ABSTRACT
Objective To investigate the effect of 5-AZA-2'-dC on Angiotensin Ⅱ (Ang Ⅱ)-induced cardiomyocyte hypertrophy.Methods Cultured cells derived from neonatal heart of rat were divided into 5 groups:normal control,hypertrophic group,5-AZA-2'-dC treatment group,and 5-AZA-2'-dC pretreatment group.Neonatal rat cardiomyocyte hypertrophic response was assayed by the size of cardiomyocytes and atrial natriuretic polypeptide (ANP) expressive level.The level of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),total calmodulin kinase Ⅱ (CaMK Ⅱ) and phospho-CaMK Ⅱ (p-CaMK Ⅱ) detected by Western blot.The intracellular calcium changes of cardiomyocytes were imaged by confocal fluorescent microscopy.Results Cells treated with Ang Ⅱ at 10-6 mol/L for 48 h were chosen as hypertrophic cardiomyocyte model.The mRNA expression and protein level of ANP were significantly decreased in the treatment and pretreatment groups compared with hypertrophic group.The protein level of SERCA2a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group.The protein level of SERCA2a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group,whereas phospho-CaMK Ⅱ showed an opposite change tendency.The time required for increasing and declining to half of the intracellular calcium peak value were both delayed in hypertrophic group,as the treatment and pretreatment groups showed shorter time required compared with hypertrophic group.Conclusion 5-AZA-2'-dC could inhibit Ang Ⅱ-induced cardiomyocyte hypertrophy which might be related to regulate SERCA2a expression.Increased SERCA2a expression may maintain the calcium homeostasis through shortening the time of transfer Ca2+ from the cytosol of the cell to the lumen of the sarcoplasmic reticulum.
ABSTRACT
Objective To study the effect of DNA methylation regulation on the toxic effect of paraquat on the sensitized V79 cells t pretreated with 5-aza-2'-deoxycytidine.Methods V79 cells were treated by 5-aza-2'-deoxycytidine (5-Aza-2'-dc) for 12h,which is a DNA methylation inhibitor,and then treated with paraquat for 12h.The morphological changes of V79 cells were observed by microscopy and the cell viability was determined by MTT assay and trypan blue staining method.Results Microscopic examination showed that the combination of 5-Aza-2'-dc and paraquat had stronger effect in inhibiting the growth of V79 cells(the cells became smaller and poorer adhensive ability) than single 5-Aza-2'-dc or paraquat.MTT assay showed that cell viability in the combination group (54.47 ± 3.04) ％ was significantly lower than the 5-Aza-2'-dc group (95.52 ± 0.90) ％ and paraquat group (89.68 ± 4.26) ％ (P＜0.05).Trypan blue staining assay showed that the death rate of ceils in the combination group (53.58 ± 1.57) ％ was significantly higher than the 5-Aza-2'-dc group (7.44 ± 2.31) ％ and paraquat group (12.90 ± 1.21) ％ (P＜0.05) Conclusion 5-Aza-2'-dc promotes V79 cells damage caused by paraquat.
ABSTRACT
Objective To study the reactive oxygen level and the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax after treatment of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-2'-dc) and paraquat in V79 cells.Methods Cultured V79 cells were divided into 5-Aza-2'-dc treatment group (group A),paraquat treatment group (group B),5-Aza-2'-dc and paraquat treatment group (group C,V79 cells were pretreated with 5-Aza-2'-dc for 12h followed by exposure to paraquat for 12h) and control group (group D).Reactive oxygen level in V79 cells was measured by DCFH-DA flow cytometry and expression of Bcl-2 and Bax was detected by Western blot.Results Reactive oxygen levels and expression levels of Bcl-2 and Bax in V79 cells were significantly different (P＜0.05) in 5-Aza-2'-dc and paraquat treatment group (group C),compared with 5-Aza-2'-dc treatment group (group A),paraquat treatment group (group B) and control group (group D).Expression levels of Bcl-2 and the ratio of Bcl-2 and Bax were lower while reactive oxygen levels and expression levels of Bax were higher in group C than in groups A,B and D.Conclusion 5-Aza-2'-dc regulates DNA methylation by the imbalancing the reactive oxygen metabolism and apoptosis,thus up-regulating the toxic effect of paraquat on V79 cells.
ABSTRACT
Objective To investigate the role and regulatory mechanism of micro RNA-9-3 (miR-9-3)in the pathogenesis of chronic lymphocytic leukemia.Methods Using the methylation specific PCR (MSP)technology to detect 8 cases of normal bone marrow tissue and peripheral blood,78 cases of bone marrow tissue came from the chronic lymphocytic leukemia pateints newly diagnosed and the methylation level of 7 kinds of leukemia cell line.Used Western blot to detected the NF-kappa B1 signal transduction pathway activation levels of methylation positive leukemia cell line.Results The miR-9-3 of normal control group were in the negative methylation status.Only I83-E95 and WAC3CD5+ were in positive methylation status in seven kinds of leukemia cell line (the positive of MSP was 28.6%);65 cases occurred miR-9-3 methylated in 78 of chronic lymphocytic leukemia patients (the positive of MSP was 83%).I83-E95 and miR-9-3 cells of WAC3CD5+ were in the methylation state when treatment with 5-nitrogen-2’-deoxidization cytidine (5-Aza2’Dc).Conclusion The abnormal methylation of miR-9-3 were usually seenin chronic lymphocytic leukemia,it could lead to abnormal hyperplasis in cancer cells.The methylation of miR-9-3 could inhibit the activation of NF-kappa B1 signal pathway suggested that it could sup-press the apoptosis of cancer cells through this pathways to trogered the progression of disease.The inhibitor of methylation could be induced the demethylation of leukemia cell lines,so it is possible that miR-9-3 maight be a new gene targets for the treatment of chronic lymphocytic leukemia.