miR-181a调控血管紧张素Ⅱ诱导骨桥蛋白在血管平滑肌细胞的表达研究
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摘要
目的明确miRNA-181a在血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMCs)中的骨桥蛋白(OPN)的作用及相关机制。方法分离培养出的VSMCs,使用100 nM AngⅡ处理后,检测不同时间点OPN蛋白表达水平的变化,同时利用q PCR检测miR-181a的变化后,将VSMCs分为空白对照组(不做任何处理)、AngⅡ刺激组(加入100 nM AngⅡ诱导)、阴性miRNA+AngⅡ刺激组(转入阴性miRNA并加入100 nM AngⅡ)以及miR-181a模拟剂+AngⅡ刺激组(转入miR-181a模拟剂并加入100 nM AngⅡ)。48 h后Western blotting检测OPN在蛋白水平的变化,并使用Annexin V/PI方式检测凋亡变化、transwell检测迁移的变化;随后VSMCs被分为CON与miR181a inhibitor组,48 h后使用Western blotting检测p53、Bax、p21变化。结果使用100 nM AngⅡ处理后,VSMCs的OPN蛋白表达水平不断上升;使用100 nM AngⅡ处理24 h后,miRNA-181a相对表达量100 n M组显著低于0 nM组; VSMCs在转入miR-181a模拟剂48 h后,miR-181a模拟剂显著高CON组;在空白对照组、AngⅡ刺激组、阴性miRNA+AngⅡ刺激组以及miR-181a模拟剂+AngⅡ刺激组中,相对于阴性对照组,AngⅡ刺激组OPN表达显著上升,而miR-181a模拟剂+AngⅡ刺激组OPN表达显著低于AngⅡ刺激组,略高于阴性对照组;在细胞凋亡中,与AngⅡ刺激组阴性组和miRNA+AngⅡ刺激组相比,miR-181a模拟剂+AngⅡ刺激组Annexin V阳性细胞比率明显减少,说明能够明显抑制细胞凋亡。在细胞迁移中,miR-181a模拟剂+AngⅡ刺激组能够明显抑制细胞迁移。VSMCs在转入miR-181a inhibitor后,相对于CON组,p53、Bax、p21表达均有明显的上升。结论 miR181-a抑制了AngⅡ诱导VSMCs的OPN,同时抑制了AngⅡ诱导的凋亡与增殖,而miR181-a的抑制所诱导的凋亡与p53通路相关。
Objective To clarify the role of miRNA-181 a in angiotensinⅡ( AngⅡ)-induced osteopontin( OPN) regulation. Methods Vascular smooth muscle cells( VSMCs) were isolated and cultured. On detecting the change of miR-181 a by q PCR,the VSMCs were divided into4 groups,namely blank control group,AngⅡgroup,miR-181a mimic + AngⅡgroup and blank miRNA + AngⅡgroup. The OPN level was detected by western blotting 48 h After 100 nM AngⅡtreatment. Cell apoptosis was detected by Annexin V/PI method and Cell migration was by transwell method. VSMCs were subsequently divided into CON group and miR181a inhibitor group. After 48 h,p53,Bax,and p21 changes were detected by western blotting. Results After treatment with 100 nM AngⅡ,the expression level of OPN protein in VSMCs increased continuously. After treatment with 100 n M AngⅡfor 24 h,the relative expression of miRNA-181a was significantly lower in AngⅡ group than in control group. VSMCs were transferred with miR-181 a mimic. After 48 h,miR-181a mimic was significantly higher in CON group than in miR181a inhibitor group. OPN level:AngⅡgrou P > miR-181 a mimic + AngⅡgrou P > blank control group. Regarding apoptosis,the Annexin V-positive cells in miR-181a mimic +AngⅡ group significantly decreased,indicating apoptosis inhibition. Cell migration was also inhibited in miR-181a mimic + AngⅡ group. As for p53,Bax and p21 levels: miR181a inhibitor grou P > CON group. Conclusion miR-181 a inhibits AngⅡ-induced OPN of VSMCs and inhibits AngⅡ-induced apoptosis and migration. Apoptosis induced by miR-181a inhibition is associated with p53 pathway.
Objective To clarify the role of miRNA-181 a in angiotensinⅡ( AngⅡ)-induced osteopontin( OPN) regulation. Methods Vascular smooth muscle cells( VSMCs) were isolated and cultured. On detecting the change of miR-181 a by q PCR,the VSMCs were divided into4 groups,namely blank control group,AngⅡgroup,miR-181a mimic + AngⅡgroup and blank miRNA + AngⅡgroup. The OPN level was detected by western blotting 48 h After 100 nM AngⅡtreatment. Cell apoptosis was detected by Annexin V/PI method and Cell migration was by transwell method. VSMCs were subsequently divided into CON group and miR181a inhibitor group. After 48 h,p53,Bax,and p21 changes were detected by western blotting. Results After treatment with 100 nM AngⅡ,the expression level of OPN protein in VSMCs increased continuously. After treatment with 100 n M AngⅡfor 24 h,the relative expression of miRNA-181a was significantly lower in AngⅡ group than in control group. VSMCs were transferred with miR-181 a mimic. After 48 h,miR-181a mimic was significantly higher in CON group than in miR181a inhibitor group. OPN level:AngⅡgrou P > miR-181 a mimic + AngⅡgrou P > blank control group. Regarding apoptosis,the Annexin V-positive cells in miR-181a mimic +AngⅡ group significantly decreased,indicating apoptosis inhibition. Cell migration was also inhibited in miR-181a mimic + AngⅡ group. As for p53,Bax and p21 levels: miR181a inhibitor grou P > CON group. Conclusion miR-181 a inhibits AngⅡ-induced OPN of VSMCs and inhibits AngⅡ-induced apoptosis and migration. Apoptosis induced by miR-181a inhibition is associated with p53 pathway.
引文
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[2]Cho JR,Lee CY,Lee J,et al.MicroRNA-761 inhibits Angiotensin Ⅱ-induced vascular smooth muscle cell proliferation and migration by targeting mammalian target of rapamycin[J].Clin Hemorheol Microcirc,2015,63(1):45-56.
[3]Li B,Wang Z,Hu Z.P38 MAPK Signaling Pathway Mediates Angiotensin Ⅱ-Induced miR143/145 Gene Cluster Downregulation during Aortic Dissection Formation[J].Ann Vasc Surg,2017,40:262-273.
[4]Hu J,Wang X,Wei SM.Activin A stimulates the proliferation and differentiation of cardiac fibroblasts via the ERK1/2 and p38-MAPKpathways[J].Eur J Pharmacol,2016,789:319-327.
[5]Remus EW,Lyle AN,Weiss D,et al..miR181a protects against angiotensin Ⅱ-induced osteopontin expression in vascular smooth muscle cells[J].Atherosclerosis,2013,228(1):168-174.
[6]Han M,Wen JK,Zheng B,et al.Blockade of integrin beta3-FAKsignaling pathway activated by osteopontin inhibits neointimal formation after balloon injury[J].Cardiovasc Pathol,2007,16(5):283-290.
[7]Saito K,Nakatomi M,Ida-Yonemochi H,et al.The expression of GM-CSF and osteopontin in immunocompetent cells precedes the odontoblastdifferentiation following allogenic tooth transplantation in mice[J].JHistochem Cytochem,2011,59(5):518-529.
[8]Niu J,Xue A,Chi Y,et al.Induction of miRNA-181a by genotoxic treatments promotes chemotherapeutic resistance and metastasis in breast cancer[J].Oncogene,2016,35(10):1302-1313.
[9]Wei X,Sun Y,Wu Y,et al.Downregulation of Talin-1 expression associates with increased proliferation and migration of vascular smooth muscle cells in aortic dissection[J].BMC Cardiovasc Disord,2017,17(1):162.
[10]石凯峰,张宁,柳诗意,等.补肾活血方通过调节Bcl-2/Bax凋亡相关蛋白对慢性肾脏病大鼠血管钙化的影响[J].中华中医药杂志,2017,32(5):2188-2193.
[11]Liu W,Zeng Q,Luo R.Correlation between Serum Osteopontin and miR-181a Levels in Allergic Rhinitis Children[J].Mediators Inflamm,2016,2016:9471215.