/kg). At 14 weeks of age, diabetic rats were randomly allocated to treatment with either saline (200 µg/kg/day, n=7) or granulocyte-colony stimulating factor (G-CSF; 200 µg/kg/day, n=8). Functional and histological analysis were performed at 14 and 18 weeks.
Fig. 2Effects of granulocyte-colony stimulating factor (G-CSF) on cardiac function. (A) Left ventricular ejection fraction (LVEF). (B) Peak velocity of the early diastolic filling wave (E velocity). (C) Early mitral annulus velocity during the diastolic phase (E' velocity). (D) the ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E'). DM, diabetic rat model. aP<0.05 vs. normal, bP<0.05 vs. DM/G-CSF.
Fig. 3Granulocyte-colony stimulating factor (G-CSF) improves fibrosis and apoptosis in a rat model of diabetic cardiomyopathy. (A) Representative images of Masson's trichrome (MT) staining of heart tissue at 4 weeks after treatment in each group (×200). (B) Representative photomicrographs showing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay staining in the myocardium at 4 weeks after treatment in each group (×200). Scale bar=100 µm. (C) Results of quantitative analysis of collagen area as a ratio of fibrotic area to heart area. (D) Results of quantitative analysis of apoptotic cells. All data are expressed as mean±standard error (n=8 per group). DM, diabetic rat model. aP<0.05 vs. normal, bP<0.05 vs. DM/saline.
Fig. 4Granulocyte-colony stimulating factor (G-CSF) regulates cardiac microRNAs (miRNAs) in diabetic myocardium and H9c2 cells under high glucose (HG) condition. miRNA expression was measured in myocardium and H9c2 cells by real-time polymerase chain reaction using TaqMan (Applied BioSystems) probes after treatment. (A) miR-34a expression of myocardium at 4 weeks after treatment. miR-34a expression was significantly decreased in G-CSF treated rats compared to saline treated rats. All data are expressed as mean±standard deviation. (B) miR-34a expression of H9c2 cells after treatment. miR-34a expression was significantly decreased in G-CSF treated H9c2 cells under HG condition. All data are expressed as mean±standard deviation. (C) Expression of candidate miRNAs was measured in the myocardium. miR-15, miR-23a, miR-21, miR-23a, and miR-320 expression was not different after treatment with G-CSF. DM, diabetic rat model; NG, normal glucose; HG, high glucose. aP<0.05 vs. normal, bP<0.05 vs. DM/saline, cP<0.05 vs. NG, dP<0.05 vs. HG.
Fig. 5Anti-apoptotic effect of granulocyte-colony stimulating factor (G-CSF) on H9c2 cells can be mediated by miR-34a mimic or miR-34a inhibitor under high glucose (HG) condition. The apoptosis rate was measured by flow cytometry using annexin V/PI staining. (A, B) Flow cytometric analysis of H9c2 cell apoptosis. Apoptotic cells were significantly decreased in G-CSF treated H9c2 cells under HG condition. (C, D) Quantitative flow cytometry of H9c2 cells treated with HG and G-CSF and transfected with miR-34a inhibitor or miR-34a mimic. G-CSF treatment did not reduce apoptosis when cells were transfected with miR-34a mimic. All data were expressed as mean±standard error of the mean (n=5 per group). NG, normal glucose; FITC, fluorescein isothiocyanate. aP<0.05 vs. NG group, bP<0.05 vs. HG group, cP<0.05 vs. miR-34a inhibitor group.
Fig. 6B-cell lymphoma 2 (Bcl-2) is directly targeted by miR 34a. TargetScan software was used to predict the potential miR 34a binding site in the 3'-untranslated region (3'-UTR) of Bcl-2. (A, B) Representative Western blot analysis and quantitative analysis demonstrated that Bcl-2 protein levels was decreased in diabetic myocardium and H9c2 cells under high glucose (HG) condition. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as the control. (C) Representative Western blot analysis and quantitative analysis demonstrated that Bcl-2 protein levels was decreased by transfection with miR-34a mimic in H9c2 cells. GAPDH was used as the control. (D) Predicted pairing of target region (top) and miR-34a-5p (bottom). Dual luciferase assays were used to detect luciferase activity. Cells were co-transfected with pGL4-Bcl2-3'-UTR firefly luciferase expression construct and pRL-TK Renilla luciferase expression construct together with either miR-34 inhibitor or miR-34a mimic. All data are expressed as mean±standard deviation. DM, diabetic rat model; G-CSF, granulocyte-colony stimulating factor; NG, normal glucose. aP<0.05 vs. normal or NG, bP<0.05 vs. DM/saline or HG, cP<0.05 vs. negative control, dP<0.05 vs. negative control, eP<0.05 vs. miR-34a mimic.