| Objects:To observe the role of mi R-195 in cardiac fibrosis, and further research whether mi R-195 by targeting Chek1 inhibits cardiac fibroblasts(CF) proliferation and the transdifferentiation into cardiac myofibroblasts(CMF).Methods:(一) Cardiac fibroblasts culture: 1. Rat neonatal cardiac fibroblasts were isolated from the ventricles of 1–3 day old SD rats. Ventricles were minced and digested in 4mg/ml collagenase and 6mg/ml pancreatin containing solution at 37 °C. Neonatal cells were pre-plated for 3h on uncoated culture discs, during which cardiac fibroblasts rapidly adhered to the dishes. After pre-plating the medium containing cardiomyocytes was removed and the attached cardiac fibroblasts were washed and further cultured in CF DMEM at 37 °C and 5% CO2. These cells were referred to as passage 0(P0) cardiac fibroblast. Next day cardiac fibroblast were subcultured(1:3 dilution) to passage 1(P1). Subsequently, cells were split every 4 days and cultured up to passage 3(P3) after which the vast majority of cells acquired a myofibroblast-like phenotype. 2. The transdifferentiation into myofibroblasts was analysised by Real-time PCR, western blot and immunofluorescence assay.(二) The role of mi R-195 in cardiac fibrosis: 1. Real-time PCR analysised the m RNA level of mi R-195 in P1, P2, P3 generation cells; 2. Cells were transfected at P1 with mi R-195 agomir or mi R-195 antagomir. After 48 h cells were harvested for Real-time PCR, western blot and immunofluorescence assay. Real-time PCR analysised the m RNA level of Chek1, Cdc2 a, Birc5, Nusap1 and Spag5 and western blot detected the expression of Chek1.(三) The role of Chek1 in cardiac fibroblasts proliferation and the transdifferentiation into myofibroblasts: Cells were transfected at P1 with si RChek1. After 72 h cells were harvested to evaluate the m RNA level of Chek1. Real-time PCR and immunofluorescence analysised cell proliferation and the transdifferentiation.(四) Data are presented as mean±SEM(standard error of the mean) and reflect duplicate or triplicate measurements from at least two separate cell isolations. Two-way ANOVA was performed to compare values in multiple group experiments with two main treatments using SPSS 17.0. Differences were considered significant at p< 0.05.Results:(一) Neonatal rat cardiac fibroblasts cultured on rigid substrate showed progressive fibroblast to myofibroblast differentiation: Indeed, from passage 1(P1) to passage 3(P3) expression of the myofibroblast marker α-SMA markedly increased at the m RNA level(p<0.05).(二) Upregulation of mi R-195 inhibited cardiac fibroblasts proliferation and the transdifferentiation into myofibroblasts: 1. The m RNA level of mi R-195 increased from passage 1(P1) to passage 3(P3). 2. The m RNA level of mi R-195 increased in the group of mi R-195 agomir, while declined in the group of mi R-195 antagomir. 3.The EDU and Ki67 positive cells and the level of PCNA, α-SMA were significantly reduced following mi R-195 agomir treatment, while increased following mi R-195 antagomir treatment. 4. Mi R-195 negatively regulated cell cycle genes Chek1, Cdc2 a, Birc5, Nusap1 and Spag5 m RNA level.(三) Mi R-195 inhibited cardiac fibroblasts proliferation and the transdifferentiation into myofibroblasts by negatively regulating cell cycle gene Chek1: 1. Chek1 expression in the si RChek1 group was significantly lower than the control group. 2. The EDU positive cells and the level of α-SMA expression in si RChek1 group were significantly lower than the control group and the mi R-195 antagomir and si RChek1 treatments group(p< 0.05), while no statistical difference between the si RChek1 group and the co-transfection group.Conclusions: Upregulation of the mi R-195 plays a role in arresting cardiac fibroblasts proliferation and the transdifferentiation into myofibroblasts, while the effect maybe by negatively regulating cell cycle gene Chek1. |
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