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Objective:To observe the effect of mechanical vibration on the expression of microRNA-214-3p and serum interleukin-1β (IL-1β) in the broken ends of fractured bones in ovariectomized rats.Methods:Thirty 3-month-old female Wistar rats were randomly divided into a control group, a model group, and a vibration group, each of 10. An operation of ovariotomy was performed in those of the model and vibration groups to establish osteoporosis model. Five months later a model of mid femur fracture was made with animals in all the groups. Five days after their fracture, the vibration group received 20 minutes of whole-body vibration treatment at 35Hz for 5 days a week, while the control group and the model group received natural rearing without any additional intervention At 2 and 6 weeks after the operation, the Lane-Sandhu X-ray scoring system was used to evaluate the quality of fracture healing, and reverse-transcription polymerase chain reactions and enzyme-linked immunosorbent assays were used to detect miR-214-3p in the fractured bones and the serum levels of IL-1β.Results:At 2 weeks and 6 weeks after the operation the average growth score of the broken ends in the model group was significantly lower than that of the control group, while that of the vibration group was significantly higher than the model group′s average. Compared with the control group at the same time point, the average miR-214-3p content of the model and vibration groups was significantly higher 2 and 6 weeks after the surgery. Compared with the model group, the average level of miR-214-3p of the fractured ends of the vibration group was significantly lower at 6 weeks. Two and six weeks after the surgery, the average IL-1β of the model group was significantly higher than the control group′s average, and that of the vibration group was significantly lower.Conclusion:Mechanical vibration can promote osteoporotic fracture healing by inhibiting the expression of miR-214-3p and reducing the level of IL-1β at the broken ends of fractured bones.
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Objective@#To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1.@*Methods@#H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO2, repeat passage was made after cell density reached about 80%, The 5th to 8th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with β-actin as internal reference. The secretion of IL-1β and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene.@*Results@#(1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1β and IL-18 in the cell culture medium of each group: the content of IL-1β and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1β and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 ′UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1β and IL-18 in rescue experiment: the secretions of IL-1β and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05).@*Conclusion@#miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.
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Objective To investigate the expression of plasma microRNA-214 in patients with acute myocardial infarction (AMI) and its correlation with left ventricular remodeling (LVR). Methods A total of 158 AMI patients and 85 controls were selected from Hebei province Zhuozhou City Hospital. According to the left ventricular remodeling after PCI operation, AMI patients were divided into LVR group (n=41) and non LVR group (n=105). Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the plasma microRNA-214 levels, and the brain natriuretic peptide (BNP) and C-reactive protein (CRP) levels were detected by immunoassay. Pearson correlation analysis of plasma microRNA-214 levels and the correlation between CRP and BNP were analyzed in patients with LVR. The receiver operating characteristic (ROC) curve analysis of plasma microRNA-214, BNP and CRP were used to predict the efficacy of LVR in patients with AMI after PCI. Logistic regression model was used to analyze the relationship between the above indexes and LVR. Results Compared with the control group, left ventricular ejection fraction (LVEF) and left ventricular short axis shortening (FS) were significantly decreased in LVR group and non LVR group, and which were decreased more significantly in LVR group compared with those of non LVR group (P<0.05). The left ventricular end diastolic diameter (LVDD), left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV) were significantly increased in LVR group and non LVR group, and which were increased more significantly in LVR group than those in non LVR group (P<0.05). The levels of plasma microRNA-214, BNP and CRP were significantly higher in LVR group than those in non LVR group and control group (P<0.05). Correlation analysis showed that microRNA-214, BNP and CRP were positively correlated in patients with LVR (r=0.684 and r=0.405, P<0.01). The area under ROC curve (AUC) and 95%CI predicted by plasma microRNA-214 and BNP were 0.824 (0.757-1.015) and 0.785 (0.721-0.864) in patients with AMI and LVR, which were higher than CRP [0.716 (0.645-0.837), P=0.0167]. The sensitivity and specificity of plasma microRNA-214 in predicting the occurrence of LVR were 72.6% and 86.2% in patients with AMI, respectively. Logistic regression model analysis showed that plasma microRNA-214 and BNP were risk factors of LVR. Conclusion High expression of plasma microRNA-214 is found in LVR patients, which is expected to be the biological indicator for predicting the occurrence of LVR after PCI in patients with AMI.
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AIM:To investigate the effect of microRNA-214 ( miR-214) on cardiomyocyte hypertrophy and the expression of the potential target genes .METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular cardiomyocytes ( NMVCs) .Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Western blot , respectively .RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-induced hypertrophic cardiomyocytes .Dual lu-ciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly increased in the hypertro-phic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hypertrophy-re-lated genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .