miR-936通过靶向CKS1诱导细胞周期停滞并抑制神经胶质瘤细胞增殖
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摘要
目的探讨微小RNA-936(miR-936)通过靶向细胞周期蛋白依赖性激酶调节亚基1(CKS1)诱导细胞周期停滞并抑制神经胶质瘤细胞增殖行为及其作用机制。方法流式细胞术检测miR-936对胶质瘤细胞细胞周期的影响;细胞增殖-毒性检测试剂盒(CCK-8)和集落形成实验检测miR-936对胶质瘤细胞的增殖行为的影响,miR-936对细胞周期蛋白和通路蛋白的影响情况,双荧光素酶实验检测miR-936和CKS1在神经胶质瘤细胞中的相互作用,miR-936对裸鼠体中的肿瘤生长及肿瘤中的Ki67蛋白水平的影响。结果 miR-936的表达下调可以调控胶质瘤细胞细胞周期抑制胶质瘤细胞增殖行为,抑制miR-936的表达可以抑制胶质瘤细胞的增殖行为,miR-936的异位表达显着抑制U87细胞中的细胞分裂周期蛋白2(CDC2),细胞分裂蛋白激酶2(CDk2),细胞分裂蛋白激酶4(CDK4)和细胞周期蛋白E1(Cyclin E1)的表达,miR-936能与CKS1的3'UTR特异性结合,调控CKS1的表达活性;miR-936的表达下调可以抑制裸鼠体内胶质瘤细胞的生长,并降低Ki67的表达水平。结论 miR-936在胶质瘤的发生发展过程中起重要作用,通过靶向调节CKS1的表达影响胶质瘤细胞的增殖和迁移。
Objective To investigate the role of microRNA-936(miR-936)on cell cycle arrest and glioma cell proliferation induced by cyclin-dependent kinase-regulated subunit 1(CKS1).Method The effect of Mir-936 on the cell cycle of glioma cells was detected by flow cytometry.The proliferation of glioma cells was detected by the proliferation assay and cell proliferation and toxicity test kit(CCK-8).The effect of miR-936 on the expression of cyclin and pathway proteins; the dual luciferase assay was used to detect the interaction between miR-936 and CKS1 in glioma cells,effect of miR-936 on tumor growth and Ki67 in tumor in nude mice.Results The downregulation of miR-936 could regulate the cell cycle inhibition of glioma cell proliferation in glioma cells.Inhibition of miR-936 expression can inhibit the proliferation of glioma cells;ectopic expression of miR-936 significantly inhibited the expression of CDC2,CDk2,CDK4 and Cyclin E1 in U87 cells;miR-936 specifically binds to the 3'UTR of CKS1 and regulates the expression of CKS1.Down-regulation of miR-936 can inhibit the growth of glioma cells and decrease the expression of Ki67 in nude mice.Conclusion MiR-936 plays an important role in the development and progression of glioma,which may affect the proliferation and migration of glioma cells by regulating the expression of CKS1.
Objective To investigate the role of microRNA-936(miR-936)on cell cycle arrest and glioma cell proliferation induced by cyclin-dependent kinase-regulated subunit 1(CKS1).Method The effect of Mir-936 on the cell cycle of glioma cells was detected by flow cytometry.The proliferation of glioma cells was detected by the proliferation assay and cell proliferation and toxicity test kit(CCK-8).The effect of miR-936 on the expression of cyclin and pathway proteins; the dual luciferase assay was used to detect the interaction between miR-936 and CKS1 in glioma cells,effect of miR-936 on tumor growth and Ki67 in tumor in nude mice.Results The downregulation of miR-936 could regulate the cell cycle inhibition of glioma cell proliferation in glioma cells.Inhibition of miR-936 expression can inhibit the proliferation of glioma cells;ectopic expression of miR-936 significantly inhibited the expression of CDC2,CDk2,CDK4 and Cyclin E1 in U87 cells;miR-936 specifically binds to the 3'UTR of CKS1 and regulates the expression of CKS1.Down-regulation of miR-936 can inhibit the growth of glioma cells and decrease the expression of Ki67 in nude mice.Conclusion MiR-936 plays an important role in the development and progression of glioma,which may affect the proliferation and migration of glioma cells by regulating the expression of CKS1.
引文
[1]Trabelsi S,Brahim D H,Ladib M,et al.Glioma epidemiology in the central tunisian population[J].Asian Pac Jo Cancer Prev,2014,15(20):8753-8757.
[2]Rasmussen B K,Hansen S,Laursen R J,et al.Epidemiology of glioma:clinical characteristics,symptoms,and predictors of glioma patients grade I-IV in the the danish neuro-oncology Registry[J].J Neurooncol,2017,135(3):571-579.
[3]Que T,Song Y,Liu Z,et al.Decreased miRNA-637 is an unfavorable prognosis marker and promotes glioma cell growth,migration and invasion via direct targeting Aktl[J].Oncogene,2015,34(38):4952-4963.
[4]Siegal T,Charbit H,Paldor I,et al.Dynamics of circulating hypoxiamediated miRNAs and tumor response in patients with high-grade glioma treated with bevacizumab[J].J Neurosur,2016,125(4):1008-1015.
[5]Shi R,Wang P Y,Li X Y,et al.Exosomal levels of miRNA-21from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients[J].Oncotarget,2015,6(29):26971-2698 1.
[6]Schliesser M G,Claus R,Hielscher T,et al.Prognostic relevance of miRNA-155 methylation in anaplastic glioma[J].Oncotarget,2016,7(50):82028-82045.
[7]Liu S,Yin F,Zhang J,et al.Regulatory roles of miRNA in the human neural stem cell transformation to glioma stem cells[J].J Cell Biochem,2014,115(8):1368-1380.
[8]Grey W,Ivey A,Milne T A,et al.The CKS1/Cks2 axis fine-tunes Mill expression and is crucial for MLL-rearranged leukaemia cell viability[J].Biochim Biophy Acta,2018,1865(1):105-116.
[9]Tomiatti V,Istvanffy R,Pietschmann E,et al.CKS1 is a critical regulator of hematopoietic stem cell quiescence and cycling,operating upstream of Cdk inhibitors[J].Oncogene,2015,34(33):4347-4357.
[10]Amirian E S,Armstrong G N,Zhou R,et al.The glioma internationalcase-control study:a report from the genetic epidemiology of glioma international consortium[J].Am J Epidemiol,2016,183(2):85-91.
[11]Liu M,Zhou K,Cao Y.MicroRNA-944 affects cell growth by targeting EPHA7 in non-small cell lung cancer[J].Inter J Molecular Sci,2016,17(10):B493.
[12]Zhang H,Yan X.Cantharidin modulates the E2F1/MCM7-miR-106b-93/p21-PTEN signaling axis in MCF-7 breast cancer cells[J].Oncol Lett,2015,10(5):2849-2855.
[13]Henriksen M,Johnsen K B,Andersen H H,et al.MicroRNA expression signatures determine prognosis and survival in glioblastoma multiforme—a systematic overview[J]. Mol Neurobiol,2014,50(3):896-913.
[14]Wolter M,Werner T,Malzkorn B,et al.Role of microRNAs located on chromosome arm 10q in malignant gliomas[J].Brain Pathol,2016,26(3):344-358.
[15]Nolte E,Wach S,Silva I T,et al.A new semisynthetic cardenolide analog 3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin(AMANTADIG)affects G2/M cell cycle arrest and miRNA expression profiles and enhances proapoptotic survivin-2B expression in renal cell carcinoma cell lines[J].Oncotarget,2017,8(7):11676-11691.
[16]Tan X,Fu Y,Chen L,et al.miR-671-5p inhibits epithelial-tomesenchymal transition by downregulating FOXM1 expression in breast cancer[J].Oncotarget,2016,7(1):293-307.
[17]Zhao H,Lu Z,Bauzon F,et al.p27T187A knockin identifies Skp2/CKS1 pocket inhibitors for advanced prostate cancer[J].Oncogene,2017,36(1):60-70.
[18]Grey W,Ivey A,Milne T A,et al.The CKS1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability[J].Biochim Biophys Acta,2018,1865(1):105-116.
[19]Cheng C W,Leong K W,Ng Y M,et al.The peptidyl-prolyl isomerase PIN1 relieves cyclin-dependent kinase 2(CDK2)inhibition by the CDK inhibitor p27[J].J Biol Chemi,2017,292(52):21431-21441.
[20]Kukalev A,Ng Y M,Ju L,et al.Deficiency of CKS1 leads to learning and long-term memory defects and p27 dependent formation of neuronal cofilin aggregates[J].Cerebral Cortex,2017,27(1):11-23.
[2]Rasmussen B K,Hansen S,Laursen R J,et al.Epidemiology of glioma:clinical characteristics,symptoms,and predictors of glioma patients grade I-IV in the the danish neuro-oncology Registry[J].J Neurooncol,2017,135(3):571-579.
[3]Que T,Song Y,Liu Z,et al.Decreased miRNA-637 is an unfavorable prognosis marker and promotes glioma cell growth,migration and invasion via direct targeting Aktl[J].Oncogene,2015,34(38):4952-4963.
[4]Siegal T,Charbit H,Paldor I,et al.Dynamics of circulating hypoxiamediated miRNAs and tumor response in patients with high-grade glioma treated with bevacizumab[J].J Neurosur,2016,125(4):1008-1015.
[5]Shi R,Wang P Y,Li X Y,et al.Exosomal levels of miRNA-21from cerebrospinal fluids associated with poor prognosis and tumor recurrence of glioma patients[J].Oncotarget,2015,6(29):26971-2698 1.
[6]Schliesser M G,Claus R,Hielscher T,et al.Prognostic relevance of miRNA-155 methylation in anaplastic glioma[J].Oncotarget,2016,7(50):82028-82045.
[7]Liu S,Yin F,Zhang J,et al.Regulatory roles of miRNA in the human neural stem cell transformation to glioma stem cells[J].J Cell Biochem,2014,115(8):1368-1380.
[8]Grey W,Ivey A,Milne T A,et al.The CKS1/Cks2 axis fine-tunes Mill expression and is crucial for MLL-rearranged leukaemia cell viability[J].Biochim Biophy Acta,2018,1865(1):105-116.
[9]Tomiatti V,Istvanffy R,Pietschmann E,et al.CKS1 is a critical regulator of hematopoietic stem cell quiescence and cycling,operating upstream of Cdk inhibitors[J].Oncogene,2015,34(33):4347-4357.
[10]Amirian E S,Armstrong G N,Zhou R,et al.The glioma internationalcase-control study:a report from the genetic epidemiology of glioma international consortium[J].Am J Epidemiol,2016,183(2):85-91.
[11]Liu M,Zhou K,Cao Y.MicroRNA-944 affects cell growth by targeting EPHA7 in non-small cell lung cancer[J].Inter J Molecular Sci,2016,17(10):B493.
[12]Zhang H,Yan X.Cantharidin modulates the E2F1/MCM7-miR-106b-93/p21-PTEN signaling axis in MCF-7 breast cancer cells[J].Oncol Lett,2015,10(5):2849-2855.
[13]Henriksen M,Johnsen K B,Andersen H H,et al.MicroRNA expression signatures determine prognosis and survival in glioblastoma multiforme—a systematic overview[J]. Mol Neurobiol,2014,50(3):896-913.
[14]Wolter M,Werner T,Malzkorn B,et al.Role of microRNAs located on chromosome arm 10q in malignant gliomas[J].Brain Pathol,2016,26(3):344-358.
[15]Nolte E,Wach S,Silva I T,et al.A new semisynthetic cardenolide analog 3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin(AMANTADIG)affects G2/M cell cycle arrest and miRNA expression profiles and enhances proapoptotic survivin-2B expression in renal cell carcinoma cell lines[J].Oncotarget,2017,8(7):11676-11691.
[16]Tan X,Fu Y,Chen L,et al.miR-671-5p inhibits epithelial-tomesenchymal transition by downregulating FOXM1 expression in breast cancer[J].Oncotarget,2016,7(1):293-307.
[17]Zhao H,Lu Z,Bauzon F,et al.p27T187A knockin identifies Skp2/CKS1 pocket inhibitors for advanced prostate cancer[J].Oncogene,2017,36(1):60-70.
[18]Grey W,Ivey A,Milne T A,et al.The CKS1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability[J].Biochim Biophys Acta,2018,1865(1):105-116.
[19]Cheng C W,Leong K W,Ng Y M,et al.The peptidyl-prolyl isomerase PIN1 relieves cyclin-dependent kinase 2(CDK2)inhibition by the CDK inhibitor p27[J].J Biol Chemi,2017,292(52):21431-21441.
[20]Kukalev A,Ng Y M,Ju L,et al.Deficiency of CKS1 leads to learning and long-term memory defects and p27 dependent formation of neuronal cofilin aggregates[J].Cerebral Cortex,2017,27(1):11-23.