MicroRNA-21, Induced By High Glucose, Modulates Macrophage Apoptosis

BackgroundMicroRNAs (miRNAs) are small, endogenous, noncoding RNAs of about 22 nucleotides that have a broad impact on gene expression through translational repression or post-transcriptional suppression. So far, miRNAs have been shown to play crucial roles in various diseases, such as cancer, infectious diseases, diabetes and cardiovascular diseases, etc. Recently, the microRNA-21 (miR-21) which was reported to be involved in many forms of cardiovascular diseases has started to attract the attention of researchers. The expression of miR-21 was significantly up-regulated in atherosclerotic arteries. However, the mechanisms of miR-21 involved in atherosclerosis remain to be elucidated. miR-21 is shown to target and down-regulate programmed cell death 4 (PDCD4) and promote cell proliferation. Moreover, overexpression of miR-21 represses normal apoptotic signaling but inhibition of miR-21 increases cell apoptosis. Therefore, cell apoptosis regulated by miR-21 may be a potential mechanism of atherosclerosis and miR-21 may be involved in macrophage apoptosis. We hypothesize that miR-21 can resist macrophage apoptosis induced by high glucose through PDCD4. In the present study, to investigate the effect of miR-21 on macrophage apoptosis, Raw 264.7 macrophages were stimulated with high glucose and transfected with anti-miR-21 inhibitor, then the expression of miR-21 and PDCD4 and cell apoptosis were detected.Objectives1. To investigate the expression of miR-21 on Raw 264.7 macrophage which were stimulated with high glucose.2. To investigate the possible mechanism of the protect effect of miR-21 on macrophage apoptosis.Methods1. Cell culture and treatmentThe murine monocytic cell line, Raw264.7, was routinely maintained in DMEM (5,5mM D-glucose) supplemented with 10% fetal bovine serum (FBS) and cultured at 37℃ in a water-saturated containing 5% CO2 atmosphere. Cells were passaged every 2-3 days to maintain exponential growth. Treatment of Raw264.7 macrophages with 5.5 mM glucose (normal glucose, NG),25 mM glucose (high glucose, HG), or 25 mM mannose (osmotic control, OC) were performed in serum-free media.2. qRT-PCR analysis of miR-21 and PDCD4 mRNA expressionTotal RNA was extracted from treated cells using the mirVana miRNA Isolation Kit (Ambion, Inc., Austin, TX) or Trizol (Invitrogen, Carlsbad, CA, USA). For analysis the expression of miR-21,10 ng total RNA was reverse transcribed into cDNA by MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA). Real-time PCR was performed using a standard TaqMan PCR protocol according to manufacturer’s protocols (Applied Biosystems), then the relative miR-21 expression level was calculated by the 2(-△△CT) method and normalized to the expression of U6. Relative levels of PDCD4 mRNA were examined by SYBR green real-time quantitative reverse transcription-PCR (qRT-PCR) (Applied Biosystems) and normalized to β-actin. The specific primers as follows:PDCD4:forward primer 5’-GGT GGA TGT GAA AGA TCC AAA-3’, reverse primer 5’-CAT CCA GGG GCA AAA CTA CA-3’; β-actin:forward primer 5’-CAA CTT GAT GTA TGA AGG CTT TGG T-3’, reverse primer 5’-ACT TTT ATT GGT CTC AAG TCA GTG TAC AG-3’.3. Oligonucleotide transfectionFor miR-21 inhibition, cells were plated in 12-well plates at 3×105 cells/ml and incubated at 37℃ in a 5% CO2 atmosphere. Cells at 50% to 70% confluence were transfected with 60nM of anti-miR-21 inhibitor by use of Lipofectamine 2000 reagent (Invitrogen). Cells were incubated for 24 hr post-transfection before treatment with NG or HG. The anti-miRTM miRNA Inhibitor (P/N:AM17000) and the Negative Control (P/N:AM17010) were purchased from Applied Biosystems.4. Western blot analysisTotal protein from cells was extracted by use of RIPA buffer (1 mM MgCl2, lOmM Tris-HCl pH 7.4,1% Triton X-100,0.1% SDS,1% NP-40). Proteins were separated on NuPAGE 4%-12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membranes, and incubated with PDCD4 antibody (Santa Cruz Biotechnology, CA), caspase-3 antibody or β-actin antibody (Abcam, Cambridge, UK), then horseradish peroxidase-conjugated secondary antibody (Abcam). Blots were developed with use of Supersignal West Dura Extended Duration Substrate (Perbio, Cheshire, UK). Images were captured by use of a Chemigenius imaging system (Syngene, Cambridge, UK).5. Flow cytometry analysis of cell apoptosis by PI-Annexin Ⅴ stainingCells plated in 12-well plates were transfected with anti-miR-21 inhibitor or negative control for 24 h, and then treated with NG or HG for 48h. The apoptosis ratio was analyzed with Annexin V FITC Apoptosis Detection Kit (Bipec Biopharma Corporation, USA) on a BD FACSCalibur TM system (Becton Dickson, San Jose, CA, USA) according to the manufacture’s instructions. Tests were repeated in triplicate.Results1. To examine whether the expression of miR-21 in macrophages was regulated by high glucose, Raw264.7 macrophages were treated with 5.5 mM glucose (NG), 25mM glucose (HG) or 25mM mannose (OC) for various times. TaqMan real-time PCR showed the expression of miR-21 mRNA significantly upregulated by HG, but reduced by OC. HG increased miR-21 expression in a time-dependent manner compared to the NG control cells. miR-21 expression peaked at 6h, and began to reduce at 12h.2. Since cell apoptosis increased with increased miR-21 expression, we investigated the role of miR-21 on high-glucose-induced macrophage apoptosis. Cells were transfected with anti-miR-21 inhibitor or negative control and incubated for an additional 48 h with NG or HG. Apoptosis of cells was detected by flow cytometry. miR-21 mRNA expression was significantly decreased after anti-miR-21 inhibitor transfection. The results of flow cytometry analysis showed that apoptosis of macrophages transfected with anti-miR-21 inhibitor was significantly higher than in those transfected with negative control incubated with NG. In addition, miR-21-inhibited cells treated with HG showed an obvious increase in apoptosis compared to HG-treated negative control cells or cells with miR-21 inhibition alone. The results indicated that miR-21 had a protective effect against the high-glucose-induced macrophage apoptosis.Western blot analysis showed an increased activated caspase-3 protein level in miR-21-inhibited cells as compared with control cells, an increased level with HG-treated miR-21-inhibited cells as compared with HG-treated controls, and an increased level as compared with miR-21-inhibited alone.3. Raw264.7 macrophages were treated with NG, HG or OC for various times, and then the expression of PDCD4 mRNA was determined. As shown in Figure 4A, macrophages treated with HG showed markedly reduced the expression of PDCD4 mRNA at 12 h, which was increased beginning at 24 h compared to NG control cells; however, PDCD4 mRNA expression was significantly increased with 6-h high-osmolarity treatment and began to decrease at 12 h, but the differences were not significant. PDCD4 protein level was also significantly reduced with HG treatment for 12 h.4. To verify PDCD4 as a target gene of miR-21 in high-glucose-incubated macrophages, the anti-miR-21 inhibitor was transfected into macrophages to inhibit miR-21 expression. With NG treatment, the mRNA and protein expression of PDCD4 in macrophages transfected with anti-miR-21 inhibitor was higher than in those transfected with negative control, which indicated that in macrophages, miR-21 inhibition promotes PDCD4 expression and that PDCD4 is the target gene of miR-21. With HG treatment, PDCD4 expression of miR-21-inhibited macrophages was still higher than in cells transfected with negative control, but lower than for miR-21-inhibited cells followed by treated with NG, which indicates that high glucose may reduce PDCD4 expression of macrophages via a miR-21 pathway.ConclusionsIn conclusion, high glucose induces the expression of miR-21 in Raw264.7 macrophages. miR-21 participates in macrophage apoptosis induced by high glucose via PDCD4.BackgroundCardiovascular disease was the leading cause of death in our country. Recently,the incidence and mortality of coronary heart disease was gradually increased. Many studies showed that the elevation of serum total cholesterol(TC) and low-density cholesterol level was one of the independent risk factor of coroonary heart disease and ischmic stroke. hcrcforc,should pay more attentioon to the prcvsontioon and treatment of hypercholesterolemia patients.The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or "Latins" e agents that ibit the rate-limiting enzyme of cholesterol synthesis in the liver,thereby decreasing the hepatic production of cholesthrol,increasing the expression of low-density lipoprotein receptors, and lowering the circulating levels of low-density lipoprotein cholesterol. Although it is widely accepted that the majority of clinical benefit obtained with statins is a direct result of their lipid-lowering properties, these agents appear to display additional cholesterol independent or ’pleiotropic’ effects on various aspects of cardiovascular disease. These effects include improving or restoring endothelial flinctioon,enhancing the staWlity of atherosclerotic plaques,decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response in the vascular wall.The incidence of atrial fibrillation was increased in patients with hypcholesterolemihe left atrium through the three fiinctioons modulates left ventricular onlling and cardiovascular performance—-reservoir,conduit and booster pximp function.Several studies showed that independent of cardiac translation and tethering effect,strain/strain rate imaging(SRI) enables quantitative measurement of leatrial flmction in patients with hypertension,metabolic syndrome and obesity.In our study,the left atrial fUnctioon of hypercholesterolemia patients by strain/SRI was studied. And we investigated the impact of pitavastatin on lefi atrial fonction of hypercholesterolemia patients.Objectives(1)To investigate the left atrial function of hypercholesterolemia patients by strain/SRI.(2)To investigate the impact of pitavastatin on left atrial function of hypercholesterolemia patients.MethodsThe experimental design was a double-blind randomized study. 40 patients with hypei:cholesterolemia weire enrolled in our study. The patients weie further vided into 1 mg dose group or 2mg dose group accordiong th the dose of pitavastatin. The diagnostic criteria of hypercholesterolemia was: fasting serum thtal cholesterol(TC)5.72mmol/LD-C<12.7mmol/L and/or fasting low-density lipoproteion cholesterol(LDLC) 3.64mmol/L5LDLC￡6.5mmol/L。We also enrolled 30 age- and sex-matched normal subjects.All subjects underwent thorough review of their medical history, clinical examination and electrocardiography(BCG) for deteclioon of clinical events. And all subjects were in sinus rhythm. A commercially available trasound machine(Simeoon5 Sequoia c512) with a 2-5-MHz variable-frequency transducer was used for all echocardiographic studies. All echocardiographic data were measured using the offline workstatioon of Syngo US Workplace.Statistical analyses involved use of SPSS 17.0. Data are piesented as mean ± SD for continuous variables and as proportions for categorical variables.Independent-samples Student,s t thsts was used to assess differences ion continuous variables between 2 groups. Categorical variables were analyzed by the chiquare test. The comparison of the left atrial fimction between the two fferent doth groups was analyzed by general Knear model analysis of variance. The independent correlation of LA fbnction wmetabolic factors and LV fiinction was investigated by partial correlation analysis. Variables with P < 0.10 in univariate analysis wee entered into the multivariate model with a stepwise regression analysis.Results1.There was no significant difference on gender,age, height, waist-to-hdp rate, pulse pressure,heart rate,ALT,AST, rGGT,AKP,AG,Tbil,HDIv-C and BUN between normal control group with hypercholesterolemia group. Significant differences were showed on weight, BMI, waist circumference, hip circumference, systolic blood pressure,diastolic blood pressure,mean arterial pressure,TP ALB,GLB,TC,TG,LDI-C,GLU and Cr between the two groups.(P<0.05).2.Compared with normal control group, the traditional echocardiography data of hypercholesterolemia group showed no significant difference。3.Strain/strain rate technique was used to assess the left atrial function. Patients with hypercholesterolemia showed significantly decreased mean strain, mean peakstolic SR SR) and mean peak early diastolic SSR_)(P<0.05 for aU),wkh no difference in mean peak late astolic S(ASR).4.After treating with pitavastation for 8 weeks, hypercholesterolemi patients showed significantly lower TC,TG and LDLC level。5.Patients with hypercholesterolemia showed increased mean peak early diastolic SS(P<0.05 fbr all) after treating with pitavastatin for 8 weeks, withono difference in mean strain,mean peak systolic S(SSR) and mean peak latethastolic SR(ASR). Further comparison of pitavastatin calcium 1 mg and 2mg doth group, left atrial function showed no significant difference.Conclusions1. In cholesterolemia patients, imagiong of strain and SR can be used th show decreased LA reservoir and conduit functions as well as maintaioned booer ftmction.2,After treating with pitavation for 8 weeks,hypercholesterolemia patieonts showed significantly lower TC,TG and LDL-C level。3.Patients with hypercholesterolemia showed increased conduit nctions afttreating with pitavastatin for 8 weeks. Further comparison of pitavastatin calcium1 mg and 2mg dose group, left atrial function showed no signicant difference.