miR-17-5p诱导小鼠肾足细胞系凋亡
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摘要
目的探讨miR-17-5p在小儿肾病综合征(NS)发病中的作用及其机制。方法将miR-17-5p mimic转染入小鼠肾足细胞系MPC5 24 h后收集细胞。通过microRNA.org、Target Scan和PicTar在线预测miR-17-5p对蛋白酪氨酸磷酸酶受体(PTPRO)-3′UTR区的调控作用。用RT-PCR和Western blot检测PTPRO mRNA以及蛋白的表达, Fluo-3-Am特异性Ca~(2+)荧光指示剂检测足细胞内游离钙离子浓度([Ca~(2+)]), annexin V/PI双染色流式细胞计量术检测足细胞凋亡率。结果生物信息学技术预测,小鼠源及人源miR-17-5p都有多个位点结合于相应的PTPRO-3′UTR区,并抑制PTPRO蛋白及mRNA的表达。PTPRO过表达抑制miR-17-5p诱导的足细胞[Ca~(2+)]升高,PTPRO过表达减缓miR-17-5p mimic诱导的肾小球足细胞凋亡。结论 miR-17-5p抑制小鼠肾足细胞内PTPRO表达,并激发肾小球足细胞[Ca~(2+)]升高,诱导肾小球足细胞凋亡。
Objective To explore the role and mechanisms of miR-17-5 p in the pathogenesis of childhood nephrotic syndrome. Methods miR-17-5 p mimic were transfected into podocytes in kidney of mice(MPC5) for 24 h and then harvested the cells. The potential targets of miR-117-5 P were searched using the prediction programs microRNA.org, TargetScan and PicTar. RT-PCR and Western blot were used to detect expression of protein tyrosine phosphatase receptor type O(PTPRO) mRNA and protein. Ca~(2+) fluorescence indicator with Fluo-3-Amspecificity was used to measure intracellular free calcium concentration([Ca~(2+)]) in podocytes. Annexin V/PI flow cytomeutry was used to test apoptosis rate of podocytes. Results According to the bioinformatics technology prediction, miR-17-5 p derived from human or mouse can all bind to multiple sites of the corresponding PTPRO-3′UTR region and inhibit the expression of PTPRO protein and mRNA. PTPRO overexpression inhibited miR-17-5 p-induced increase of [Ca~(2+)] in podocytes in kidney of mice. PTPRO over-expression alleviated miR-17-5 p-induced apoptosis of podocytes in kidney of mice. Conclusions miR-17-5 p inhibits expression of PTPRO in podocytes in kidney of miceand stimulates increase of intracellular free calcium concentration([Ca~(2+)]) in podocytes, inducing apoptosis of podocytes in kidney of mice.
Objective To explore the role and mechanisms of miR-17-5 p in the pathogenesis of childhood nephrotic syndrome. Methods miR-17-5 p mimic were transfected into podocytes in kidney of mice(MPC5) for 24 h and then harvested the cells. The potential targets of miR-117-5 P were searched using the prediction programs microRNA.org, TargetScan and PicTar. RT-PCR and Western blot were used to detect expression of protein tyrosine phosphatase receptor type O(PTPRO) mRNA and protein. Ca~(2+) fluorescence indicator with Fluo-3-Amspecificity was used to measure intracellular free calcium concentration([Ca~(2+)]) in podocytes. Annexin V/PI flow cytomeutry was used to test apoptosis rate of podocytes. Results According to the bioinformatics technology prediction, miR-17-5 p derived from human or mouse can all bind to multiple sites of the corresponding PTPRO-3′UTR region and inhibit the expression of PTPRO protein and mRNA. PTPRO overexpression inhibited miR-17-5 p-induced increase of [Ca~(2+)] in podocytes in kidney of mice. PTPRO over-expression alleviated miR-17-5 p-induced apoptosis of podocytes in kidney of mice. Conclusions miR-17-5 p inhibits expression of PTPRO in podocytes in kidney of miceand stimulates increase of intracellular free calcium concentration([Ca~(2+)]) in podocytes, inducing apoptosis of podocytes in kidney of mice.
引文
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[11] Tian J,Wang HP,Mao YY,et al.Reduced glomerular epithelial protein 1 expression and podocyte injury in immunoglobulin A nephropathy [J].J Int Med Res,2007,35:338-345.
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[13] Charba DS,Wiggins RC,Goyal M,et al.Antibodies to protein tyrosine phosphatase receptor type O (PTPro) increase glomerular albumin permeability (P(alb)) [J].Am J Physiol Renal Physiol,2009,297:F138-144.
[14] Ozaltin F,Ibsirlioglu T,Taskiran EZ,et al.Disruption of PTPRO causes childhood-onset nephrotic syndrome [J].Am J Hum Genet,2011,89:139-147.
[15] 湛颖.淋巴瘤细胞中PTPROt基因的甲基化状态及其意义 [D].石家庄:河北医科大学,2010:28-29.
[2] 赵芸,苏白鸽,肖慧,等.原发肾病综合征患儿糖皮质激素治疗致眼科不良反应临床特点分析[J].北京大学学报医学版,2017,49:794-797.
[3] Fogo AB.The targeted podocyte [J].J Clin Invest,2011,121:2142-2145.
[4] Fufaa GD,Weil EJ,Lemley KV,et al.structural predictors of loss of renal function in American Indians with type 2 diabetes [J].Clin J Am Soc Nephrol,2016,11:254-261.
[5] Schwartzman M,Reginensi A,Wong JS,et al.podocyte-specific deletion of yes-associated protein causes FSGS and progressive renal failure [J].J Am Soc Nephrol,2016,27:216-226.
[6] Ma L,Qu L.The function of microRNAs in renal development and pathophysiology [J].J Genet Genomics,2013,40:143-152.
[7] Berridge MJ,Bootman MD,Roderick HL,et al.Calcium signalling:dynamics,homeostasis and remodeling[J].Nat Rev,2003,4:517-529.
[8] Canaud G,Bienaime F,Viau A,et al.AKT2 is essential to maintain podocyte viability and function during chronic kidney disease [J].Nat Med,2013,19:1288-1296.
[9] Hao J,Wei Q,Mei S,et al.Induction of microRNA-17-5p by p53 protects against renal ischemia-reperfusion injury by targeting death receptor 6 [J].Kidney Int,2017,91:106-118.
[10] Sharif K,Goyal M,Kershaw D,et al.Podocyte phenotypes as defined by expression and distribution of GLEPP1 in the developing glomerulus and in nephrotic glomeruli from MCD,CNF,and FSGS.A dedifferentiation hypothesis for the nephrotic syndrome [J].Exp Nephrol,1998,6:234-244.
[11] Tian J,Wang HP,Mao YY,et al.Reduced glomerular epithelial protein 1 expression and podocyte injury in immunoglobulin A nephropathy [J].J Int Med Res,2007,35:338-345.
[12] Clement LC,Liu G,Perez-Torres I,et al.Early changes in gene expression that influence the course of primary glomerular disease [J].Kidney Int,2007,72:337-347.
[13] Charba DS,Wiggins RC,Goyal M,et al.Antibodies to protein tyrosine phosphatase receptor type O (PTPro) increase glomerular albumin permeability (P(alb)) [J].Am J Physiol Renal Physiol,2009,297:F138-144.
[14] Ozaltin F,Ibsirlioglu T,Taskiran EZ,et al.Disruption of PTPRO causes childhood-onset nephrotic syndrome [J].Am J Hum Genet,2011,89:139-147.
[15] 湛颖.淋巴瘤细胞中PTPROt基因的甲基化状态及其意义 [D].石家庄:河北医科大学,2010:28-29.