miR-222调控PARP-1在高糖致人肾脏系膜细胞tPA/PAI-1紊乱中的作用
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摘要
目的探讨微小RNA-222(miR-222)调控多聚二磷酸腺苷核糖聚合酶-1(PARP-1)在高糖诱导人肾脏系膜细胞(HMC)组织型纤溶酶原激活物/纤溶酶原激活物抑制物-1(tPA/PAI-1)紊乱中的作用。方法体外培养HMC,实时定量PCR分别检测正常糖(5 mmol/L)及高糖(25 mmol/L)刺激下miR-222表达;另将体外培养的HMC分为正常糖组、25 mmol/L高糖组、高糖+miR-222 mimics阴性对照组、高糖+miR-222 mimics组4组,运用蛋白免疫印迹法检测PARP-1蛋白表达,ELISA法测定细胞上清液tPA、PAI-1的分泌蛋白。结果实时定量PCR显示,高糖组HMC在24 h时miR-222mRNA表达水平是正常糖组的2.537倍。高糖组HMC中PARP-1蛋白表达高于正常糖组(P<0.05),高糖+miR-222 mimics组HMC中PARP-1蛋白表达低于高糖组(P<0.05),而高糖+miR-222 mimics阴性对照组与高糖组比较差异无统计学意义(P>0.05)。与正常糖组比较,高糖组的tPA和tPA/PAI-1降低、PAI-1升高(P均<0.05);与高糖组比较,高糖+mimics组的tPA和tPA/PAI-1升高、PAI-1降低(P均<0.05)。高糖+miR-222 mimics阴性对照组与高糖组tPA、PAI-1及tPA/PAI-1比较差异均无统计学意义(P均>0.05)。结论高糖可增强HMC PARP-1及分泌型PAI-1的蛋白表达、减弱分泌型tPA的蛋白表达,下调tPA/PAI-1比值。miR-222可有效下调靶蛋白PARP-1的表达,并有效改善高糖诱导的tPA/PAI-1紊乱。
Objective To investigate the role of the regulation of poly( ADP-ribose) polymerase-1( PARP-1) by microRNA-222( miR-222) in the disturbance of tissue-type plasminogen activator/plasminogen activator inhibitor-1( tPA/PAI-1) induced by high glucose in human mesangial cells( HMCs). Methods HMCs were cultured in vitro. Real-time PCR was adopted to detect the expression levels of miR-222 under the stimulation of normal glucose( 5 mmol/L) and high glucose( 25 mmol/L). The HMCs cultured in vitro were divided into the normal glucose,25 mmol/L high glucose,high glucose + miR-222 mimics negative control and high glucose + miR-222 mimics groups. Expression of PARP-1 protein was measured by western blot. Secretory proteins of tPA and PAI-1 in the cell supernatants was detected by ELISA assay. Results Real-time quantitative PCR demonstrated that the expression level of miR-222 mRNA of HMCs in the high glucose group was 2. 537 times that in the normal glucose group at 24 h. The expression of PARP-1 protein of HMCs in the high glucose group was significantly higher than that of normal glucose group( P < 0. 05). In the high glucose+ miR-222 mimics group,the expression level of PARP-1 protein of HMCs was considerably lower than that in the high glucose group( P < 0. 05). No statistical significance was noted between the high glucose + miR-222 mimics negative control and the high glucose groups( P > 0. 05). Compared with the normal glucose group,tPA and tPA/PAI-1 ratio were significantly decreased,whereas PAI-1 was evidently increased in the high glucose group( all P < 0. 05). Compared with the high glucose group,tPA and tPA/PAI-1 ratio were significantly elevated,whereas PAI-1 was significantly decreased in the high glucose + mimics group( all P < 0. 05). No statistical significance was observed in tPA,PAI-1 and tPA/PAI-1 ratio between the high-glucose + miR-222 mimics negative control and the high glucose groups( all P > 0. 05). Conclusions High glucose can up-regulate the expression levels of secretory proteins of PARP-1 and PAI-1 in HMCs,down-regulate the expression of secretory protein of PAI-1 and decrease the tPA/PAI-1 ratio. miR-222 can effectively down-regulate the expression of target protein of PARP-1 and significantly mitigate the high glucose-induced tPA/PAI-1 disturbance.
Objective To investigate the role of the regulation of poly( ADP-ribose) polymerase-1( PARP-1) by microRNA-222( miR-222) in the disturbance of tissue-type plasminogen activator/plasminogen activator inhibitor-1( tPA/PAI-1) induced by high glucose in human mesangial cells( HMCs). Methods HMCs were cultured in vitro. Real-time PCR was adopted to detect the expression levels of miR-222 under the stimulation of normal glucose( 5 mmol/L) and high glucose( 25 mmol/L). The HMCs cultured in vitro were divided into the normal glucose,25 mmol/L high glucose,high glucose + miR-222 mimics negative control and high glucose + miR-222 mimics groups. Expression of PARP-1 protein was measured by western blot. Secretory proteins of tPA and PAI-1 in the cell supernatants was detected by ELISA assay. Results Real-time quantitative PCR demonstrated that the expression level of miR-222 mRNA of HMCs in the high glucose group was 2. 537 times that in the normal glucose group at 24 h. The expression of PARP-1 protein of HMCs in the high glucose group was significantly higher than that of normal glucose group( P < 0. 05). In the high glucose+ miR-222 mimics group,the expression level of PARP-1 protein of HMCs was considerably lower than that in the high glucose group( P < 0. 05). No statistical significance was noted between the high glucose + miR-222 mimics negative control and the high glucose groups( P > 0. 05). Compared with the normal glucose group,tPA and tPA/PAI-1 ratio were significantly decreased,whereas PAI-1 was evidently increased in the high glucose group( all P < 0. 05). Compared with the high glucose group,tPA and tPA/PAI-1 ratio were significantly elevated,whereas PAI-1 was significantly decreased in the high glucose + mimics group( all P < 0. 05). No statistical significance was observed in tPA,PAI-1 and tPA/PAI-1 ratio between the high-glucose + miR-222 mimics negative control and the high glucose groups( all P > 0. 05). Conclusions High glucose can up-regulate the expression levels of secretory proteins of PARP-1 and PAI-1 in HMCs,down-regulate the expression of secretory protein of PAI-1 and decrease the tPA/PAI-1 ratio. miR-222 can effectively down-regulate the expression of target protein of PARP-1 and significantly mitigate the high glucose-induced tPA/PAI-1 disturbance.
引文
[1]Marshall CB.Rethinking glomerular basement membrane thickening in diabetic nephropathy:adaptive or pathogenic?Am J Physiol Renal Physiol,2016,311(5):F831-F843.
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[3]Mishra M,Kowluru RA.Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy.Biochim Biophys Acta,2017,1863(7):1761-1769.
[4]Zakaria EM,El-Bassossy HM,El-Maraghy NN,Ahmed AF,Ali AA.PARP-1 inhibition alleviates diabetic cardiac complications in experimental animals.Eur J Pharmacol,2016,791:444-454.
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[7]朱恒梅,祝胜郎,陈结慧,叶玲,蒋莹,李向阳.多聚二磷酸腺苷(ADP)核糖聚合酶-1(PARP-1)在高糖致大鼠肾脏系膜细胞细胞外基质沉积中的作用.中国医药导报,2012,9(31),8-11.
[8]Zhu H,Jiang Z,Lei P,Huang W,Yu X.Poly(ADP-ribose)polymerase-1 mediates angiotensin II-induced expression of plasminogen activator inhibitor-1 and fibronectin in rat mesangial cells.Kidney Blood Press Res,2011,34(5):320-327.
[9]Kato M,Dang V,Wang M,Park JT,Deshpande S,Kadam S,Mardiros A,Zhan Y,Oettgen P,Putta S,Yuan H,Lanting L,Natarajan R.TGF-βinduces acetylation of chromatin and of Ets-1to alleviate repression of miR-192 in diabetic nephropathy.Sci Signal,2013,6(278):ra43.
[10]Kato M,Natarajan R.MicroRNAs in diabetic nephropathy:functions,biomarkers,and therapeutic targets.Ann N Y Acad Sci,2015,1353:72-88.
[11]Lai JY,Luo J,O'Connor C,Jing X,Nair V,Ju W,Randolph A,Ben-Dov IZ,Matar RN,Briskin D,Zavadil J,Nelson RG,Tuschl T,Brosius FC 3rd,Kretzler M,Bitzer M.MicroRNA-21in glomerular injury.J Am Soc Nephrol,2015,26(4):805-816.
[12]何凤,周姗姗,关昌杰,黄俊,陈浩雄,刘日光,傅君舟.微小RNA135b调控Smad5在糖尿病肾病发病中的作用研究.新医学,2017,48(5),317-322.
[13]Allegri L,Rosignolo F,Mio C,Filetti S,Baldan F,Damante G.Effects of nutraceuticals on anaplastic thyroid cancer cells.J Cancer Res Clin Oncol,2018,144(2):285-294.
[14]Shi Z,Zhao C,Guo X,Ding H,Cui Y,Shen R,Liu J.Differential expression of microRNAs in omental adipose tissue from gestational diabetes mellitus subjects reveals miR-222 as a regulator of ERαexpression in estrogen-induced insulin resistance.Endocrinology,2014,155(5):1982-1990.
[15]Lightell DJ Jr,Moss SC,Woods TC.Upregulation of miR-221and-222 in response to increased extracellular signal-regulated kinases 1/2 activity exacerbates neointimal hyperplasia in diabetes mellitus.Atherosclerosis,2018,269:71-78.
[16]Neijenhuis S,Bajrami I,Miller R,Lord CJ,Ashworth A.Identification of miRNA modulators to PARP inhibitor response.DNA Repair(Amst),2013,12(6):394-402.
[17]Sambola A,Ruiz-Meana M,Barba I Del Blanco BG,Barrabés JA,Lip GY,Vilardosa U',Sansaloni S,Rello P,García-Dorado D.Glycative and oxidative stress are associated with altered thrombus composition in diabetic patients with ST-elevation myocardial infarction.Int J Cardiol,2017,243:9-14.
[18]de Candia P,Spinetti G,Specchia C,Sangalli E,La Sala L,Uccellatore A,Lupini S,Genovese S,Matarese G,Ceriello A.A unique plasma microRNA profile defines type 2 diabetes progression.PLo S One,2017,12(12):e0188980.
[19]Pheiffer C,Dias S,Rheeder P,Adam S.Decreased expression of circulating miR-20a-5p in south african women with gestational diabetes mellitus.Mol Diagn Ther,2018,22(3):345-352.
[20]Villard A,Marchand L,Thivolet C,Rome S.Diagnostic value of cell-free circulating microRNAs for obesity and type 2 diabetes:a meta-analysis.J Mol Biomark Diagn,2015,6(6).pii:251.
[21]Li T,Yang GM,Zhu Y,Wu Y,Chen XY,Lan D,Tian KL,Liu LM.Diabetes and hyperlipidemia induce dysfunction of VSMCs:contribution of the metabolic inflammation/miRNA pathway.Am J Physiol Endocrinol Metab,2015,308(4):E257-E269.
[22]Tsukita S,Yamada T,Takahashi K,Munakata Y,Hosaka S,Takahashi H,Gao J,Shirai Y,Kodama S,Asai Y,Sugisawa T,Chiba Y,Kaneko K,Uno K,Sawada S,Imai J,Katagiri H.MicroRNAs 106b and 222 improve hyperglycemia in a mouse model of insulin-deficient diabetes via pancreaticβ-cell proliferation.EBio Medicine,2017,15:163-172.
[23]Sisay M,Edessa D.PARP inhibitors as potential therapeutic agents for various cancers:focus on niraparib and its first global approval for maintenance therapy of gynecologic cancers.Gynecol Oncol Res Pract,2017,4:18.
[2]Malgorzewicz S,Skrzypczak-Jankun E,Jankun J.Plasminogen activator inhibitor-1 in kidney pathology(Review).Int J Mol Med,2013,31(3):503-510.
[3]Mishra M,Kowluru RA.Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy.Biochim Biophys Acta,2017,1863(7):1761-1769.
[4]Zakaria EM,El-Bassossy HM,El-Maraghy NN,Ahmed AF,Ali AA.PARP-1 inhibition alleviates diabetic cardiac complications in experimental animals.Eur J Pharmacol,2016,791:444-454.
[5]Zakaria EM,El-Maraghy NN,Ahmed AF,Ali AA,El-Bassossy HM.PARP inhibition ameliorates nephropathy in an animal model of type 2 diabetes:focus on oxidative stress,inflammation,and fibrosis.Naunyn Schmiedebergs Arch Pharmacol,2017,390(6):621-631.
[6]朱恒梅,祝胜郎,陈结慧,叶玲,蒋莹,李向阳.多聚二磷酸腺苷(ADP)核糖聚合酶-1介导高糖致大鼠肾脏系膜细胞t PA/PAI-1的紊乱.海南医学,2012,23(20),18-21.
[7]朱恒梅,祝胜郎,陈结慧,叶玲,蒋莹,李向阳.多聚二磷酸腺苷(ADP)核糖聚合酶-1(PARP-1)在高糖致大鼠肾脏系膜细胞细胞外基质沉积中的作用.中国医药导报,2012,9(31),8-11.
[8]Zhu H,Jiang Z,Lei P,Huang W,Yu X.Poly(ADP-ribose)polymerase-1 mediates angiotensin II-induced expression of plasminogen activator inhibitor-1 and fibronectin in rat mesangial cells.Kidney Blood Press Res,2011,34(5):320-327.
[9]Kato M,Dang V,Wang M,Park JT,Deshpande S,Kadam S,Mardiros A,Zhan Y,Oettgen P,Putta S,Yuan H,Lanting L,Natarajan R.TGF-βinduces acetylation of chromatin and of Ets-1to alleviate repression of miR-192 in diabetic nephropathy.Sci Signal,2013,6(278):ra43.
[10]Kato M,Natarajan R.MicroRNAs in diabetic nephropathy:functions,biomarkers,and therapeutic targets.Ann N Y Acad Sci,2015,1353:72-88.
[11]Lai JY,Luo J,O'Connor C,Jing X,Nair V,Ju W,Randolph A,Ben-Dov IZ,Matar RN,Briskin D,Zavadil J,Nelson RG,Tuschl T,Brosius FC 3rd,Kretzler M,Bitzer M.MicroRNA-21in glomerular injury.J Am Soc Nephrol,2015,26(4):805-816.
[12]何凤,周姗姗,关昌杰,黄俊,陈浩雄,刘日光,傅君舟.微小RNA135b调控Smad5在糖尿病肾病发病中的作用研究.新医学,2017,48(5),317-322.
[13]Allegri L,Rosignolo F,Mio C,Filetti S,Baldan F,Damante G.Effects of nutraceuticals on anaplastic thyroid cancer cells.J Cancer Res Clin Oncol,2018,144(2):285-294.
[14]Shi Z,Zhao C,Guo X,Ding H,Cui Y,Shen R,Liu J.Differential expression of microRNAs in omental adipose tissue from gestational diabetes mellitus subjects reveals miR-222 as a regulator of ERαexpression in estrogen-induced insulin resistance.Endocrinology,2014,155(5):1982-1990.
[15]Lightell DJ Jr,Moss SC,Woods TC.Upregulation of miR-221and-222 in response to increased extracellular signal-regulated kinases 1/2 activity exacerbates neointimal hyperplasia in diabetes mellitus.Atherosclerosis,2018,269:71-78.
[16]Neijenhuis S,Bajrami I,Miller R,Lord CJ,Ashworth A.Identification of miRNA modulators to PARP inhibitor response.DNA Repair(Amst),2013,12(6):394-402.
[17]Sambola A,Ruiz-Meana M,Barba I Del Blanco BG,Barrabés JA,Lip GY,Vilardosa U',Sansaloni S,Rello P,García-Dorado D.Glycative and oxidative stress are associated with altered thrombus composition in diabetic patients with ST-elevation myocardial infarction.Int J Cardiol,2017,243:9-14.
[18]de Candia P,Spinetti G,Specchia C,Sangalli E,La Sala L,Uccellatore A,Lupini S,Genovese S,Matarese G,Ceriello A.A unique plasma microRNA profile defines type 2 diabetes progression.PLo S One,2017,12(12):e0188980.
[19]Pheiffer C,Dias S,Rheeder P,Adam S.Decreased expression of circulating miR-20a-5p in south african women with gestational diabetes mellitus.Mol Diagn Ther,2018,22(3):345-352.
[20]Villard A,Marchand L,Thivolet C,Rome S.Diagnostic value of cell-free circulating microRNAs for obesity and type 2 diabetes:a meta-analysis.J Mol Biomark Diagn,2015,6(6).pii:251.
[21]Li T,Yang GM,Zhu Y,Wu Y,Chen XY,Lan D,Tian KL,Liu LM.Diabetes and hyperlipidemia induce dysfunction of VSMCs:contribution of the metabolic inflammation/miRNA pathway.Am J Physiol Endocrinol Metab,2015,308(4):E257-E269.
[22]Tsukita S,Yamada T,Takahashi K,Munakata Y,Hosaka S,Takahashi H,Gao J,Shirai Y,Kodama S,Asai Y,Sugisawa T,Chiba Y,Kaneko K,Uno K,Sawada S,Imai J,Katagiri H.MicroRNAs 106b and 222 improve hyperglycemia in a mouse model of insulin-deficient diabetes via pancreaticβ-cell proliferation.EBio Medicine,2017,15:163-172.
[23]Sisay M,Edessa D.PARP inhibitors as potential therapeutic agents for various cancers:focus on niraparib and its first global approval for maintenance therapy of gynecologic cancers.Gynecol Oncol Res Pract,2017,4:18.