MicroRNA-21 Mediates High Phosphate-induced Endothelial Cell Apoptosis

Objective:Serum phosphate range normally is within 3.0-4.5mg/dL.As one of the most important nutrient of human beings,phosphate participates in many regulation and metabolism progress.Hyperphosphatemia,which means an increased level of serum inorganic phosphate,is associated with the increased morbidity and mortality of cardiovascular diseases.Some research has shown that elevated phosphate will advance the progress of cardiovascular diseases in patients with chronic kidney diseases.Taking too much phosphate in daily diet can also increase the mortality and morbidity of cardiovascular diseases among healthy people.Elevated phosphate will induce vascular smooth muscle cell transform to osteogenic cell and become vascular calcification at the end.Although some research have shown that high phosphate can induce endothelial cells apoptosis,there is not much research has been done to investigate the mechanisms underlying it.Some data have shown that microRNA can regulate the growth and apoptosis of cells,and the changes of its expression can induce some abnormal regulation that affect the outcomes of cardiovascular disease.This project has been studying about high phosphate induce endothelial cell apoptosis and how microRNA-21 is involving in this whole progress.Methods and materials:1.GroupingWe used mouse myocardial endothelial cells(MECs)in our project.After treated the cells with DMEM medium contained 0,0.1,1.0 and 5.0mmol/L phosphate,we did cell growth experiment and microarray.In order to confirm the apoptosis and protein expression,the cells were treated under two different conditions.The control groups were treated by normal DMEM medium contained 1.0mmol/L phosphate,and the experiment groups were treated by high phosphate DMEM medium contained5.0mmol/L phosphate.After 72 hours,we test the apoptosis,protein expression,RNA and microRNA expressions.In microRNA-21 signal experiment,we did cell transfection and set up 6 groups that contained control group,experiment group,miR-21 mimic group,mimic negative control group,miR-21 inhibitor group and inhibitor negative control group.After treated the cells with different conditions,we confirmed the apoptosis and protein expression.In ERK1/2 signal experiment,we set up 4 groups that contained control group,experiment group,ERK1/2 inhibitor group and inhibitor control group.After treated the cells for 72 hours,we test the changes of protein expression.2.xCELLigence cell growth curve to analyze cell growthWe use xCELLigence Real-time Cell Electronic sensing technic to analyze cell growth.Cells were treated in DMEM medium contained 0,0.1,1.0 and 5.0mM Pi.Cell growth was monitored every 1 hour.At the end of the experiment,cell growth curves were generated.3.Immunofluorescence microscopy to confirm cell apoptosisCells were treated with normal and high phosphate medium for 72 hours.We used In Situ Cell Death Detection Kit to label the cells and analyzed under a fluorescence microscope.4.Flow cytometry to test cell apoptosisCulture the cells with relative methods.Stain the cells with Annexin V-FITC Apoptosis Detection Kit.According to the kit handbook,added Annexin V-FITC and propidium iodide to each sample.Incubated the samples at room temperature for 5min in the dark.Analyzed the apoptosis by flow cytometry.The apoptotic cells were the ones that were positive of Annexin V-FITC and negative of PI.5.Affymetrix mouse gene expression array profiling to test the microRNAs expressionMEC cells were cultured in DMEM media containing 0,0.1,1.0 and 5.0mM Pi and treated for 72 hours.Total RNA samples were submitted to UNC micro-array facility for hybridization and data analysis.Significance analysis of microarray(SAM)was performed for microRNA profiling.6.Cell transfection experiment to exam apoptosis and PDCD4,NF-?B expressionAccording to the grouping,microRNA21 cell transfection was performed as the RNAiMAX kit recommended and treated with different medium for 72 hours.We use flow cytometry to test the apoptosis and western blot to test PDCD4 expression.7.EKR1/2 transit activation experiment to confirm ERK1/2 expressionCulture the cells with high phosphate medium,and treated for 0min,1min,5min,10min,30min and 60min.Test the changes of ERK1/2 and p-ERK1/2 expression by western blot.8.Western Blot to confirm PDCD4,PTEN,PARP,NF-?B expressionAfter culture the cells with respectively methods,do western blot.Ran for 30min,transferred for 1hr,and blocked for 1 hour by using 10ml TBS with 5%non-fat milk at room temperature.The primary antibodies were rabbit anti-cleaved caspase-3,rabbit anti-PDCD4,rabbit anti-PTEN,rabbit anti-PARP,rabbit anti-ERK1/2,rabbit anti-p-ERK1/2,rabbit anti-NF-KB(relA),rabbit anti-NF-KB1,rabbit anti-NF-KB2,mouse anti-?-actin,and the membranes were incubated at 4?overnight.After finish incubating the second antibody,the ECL select WB detection reagent was used to develop the membrane.ImageJ software was used to analyze the data.9.RT-PCR to test expression of RNA and microRNAFor the microRNA part,total RNA purification was done by miRNeasy Mini Kit,reverse transcription was done by using the miScript II RT kit and RT-PCR was done by using miScript SYBR Green PCR kit following the manufacturer's protocol.For the PDCD4,PTEN,PARP and NF-?B part,total RNA purification was done by RNeasy Mini Kit,reverse transcription to cDNA was done by iScript cDNA Synthesis Kit and RT-PCR was done by using SsoAdvanced Universal SYBR Green Supermix following the manufacturer's protocol.And use QuantStudio 6 Flex PCR machine to do RT-PCR by respectively RT-PCR steps.Results were analyzed by 2~-??CT??CT methods.10.ERK inhibitor experiment to confirm PDCD4 and NF-?B expressionTreated the cells under different grouping conditions,after 72 hours,test ERK1/2,p-ERK1/2,PDCD4 and NF-?B expression.11.Statistical AnalysisAll numerical data were presented as means±SD and the comparison between groups were analyzed by t-test.P value of less than 0.05 was considered as statistically significant.All data were analyzed using SPSS 19.0.Results:1.High phosphate will induce endothelial cell apoptosis(1)The xCelligence system showed:In the Pi 0mM medium,cells grew slowly.In the first 48 hours,the cells treated with Pi 5.0mM medium grew as fast as Pi 0.1mM and Pi1mM groups.On the third day,however,the Pi 5.0mM group showed downward deviation.(2)The results of immunofluorescence microscopy and flow cytometry showed:In high phosphate group,the apoptosis number of cells was more than that of normal phosphate group(p<0.05).(3)We did western blot to test the expression of cleaved caspase-3.Compared with normal medium group,the expression of cleaved caspase-3 is significantly higher in high phosphate medium group(p<0.05).2.High phosphate could upregulate the expression of microRNA-21(1)We used Affymetrix gene expression array profiling technic to test 547 microRNAs.Compared with the normal phosphate medium group,in the high phosphate medium groups,there were 9 microRNAs expression increased,and 65 of the microRNAs decreased.Among those 74 microRNAs,if we looked into the comparison between1.0mM and 5.0mM groups with 0mM and 1.0mM groups,there were 33 of them had significant changes.MicroRNA-21 expression was increased in high phosphate group.MicroRNA-145,-143,-143hg,-22,-5123 and-1938 expressions were all decreased in high phosphate medium.MicroRNA-125 and other 24 microRNAs were increased or decreased in both 0mM groups and 5.0mM groups.(2)We used RT-PCR to exam some highlighted microRNA expression.Compared with normal phosphate group,microRNA-21,-125 increased in high phosphate groups.While microRNA-142,-143 decreased in high phosphate groups(p<0.05).3.MicroRNA directly mediated high phosphate induced endothelial apoptosisTransfected the cells with microRNA-21 mimic and inhibitor and then treated them under different conditions.Compared with normal medium,the one added miR-21 mimic and treated with normal medium,and also the one treated with high phosphate medium had more apoptosis cells(p<0.05).Compared with high phosphate group,the one added miR-21 inhibitor and treated under high phosphate medium had less apoptosis cells(p<0.05).4.MicroRNA-21 regulated high phosphate induced PDCD4 downregulation(1)We used western blot to exam the target proteins(PDCD4,PTEN and PARP)of microRNA-21.Compared with normal medium,those target proteins all decreased in high phosphate medium(p<0.05).(2)Using RT-PCR to test the expression of PDCD4,PTEN and PARP.Compared with normal medium,their RNA levels decreased under high phosphate condition(p<0.05).(3)Using microRNA-21 mimic and inhibitor experiment to exam PDCD4 expression.Compared with normal medium,in the high phosphate group and mimic with normal medium group,the PDCD4 expression decreased(p<0.05).Compared with high phosphate medium,inhibitor with high phosphate medium,the PDCD4 expression increased(p<0.05).5.High phosphate activated ERK1/2 pathway and regulated PDCD4(1)Under the stimulation of high phosphate,ERK can be phosphorylate,p-ERK1/2 was increasing in a short time.(2)Treated the endothelial cells with high phosphate medium for a long period(72hours).Compared with normal medium,it showed no changes in ERK1/2 expression,but p-ERK1/2 increased in high phosphate medium(p<0.05).(3)Using ERK inhibitor experiment to test PDCD4 expression.Compared with normal medium,PDCD4 decreased in high phosphate medium(p<0.05).And also,for the group using ERK inhibitor,there was down regulation of p-ERK(p<0.05),and the PDCD4expression increased(p<0.05).6.ERK1/2 mediated high phosphate induced NF-?B downregulation(1)After treating the cells under medium with different concentrations of phosphate(1.0mM and 5.0mM)for 72 hours,we did western blot to test the subunits of NF-?B(RelA,NF-?B1 and NF-?B2).Compared with normal medium groups,RelA(p65)and NF-?B1 decreased significantly in high phosphate medium(p<0.05),yet NF-?B2 showed no changes.(2)We used ERK1/2 inhibitor experiment to exam the expression of NF-?B(RelA).Compared with normal group,in high phosphate group,RelA decreased(p<0.05);compared with high phosphate group,RelA increased in ERK1/2 inhibitor group(p<0.05).(3)We did the miR-21 mimic and inhibitor experiment and did the western blot.NF-?B(RelA)showed no changes under those conditions.Conclusion:1.High phosphate can increase endothelial cell apoptosis in vitro.2.MicroRNA-21 can mediate high phosphate-induced apoptosis,and downregulated the expression of its target protein PDCD4.3.ERK1/2/PDCD4 pathway is activated during this progress.4.High phosphate can decrease the expression of NF-?B,this pathway is ERK1/2 dependent and microRNA-21 independent.