| Part Ⅰ Effect of miR-425-5p on monocarboxylate transporter 4 expression and the biological behavior of human umbilical vein endothelial cellsObjective:Diabetic vascular complications remain the leading cause of morbidity and mortality in patients with type 2 diabetes.Endothelial cells(ECs)dysfunction and apoptosis caused by hyperglycemia lead to vascular endothelial injury,which is the critical and initiating factor of diabetic vascular complications.ECs depend on glycolysis for their energy metabolism,and the final product of glycolysis-lactateefflux in glycolytic cells mediated by monocarboxylate transporter 4(MCT4).Besides,down-regulation of MCT4 is involved in vascular endothelial injury.However,the mechanisms underlying MCT4 downregulation remain poorly understood.The aim of this part is to explore the role and mechanism of miRNA in the regulation of MCT4 in diabetes.Methods:By using computational prediction programs such as PicTar,TargetScan and miRcode,it was found that miR-425-5p has potential binding sites in the 3’-UTR of the MCT4.And the miR-425-5p expression in blood samples of DM patients and healthy volunteers were detected.Human umbilical vein endothelial cells(HUVECs)were obtained for culture and identification.q-PCR was used to detect miR-425-5p expression in HUVECs after treated with high glucose(HG)(30 mM)and interleukin 1β(IL-1β)(10 ng/mL).The luciferase reporter assay was used to verify the binding of miR-425-5p to the 3’-UTR of MCT4.HUVECs were treated with miR-425-5p mimics(80 nM),miR-425-5p inhibitor(80 nM)and its negative control(80 nM)for 48 hours.After the treatment,q-PCR,Western blot,immunofluorescence,lactate detection kit,pH detection kit,flow cytometry,cell counting kit-8(CCK-8)assay and wound healing assay were used to detect the effect of miR-425-5p on MCT4 expression,intracellular lactate accumulation,pH changes,apoptosis,proliferation and migration of HUVECs.To further explore the effect of the miR-425-5p inhibitor on HUVECs apoptosis induced by HG and IL-1β,HUVECs were transfected with miR-425-5p inhibitor(80 nM)and its negative control(80 nM),respectively.And after transfected for 24 hours,HUVECs were treated with HG(30 mM)and IL-1β(10 ng/mL).After treatment for 48 hours,Western blot was used to detect the expression of MCT4 and Bcl-2,Bax,Caspase-3,Cleaved Caspase-3,and flow cytometry was used to detect the apoptosis of HUVECs；Results:(1)Compared with the control group,the expression of miR-425-5p in HUVECs treated with HG and IL-1β was significantly up-regulated(P<0.05).And compared with healthy volunteers,the expression of miR-425-5p in plasma and peripheral blood mononuclear cells(PBMCs)of DM patients was significantly upregulated(P<0.05).(2)The luciferase reporter assay showed that MCT4 is a target gene of miR-425-5p.(3)Compared with the control group,the expression of MCT4 in the miR-425-5p mimics group were significantly down-regulated,while the expression of MCT4 in the miR-425-5p inhibitor group were significantly upregulated(P<0.05).(4)Compared with the control group,the intracellular lactate level of HUVECs in the miR-425-5p mimics group was significantly increased,while the intracellular lactate level of HUVECs in the miR-425-5p inhibitor group was significantly decreased(P<0.05).And compared with the control group,the intracellular pH of HUVECs in the miR-425-5p mimics group was significantly decreased,while the intracellular pH of HUVECs in the miR-425-5p inhibitor group increased,but there was no statistical difference(P>0.05).(5)Compared with the control group,the expression of Bcl-2 in HUVECs in the miR-425-5p mimics group was significantly down-regulated,the expression of Bax and Cleaved Caspase-3 was significantly up-regulated,while the expression of Bcl-2 in HUVECs in the miR-4255p inhibitor group was significantly up-regulated,the expression of Bax and Cleaved Caspase-3 was significantly down-regulated(P<0.05).Similarly,the results of flow cytometry also showed that the apoptosis of HUVECs in the miR-425-5p mimics group was significantly increased when compared with the control group.And the apoptosis of HUVECs in the miR-425-5p inhibitor group was significantly reduced(P<0.05).(6)Compared with the control group,the migration and proliferation of HUVECs in the miR-425-5p mimics group were significantly decreased,while the migration and proliferation of HUVECs in the miR-425-5p inhibitor group were significantly increased(P<0.05).(7)Compared with the control group,the expressions of MCT4 and Bcl-2 of HUVECs in the miR-425-5p inhibitor group were significantly up-regulated,and the expressions of Bax and Cleaved Caspase-3 were significantly down-regulated(P<0.05).Similarly,the results of flow cytometry also showed that the apoptosis of HUVECs in the miR-425-5p inhibitor group was significantly reduced when compared with the control group(P<0.05).Conclusion:Diabetes promotes the expression of miR-425-5p in HUVECs.MCT4 is the target gene of miR-425-5p,and miR-425-5p inhibits MCT4 expression of HUVECs.Diabetes up-regulates miR-425-5p to regulate the expression of MCT4 in HUVECs,which leads to the accumulation of intracellular lactate and increased apoptosis of HUVECs.Besides,miR-425-5p inhibits the migration and proliferation of HUVECs.miR-425-5p inhibitor reduces the HUVECs apoptosis induced by HG and IL-1β through up-regulating the expression of MCT4.Part Ⅱ Role of the NF-κB/miR-425-5p/MCT4 axis in apoptosis of HUVECsObjective:Nuclear factor kappa B(NF-κB)is activated in diabetes and plays a key role in diabetic endothelial injury.And NF-κB signaling activation can not only regulate the release of cytokines and chemokines but also regulate the expression of miRNAs.Previous studies have shown that NF-κB has three binding sites in the miR425-5p promoter.And the first part of our study showed that the expression of miR425-5p was up-regulated in diabetes,which inhibited the expression of MCT4,lead to the accumulation of intracellular lactate and increased the apoptosis of ECs.However,in diabetes,whether NF-κB signaling activation is directly related to the up-regulation of miR-425-5p of ECs and the followed biological behavior are still unknown.The aim of this part was to explore the role of the NF-κB/miR-425-5p/MCT4 axis in the apoptosis of HUVECs.Methods:HUVECs were cotransfected with pcDNA3.1-NF-κB or pcDNA3.1 and PGL3-miR-425-5p or PGL3-Basic.After transfection for 24 hours,luciferase activity was measured by a dual-luciferase reporter assay kit.HUVECs were treated with HG(30 mM),IL-1β(10 ng/mL)and/or SN50(50 μg/mL),an NF-κB inhibitor.After treatment for 48 hours,Western blot and q-PCR were used to detect the levels of NFκB p65 and p-NF-κB p65 in the nucleus of HUVECs,and the levels of miR-425-5p,Bcl-2,Bax,caspase-3,and cleaved caspase-3 in the HUVECs.A lactate detection kit and pH detection kit were used to detect the intracellular lactate accumulation and pH changes in the HUVECs.Flow cytometry was used to detect the apoptosis of HUVECs.Results:(1)Compared with the control group,the level of p-NF-κB p65 and miR425-5p in HUVECs of the HG and IL-1β group was significantly increased,while SN50 can significantly rescue the up-regulation of p-NF-κB p65 and miR-425-5p(P<0.05).(2)The luciferase reporter assay showed that overexpression of NF-κB dramatically increased the luciferase activity of the HUVECs.(3)Compared with the control group,the MCT4 expression in HUVECs of the HG and IL-1β group was significantly reduced,while SN50 can significantly rescue the down-regulation of MCT4(P<0.05).(4)Compared with the control group,the intracellular lactate level in HUVECs of the HG and IL-1β group was significantly increased,while SN50 can significantly inhibit the increase of intracellular lactate level(P<0.05).Compared with the control group,the intracellular pH in HUVECs of the HG and IL-1β group was significantly reduced,while SN50 can significantly inhibit the reduction of intracellular pH(P<0.05).(5)Compared with the control group,the Bcl-2 expression in HUVECs of the HG and IL-1β group was significantly reduced and the expression of Bax and Cleaved Caspase-3 were significantly increased,while SN50 can significantly inhibit the down-regulation of Bcl-2 and the up-regulation of Bax and Cleaved Caspase-3(P<0.05).Similarly,the results of flow cytometry also showed that the apoptosis of HUVECs in the HG and IL-1β group was significantly increased when compared with the control group,while SN50 can significantly inhibit the increase of apoptosis(P<0.05).Conclusion:Activation of NF-κB signaling increases the miR-425-5p levels and inhibition of NF-κB signaling decreases the miR-425-5p levels in the HUVECs.The miR-425-5p promoter contained a target sequence of NF-κB.Activation of NF-κB signaling significantly inhibits the MCT4 expression of HUVECs and promotes the increase of intracellular lactate level and the decrease of intracellular pH of HUVECs.Activation of NF-κB signaling significantly promotes the apoptosis of HUVECs.The activation of the NF-κB/miR-425-5p/MCT4 axis plays an important role in ECs apoptosis and diabetic endothelial injury.Part III Effect of miR-425-5p on the vascular endothelial injury in diabetic miceObjective:The results of the first and second parts showed that diabetes inhibited the expression of MCT4 in HUVECs by up-regulating miR-425-5p,which lead to the accumulation of intracellular lactate and increased the apoptosis of ECs.Besides,the activation of the NF-κB/miR-425-5p/MCT4 axis played an important role in ECs apoptosis and diabetic endothelial injury.And SN50,an NF-κB inhibitor,inhibited ECs apoptosis induced by HG and IL-1β through down-regulating the expression of miR-425-5p.The aim of this part was to investigate the effect of miR-425-5p on the vascular endothelial injury in diabetic mice.Methods:After adaptive feeding for one week,male C57BL/6 mice(4-5 weeks old)were randomly divided into the diabetes mellitus group(DM)and the negative control group(NC).The DM group was given a high-carbohydrate and high-fat diet,and the NC group was given a normal diet.After 4 weeks,the mice were fasted for 12h,and then the mice in the DM group were injected with STZ(30mg/Kg)for 4 days.And the mice in the NC group were injected with the sterile citrate buffer of equal dose(0.1mol/L).If the fasting blood glucose is greater than 16.7mmol/L for three times consecutively,the DM model is considered to be successful.The mice in the DM group(N=12)that were successfully modeled and the NC group(N=12)continued to be reared for 10 weeks.Each group of mice was divided into two groups,and AAV9miR-425-5p inhibition(100μL,4.78E+12 vg/ml)or an equal dose of AAV9-NC was injected into the tail vein.That is to say,mice were divided into 4 groups:DM+AAV9-NC group(N=6),DM+AAV9-miR-425-5p inhibition group(N=6),NC+AAV9-NC group(N=6)and NC+AAV9-miR-425-5p inhibition group(N=6).After 6 weeks of feeding,the fasting blood glucose and the expression of miR-425-5p in the aorta were detected in each group.Western blot,q-PCR,immunofluorescence and aortic root oil red staining were used to detect the expression of MCT4 and the CD31 level of aortic endothelium.Results:(1)Compared with the NC group,the fasting blood glucose level of the DM group was significantly increased(>16.7mmol/L).Compared with the NC and AAV9NC groups,the miR-425-5p expression in the aorta of the DM and AAV9-NC group was significantly up-regulated.While compared with the DM and AAV9-NC groups,the fasting blood glucose level of the DM and AAV9-miR-425-5p inhibition group did not change significantly,but the miR-425-5p expression of the aorta was significantly down-regulated(P<0.05).(2)Compared with the NC and AAV9-NC group,the aortic MCT4 expression of the DM and AAV9-NC groups were significantly down-regulated.And compared with the DM and AAV9-NC groups,the aortic MCT4 expression of the DM and AAV9-miR-425-5p inhibition group were significantly up-regulated(P<0.05).(3)Compared with the NC and AAV9-NC groups,the CD31 level of aortic endothelium in the DM and AAV9-NC mice were down-regulated and endothelial discontinuity appeared.While compared with the DM and AAV9-NC groups,the CD31 level of aortic endothelium in the DM and AAV9miR-425-5p inhibition group was up-regulated and endothelial continuity was good.Conclusion:DM promotes the expression of miR-425-5p in the aorta of mice.miR425-5p promotes the down-regulation of MCT4 expression in the aorta of mice and inhibiting the expression of miR-425-5p rescues the down-regulation of MCT4 caused by DM.miR-425-5p promotes the endothelial injury in the aorta of mice and inhibiting the expression of miR-425-5p can delay the endothelial injury caused by DM.DM promotes the down-regulation of MCT4 expression by up-regulating miR-425-5p and leading to endothelial injury. |
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