| BackgroundRecently, numerals studies have indicated that microRNAs (miRNAs) widely participate in the regulations of many physiological and pathological processes.The researches of miRNAs have become hot spot in life science research area. MicroRNA-21(MiRNA-21) has been played close attention in cancer field because of its marked anti-apoptosis effect on cancer cells. Programmed cells death protein 4 (PDCD4) was confirmed as one of the important target genes regulated by miRNA-21. MiRNA-21 could process its anti-apoptosis effects on cancer cells through regulating the expression of PDCD4. Recent years, miRNA-21 has been found in vascular smooth muscle cells and cardiac myocytes also. MiRNA-21 have been participated in the processes of apoptosis, proliferation and oxidative stress by regulating the PDCD4 expression in vascular smooth muscle cells and cardiac myocytes, which indicated that miRNA-21 and its target gene PDCD4 possibly have played an important role in cardiovascular system. But the expression, regulation and function of miRNA-21 in vascular endothelial cells are still not clear. Under diabetic condition, high glucose can induce apoptosis and function damage of vascular endothelial cells to destroy the barrier function of vascular endothelial cells, which was a key factor for the development of atherosclerosis induced by diabetes. Up to now, there was few research about the relationship between the miRNA-21 and the apoptosis of vascular endothelial cells in diabetic condition.ObjectiveTo study the expression of miRNA-21 in vascular endothelial cells treated with high glucose and to explore whether miRNA-21 participates in the process of cell apoptosis in vascular endothelial cells induced by high glucose through regulating the expression of PDCD4. Methods(1) HUVECs were treated with D-Glucose in 5.5mM, 11 mM, 22 mM and 33 mM respectively for 48 hours. MiRNA-21, PDCD4, Bcl-2 associated X protein (Bax), B cell lymphoma/lewkmia-2 (Bcl-2) and Caspase-3 mRNA expressions were detected by Real-time PCR. The protein expressions of PDCD4, Bax and Bcl-2 were determined by Western blot.Caspase-3 activity was detected by spectrophotometer assay. Cell apoptosis was assayed by Hoechst33342/PI staining. The Viability of cell growth was estimated by 3-(4,5-Dimethyrthiazol-2-yl)-2,5--diphenyltetrazolium bromide (MTT) method.(2) MiRNA-21 knockdown in HUVECs:MiRNA-21 knockdown in HUVECs were performed with different concentrations of miRNA-21 inhibitor 2l-O-methyl-miRNA-21(2'OMe-miRNA-21) (3nM, 10 nM, 30 nM and 100 nM respectively) transfected by Iipofectamine? 2000 in OPTI-MEM? I reduced serum medium for 5 hours. Then cell medium was changed by DMEM with 33mM glucose and 10% fetal bovine serum (FBS) and HUVECs were cultured for 48 hours. MiRNA-21, PDCD4, Bax, Bcl-2 and Caspase-3 mRNA expressions were detected by Real-time PCR. The protein expressions of PDCD4, Bax and Bcl-2 were determined by Western blot. Caspase-3 activity was detected by spectrophotometer assay. Cell apoptosis was assayed by Hoechst33342/PI staining. The Viability of cell growth was estimated by MTT method.(3) MiRNA-21 overexpression in HUVECs:MiRNA-21 overexpression in HUVECs were performed with 10 nM and 30 nM miRNA-21 mimics transfected by Lipofectamine? 2000 in OPTI-MEM? I reduced serum medium for 5 hours. Then cell medium was changed by DMEM with 5.5mM or 33mM glucose and 10% FBS and HUVECs were cultured for 48 hours. MiRNA-21, PDCD4, Bax, Bcl-2 and Caspase-3 mRNA expressions were detected by Real-time PCR. The protein expressions of PDCD4, Bax and Bcl-2 were determined by Western blot. Caspase-3 activity was detected by spectrophotometer assay. Cell apoptosis was assayed by Hoechst33342/PI staining. The Viability of cell growth was estimated by MTT method.Results(1) With the increasing of glucose concentrations (5.5 mM> 11 mM,22 mM and 33 mM) , the levels of miRNA-21 expression in HUVECs were down-regulated in dose-dependent manner (p<0.01). The PDCD4 protein expressions were up-regulated (p<0.05), however there were no differences in PDCD4 mRNA expression in HUVECs treated with different concentrations of glucose (p>0.05). High glucose down-regulated the level of Bcl-2 mRNA and protein expression (p<0.05) and up-regulated the expression of Bax mRNA and protein in dose-dependent manner (p<0.05). The ratios of Bax/Bcl-2 mRNA or protein expression were increased significantly (p<0.01). High glucose increased the levels of Caspase-3 mRNA expression, Caspase-3 protein activity and cell apoptosis in dose-dependent manner (p<0.05), and reduced the levels of cell growth at the same time (p<0.05).(2) lOnM, 30nM and 100nM of 2' OMe- miRNA-21 significantly down-regulated the expression of miRNA-21 (p<0.01) and significantly up-regulated the expressions of PDCD4 protein in dose-dependent manner in HUVECs treated with 33mM glucose (p<0.01), however the PDCD4 mRNA expressions had not been effected by difference concentrations of 2' OMe-miRNA-21 (p>0.05).(3) 10nM, 30nM and lOOnM of 2' OMe- miRNA-21 down-regulated the expressions of Bcl-2 mRNA and protein (p<0.05), and significantly up-regulated the expressions of Bax mRNA and protein in dose-dependent manner in HUVECs treated with 33mM glucose (p<0.01). The ratios of Bax/Bcl-2 mRNA or protein expression were increased significantly (p<0.01). The levels of Caspase-3 mRNA expression, Caspase-3 activity and cell apoptosis were increased in dose-dependent manner also (p<0.01), and the levels of cell growth were reduced at the same time (p<0.05). (4) Both 10nM and 30nM miRNA-21 mimics significantly up-regulated the levels of miRNA-21 expression in HUVECs treated with 5.5mM or 33mM glucose (p<0.01). The expressions of PDCD4 protein were significantly down-regulated by lOnM and 30nM miRNA-21 mimics (p<0.01), however there was no differences of PDCD4 mRNA expression between lOnM and 30nM miRNA-21 mimics groups (p>0.05). Furthermore with the same dosage of miRNA-21 mimics, the change folds of miRNA-21 and PDCD4 protein expressions were higher in HUVECs treated with 33 mM glucose than cells with 5.5 mM glucose (p<0.05).(5) 30nM miRNA-21 mimics significantly up-regulated Bcl-2 mRNA and protein expression levels and down-regulated the expressions of Bax mRNA and protein in HUVECs with both 5.5mM or 33mM glucose (p<0.01). The ratios of Bax/Bcl-2 mRNA and protein expression were also reduced significantly (p<0.01). The levels of Caspase-3 mRNA expression, Caspase-3 activity and cell apoptosis were reduced significantly (p<0.01), and the levels of cell growth were increased at the same time (p<0.05). Furthermore with the treatment by 30nM miRNA-21 mimics, the change folds of Bax/Bcl-2 mRNA and protein expressions, Caspase-3 mRNA expressions, Caspase-3 activity, cell apoptosis and growth were lower in HUVECs treated with 33 mM glucose than cells with 5.5 mM glucose (p<0.05).Conclusions(1)High glucose down-regulates miRNA-21 expression and up-regulates PDCD4 protein expression in vascular endothelial cells. MiRNA-21 can regulate PDCD4 expression post-transcriptionally, which indicates PDCD4 is a target gene of miRNA-21 in vascular endothelial cells.(2) MiRNA-21 expression participates in the process of cell apoptosis in vascular endothelial cells induced by high glucose through regulating PDCD4 expression, and influences the proliferation of vascular endothelial cells. (3) MiRNA-21 expression can effect the balance of Bax and Bcl-2 system in vascular endothelial cells, which indicates both miRNA-21 and Bax /Bcl-2 system possibly participate in the apoptosis process of vascular endothelial cells induced by high glucose together. |
PDF Full Text Request