MiR-197-3p/STAMP2对ox-LDL诱导的人脐静脉内皮细胞凋亡及炎症应激的影响
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摘要
目的探究miR-197-3p/前列腺六次跨膜蛋白(Six transmembrane protein of prostate,STAMP) 2对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)凋亡和炎症应答。方法用ox-LDL处理HUVEC复制内皮细胞损伤模型; RT-PCR检测miR-197-3p和STAMP2表达; ELISA检测细胞凋亡; Western blot用于检测STAMP2,Bax和Bcl-2的蛋白水平; ELISA检测HUVECs中细胞间黏附分子(ICAM)-1,血管内皮细胞黏附分子(VCAM)-1和E选择素(E-selectin)的表达水平;生物信息学预测miR-197-3p的靶基因;并采用双萤光素酶报告系统、qRT-PCR及Western blot验证miR-197-3p与STAMP2的靶向调控关系。结果 ox-LDL能明显上调miR-197-3p的表达(P <0. 05),同时降低STAMP2的表达(P <0. 05),且呈时间依赖性;下调miR-197-3p能够显著抑制ox-LDL诱导的细胞凋亡(P <0. 05),同时降低促凋亡蛋白Bax并增加抑凋亡蛋白Bcl-2(P <0. 05);抑制miR-197-3p明显降低ox-LDL诱导的炎症因子的表达(P <0. 05);此外,生物信息学预测提示STAMP2是miR-197-3p的靶基因,而且qRT-PCR及Western blot结果显示抑制miR-197-3p明显上调STAMP2 mRNA和蛋白水平(P <0. 05),从而证实miR-197-3p能够靶向调控STAMP2。结论 MiR-197-3p可以通过靶向调控STAMP2从而调节ox-LDL诱导的HUVECs凋亡及炎症应答,为动脉粥样硬化的靶向治疗提供理论基础。
AIM To investigate the effect of miR-197-3 p/STAMP2 on apoptosis and inflammation responses of human umbilical vein endothelial cells( HUVECs) induced by oxidized low density lipoprotein( ox-LDL). METHODS A cell injury model was induced by ox-LDL. RT-PCR was used to detect levels of miR-197-3 p and STAMP2 and ELISA analysis were used to determined cell apoptosis. The protein expression of STAMP2,Bax and Bcl-2 were measured by Western blot and expression levels of inter-cellular adhesion molecule( ICAM)-1,vasculareen adhesion molecule( VCAM)-1 and E-selectin were detected by ELISA assay. Bioinformatics analysis identified the potential target of miR-197-3 p. The targeting effect of miR-197-3 p on STAMP2 was verified by dual-luciferase reporter assay system,Western blot and qRT-PCR.RESULTS ox-LDL induced the level of miR-197-3 p and significantly down-regulated the expression of STAMP2 in a time dependent manner in HUVECs( P < 0. 05). Down-regulating miR-197-3 p inhibited ox-LDL-induced cell apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl-2( P < 0. 05),and attenuated Caspase-3 activity( P < 0. 05). Moreover,inhibiting the expression of miR-197-3 p strongly attenuated the level of inflammatory factors CAM-1,VCAM-1 and E-selectin induced by ox-LDL( P < 0. 05). The present investigation indicated that STAMP2 was a direct and functional target of miR-197-3 p. qRT-PCR and Western blot analysis showed that inhibition of miR-197-3 p upregulated the mRNA and protein expression,which validated that miR-197-3 p negatively regulated STAMP2. CONCLUSION Our findings indicate that miR-197-3 p is involved in cell apoptosis and inflammatory response of HUVECs induced by ox-LDL through targeting STAMP2,which provides a potential target for treatment of atherosclerosis in future.
AIM To investigate the effect of miR-197-3 p/STAMP2 on apoptosis and inflammation responses of human umbilical vein endothelial cells( HUVECs) induced by oxidized low density lipoprotein( ox-LDL). METHODS A cell injury model was induced by ox-LDL. RT-PCR was used to detect levels of miR-197-3 p and STAMP2 and ELISA analysis were used to determined cell apoptosis. The protein expression of STAMP2,Bax and Bcl-2 were measured by Western blot and expression levels of inter-cellular adhesion molecule( ICAM)-1,vasculareen adhesion molecule( VCAM)-1 and E-selectin were detected by ELISA assay. Bioinformatics analysis identified the potential target of miR-197-3 p. The targeting effect of miR-197-3 p on STAMP2 was verified by dual-luciferase reporter assay system,Western blot and qRT-PCR.RESULTS ox-LDL induced the level of miR-197-3 p and significantly down-regulated the expression of STAMP2 in a time dependent manner in HUVECs( P < 0. 05). Down-regulating miR-197-3 p inhibited ox-LDL-induced cell apoptosis accompanied by a decrease in the expression of Bax and an increase in the expression of Bcl-2( P < 0. 05),and attenuated Caspase-3 activity( P < 0. 05). Moreover,inhibiting the expression of miR-197-3 p strongly attenuated the level of inflammatory factors CAM-1,VCAM-1 and E-selectin induced by ox-LDL( P < 0. 05). The present investigation indicated that STAMP2 was a direct and functional target of miR-197-3 p. qRT-PCR and Western blot analysis showed that inhibition of miR-197-3 p upregulated the mRNA and protein expression,which validated that miR-197-3 p negatively regulated STAMP2. CONCLUSION Our findings indicate that miR-197-3 p is involved in cell apoptosis and inflammatory response of HUVECs induced by ox-LDL through targeting STAMP2,which provides a potential target for treatment of atherosclerosis in future.
引文
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[2]Brevetti G,Giugliano G,Brevetti L,et al. Inflammation in peripheral artery disease[J]. Circulation,2010,122(18):1862-1875.
[3]Harada-Shiba M,Kinoshita M,Kamido H,et al. Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms[J]. J Biol Chem,1998,273(16):9681-9687.
[4]Harris TA,Yamakuchi M,Ferlito M,et al. MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1[J]. Proc Natl Acad Sci U S A,2008,105(5):1516-1521.
[5]Willeit P,Zampetaki A,Dudek K,et al. Circulating microRNAs as novel biomarkers for platelet activation[J]. Circ Res,2013,112(4):595-600.
[6]Schulte C,Molz S,Appelbaum S,et al. miRNA-197 and miRNA-223 Predict Cardiovascular Death in a Cohort of Patients with Symptomatic Coronary Artery Disease[J]. PLoS One,2015,10(12):e0145930.
[7]Zampetaki A,Willeit P,Tilling L,et al. Prospective study on circulating MicroRNAs and risk of myocardial infarction[J]. J Am Coll Cardiol,2012,60(4):290-299.
[8]Kitagawa K,Matsumoto M,Sasaki T,et al. Involvement of ICAM-1in the progression of atherosclerosis in APOE-knockout mice[J].Atherosclerosis,2002,160(2):305-310.
[9] Wang ZH,Zhang W,Gong HP,et al. Expression of STAMP2 in monocytes associates with cardiovascular alterations[J]. Eur J Clin Invest,2010,40(6):490-496.
[10]ten Freyhaus H,Calay ES,Yalcin A,et al. Stamp2 controls macrophage inflammation through nicotinamide adenine dinucleotide phosphate homeostasis and protects against atherosclerosis[J]. Cell Metab,2012,16(1):81-89.
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