RANKL/RANK通路介导神经母细胞瘤细胞迁移的机制探讨
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摘要
目的:探讨RANKL/RANK通路对神经母细胞瘤(neuroblastoma,NB)SH-SY5Y细胞系细胞侵袭和转移的作用机制。方法:通过pcDNA3.1~+-RANKL和siRNA-RANKL转染NB SH-SY5Y细胞系过表达或沉默外源基因RANKL;细胞增殖实验(cell counting kit-8,CCK-8)检测外源基因RANKL转染NB SH-SY5Y细胞系对细胞增殖的影响;划痕试验检测外源基因RANKL转染NB SH-SY5Y细胞系对细胞迁移的影响;Western blot实验检测外源性基因RANKL转染NB SH-SY5Y细胞系后细胞内基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)表达变化。结果:通过pcDNA3.1~+-RANKL和siRNA-RANKL对NB SH-SY5Y细胞系转染外源性RANKL基因后三组NB细胞增殖能力无统计学差异;RANKL基因过表达后NB SH-SY5Y细胞迁移能力增强(P<0.01),RANKL沉默后NB SH-SY5Y细胞迁移能力减弱(P<0.01);RANKL基因过表达后NB SH-SY5Y细胞内MMP9、MMP2表达增强(P<0.05、P<0.001),RANKL沉默后NB SH-SY5Y细胞内MMP9、MMP2表达减弱(P<0.05、P<0.01)。结论:RANKL/RANK通路介导NB细胞迁移,并可能通过抑制细胞内MMP2、MMP9表达实现。
Objective:The aim of this study was to investigate the mechanism of cell migration activities on NB SH-SY5 Y.Methods:To evaluate possible contributions of RANKL/RANK axis to cell invasion of neuroblastoma,the over expression vectors pcDNA3.1~+-RANKL and knock down siRNA-RANKL were transiently transfected into SH-SY5 Y cells.The cell growth was evaluated by cell counting kit-8(CCK-8) assay.The transfected-cells of SH-SY5 Y migration were analyzed by wound healing assay.Expression of matrix metalloproteinase 9(MMP9),matrix metalloproteinase 2(MMP2) in transfected cells of SH-SY5 Y was detected by Western blot assay.Results:To evaluate possible contributions of RANKL/RANK signaling pathway to cell invasion/migration of neuroblastoma,the over expression vectors pcDNA3.1~+-RANKL and knock down siRNA-RANKL were transiently transfected into SH-SY5 Y cells.At first,We demonstrated that RANKL/RANK signaling pathway had no effect in SH-SY5 Y proliferation.Moreover overexpression of RANKL accelerated the cell migration,meanwhile increased the expression of MMP9,MMP2(P<0.05,P<0.001),whereas knockdown of RANKL was quite the contray(P<0.05,P<0.01).Conclusion:The effect of cell invasion and migration were inhibited by RANKL/RANK signaling pathway via down-regulation expression of MMP9 and MMP2.
Objective:The aim of this study was to investigate the mechanism of cell migration activities on NB SH-SY5 Y.Methods:To evaluate possible contributions of RANKL/RANK axis to cell invasion of neuroblastoma,the over expression vectors pcDNA3.1~+-RANKL and knock down siRNA-RANKL were transiently transfected into SH-SY5 Y cells.The cell growth was evaluated by cell counting kit-8(CCK-8) assay.The transfected-cells of SH-SY5 Y migration were analyzed by wound healing assay.Expression of matrix metalloproteinase 9(MMP9),matrix metalloproteinase 2(MMP2) in transfected cells of SH-SY5 Y was detected by Western blot assay.Results:To evaluate possible contributions of RANKL/RANK signaling pathway to cell invasion/migration of neuroblastoma,the over expression vectors pcDNA3.1~+-RANKL and knock down siRNA-RANKL were transiently transfected into SH-SY5 Y cells.At first,We demonstrated that RANKL/RANK signaling pathway had no effect in SH-SY5 Y proliferation.Moreover overexpression of RANKL accelerated the cell migration,meanwhile increased the expression of MMP9,MMP2(P<0.05,P<0.001),whereas knockdown of RANKL was quite the contray(P<0.05,P<0.01).Conclusion:The effect of cell invasion and migration were inhibited by RANKL/RANK signaling pathway via down-regulation expression of MMP9 and MMP2.
引文
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[8] Alvi S,Karadaghy O,Manalang M,et al.Clinical manifestations of neuroblastoma with head and neck involvement in children[J].Int J Pediatr Otorhinolaryngol,2017,97:157-162.
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[12] Wang Y,Song Y,Che X,et al.Caveolin 1 enhances RANKL induced gastric cancer cell migration[J].Oncol Rep,2018,40(3):1287-1296.
[13] Ahern E,Harjunp?? H,O'Donnell JS,et al.RANKL blockade improves efficacy of PD1-PD-L1 blockade or dual PD1-PD-L1 and CTLA4 blockade in mouse models of cancer[J].Oncoimmunology,2018.doi:10.1080/2162402X.2018.1431088.
[14] Dünne AA,Gr?be A,Sesterhenn AM,et al.Influence of matrix metallo proteinase 9(MMP-9) on the metastatic behavior of oropharyngeal cancer[J].Anticancer Res,2005,25(6B):4129-4134.
[15] Sugiura Y,Shimada H,Seeger RC,et al.Matrix metalloproteinases-2 and-9 are expressed in human neuroblastoma:Contribution of stromal cells to their production and correlation with metastasis[J].Cancer Res,1998,58(10):2209-2216.
[16] Takahama A Jr,R?as IN,Faustino ISP,et al.Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis[J].Int Endod J,2018,51(7):738-746.
[17] Kambayashi Y,Fujimura T,Furudate S,et al.The expression of matrix metalloproteinases in receptor activator of nuclear factor kappa-b ligand(RANKL)-expressing cancer of apocrine origin[J].Anticancer Res,2018,38(1):113-120.
[2] Armstrong JL,Martin S,Illingworth NA,et al.The impact of retinoic acid treatment on the sensitivity of neuroblastoma cells to fenretinide[J].Oncol Rep,2012,27(1):293-298.
[3] Dubois SG,Kalika Y,Lukens JN,et al.Metastatic sitesin stage Ⅳ and Ⅳs neuroblastoma correlate with age,tumor biology,and survival[J].Pediatr Hematol Oncol,1999,21:181-189.
[4] Liu Ying,Sun Yanli,Cheng Shikai,et al.Clinical analysis of pulmonary metastases at diagnosis of neuroblastoma in pediatric patients[J].Modern Oncology,2013,21(3):604-607.[刘颖,孙艳丽,程士凯,等.小儿神经母细胞瘤肺转移临床特征分析[J].现代肿瘤医学,2013,21(3):604-607.]
[5] Nakashio A,Fujita N,Tsuruo T.Topotecan inhibits VEGF-and bFGF-induced vascular endothelial cell migration via downregulation of the PI3K-Akt signaling pathway[J].Int J Cancer,2002,98(02):36-41.
[6] Aimes RT,Quigley JP.Matrix metalloproteinase-2 is an interstitial collagenase:Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4-and 1/4-length fragments[J].J Biol Chem,1995,270(11):5872-5876.
[7] Raymond SLT,Yugawa D,Chang KHF,et al.Metastatic neoplasms to the pancreas diagnosed by fine-needle aspiration/biopsy cy tology:A 15-year retrospective analysis [J].Diagn Cytopathol,2017,45(9):771-783.
[8] Alvi S,Karadaghy O,Manalang M,et al.Clinical manifestations of neuroblastoma with head and neck involvement in children[J].Int J Pediatr Otorhinolaryngol,2017,97:157-162.
[9] HaDuong JH,Blavier L,Baniwal SK,et al.Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis[J].Int J Cancer,2015,137(4):797-809.
[10] Leibbrandt A,Penninger JM.RANK/RANKL:Regulators of immune responses and bone physiology[J].Ann N Y Acad Sci,2008,11(43):123-150.
[11] Michael L Cheng,Lawrence Fong.Effects of RANKL-targeted therapy in immunity and cancer[J].Front Oncol,2014.doi:10.3389/fonc.2013.00329.
[12] Wang Y,Song Y,Che X,et al.Caveolin 1 enhances RANKL induced gastric cancer cell migration[J].Oncol Rep,2018,40(3):1287-1296.
[13] Ahern E,Harjunp?? H,O'Donnell JS,et al.RANKL blockade improves efficacy of PD1-PD-L1 blockade or dual PD1-PD-L1 and CTLA4 blockade in mouse models of cancer[J].Oncoimmunology,2018.doi:10.1080/2162402X.2018.1431088.
[14] Dünne AA,Gr?be A,Sesterhenn AM,et al.Influence of matrix metallo proteinase 9(MMP-9) on the metastatic behavior of oropharyngeal cancer[J].Anticancer Res,2005,25(6B):4129-4134.
[15] Sugiura Y,Shimada H,Seeger RC,et al.Matrix metalloproteinases-2 and-9 are expressed in human neuroblastoma:Contribution of stromal cells to their production and correlation with metastasis[J].Cancer Res,1998,58(10):2209-2216.
[16] Takahama A Jr,R?as IN,Faustino ISP,et al.Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis[J].Int Endod J,2018,51(7):738-746.
[17] Kambayashi Y,Fujimura T,Furudate S,et al.The expression of matrix metalloproteinases in receptor activator of nuclear factor kappa-b ligand(RANKL)-expressing cancer of apocrine origin[J].Anticancer Res,2018,38(1):113-120.
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