MTBP调控前列腺癌细胞的迁移和侵袭
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摘要
目的研究鼠双微体2癌基因结合蛋白(MTBP)对人前列腺癌细胞迁移及侵袭能力的影响及其作用机制。方法用Western blot检测MTBP在22RV1、DU145及Lncap三种不同前列腺癌细胞中的表达水平。采用siRNA及MTBP质粒分别转染细胞,转染48 h后,用Western blot检测MTBP蛋白的表达量,划痕实验检测细胞的平行迁移能力,Transwell实验检测细胞的垂直迁移及侵袭能力。用Western blot检测细胞上皮间质转化(EMT)标志分子E-cadherin蛋白表达量的变化。结果在前列腺癌细胞中,MTBP在转移性前列腺癌细胞DU145中表达最高,Lncap细胞次之,局限性前列腺癌细胞22RV1中最低,MTBP的表达水平与前列腺癌细胞的转移侵袭能力呈正相关。siRNA干扰及MTBP质粒转染细胞分别能显著降低或提高前列腺癌细胞中MTBP的蛋白表达水平。划痕实验结果显示抑制MTBP的表达后,前列腺癌细胞的迁移能力降低,而MTBP过表达能显著促进前列腺癌细胞的迁移(P<0.01)。Transwell实验发现抑制MTBP的表达可降低前列腺癌细胞的迁移侵袭能力,而MTBP过表达后,能显著促进前列腺癌细胞的迁移侵袭能力(P<0.01);Western blot结果显示抑制MTBP的表达可使前列腺癌细胞的Ecadherin蛋白表达水平升高,而MTBP过表达的前列腺癌细胞中E-cadherin蛋白表达水平降低。结论 MTBP可促进前列腺癌细胞的迁移和侵袭,其作用机制可能与诱导EMT有关。
Objective To investigate the role of MTBP in regulating the migration and invasion of human prostate cancer cells.Methods The baseline expressions of MTBP in 3 different human prostate cancer cells lines(22RV1, DU145 and Lncap) were detected using Western blotting. The cells were transfected with a small interfering RNA(siRNA) for MTBP knockdown or MTBP plasmid for MTBP overexpression, and 48 h later, the cells were examined for MTBP expression with Western blotting;the changes in the migration abilities of the cells were evaluated using wound healing assay and Transwell assay, and the cell invasiveness was assessed using Matrigel Transwell assay. The expression of E-cadherin protein, a marker of epithelial mesenchymal transition(EMT), was detected using Western blotting. Results MTBP expression was the highest in DU145 cells followed by Lncap cells, and was the lowest in 22RV1 cells, indicating a positive correlation of MTBP expression with the level of malignancy of human prostate cancer cells. Transfection of the cells with siRNA or MTBP plasmids efficiently lowered or enhanced the expressions of MTBP in human prostate cancer cells. Wound healing assay showed that inhibition of MTBP expression decreased the migration ability of the prostate cancer cells, and MTBP overexpression significantly promoted the migration of the cells(P<0.01). Transwell assay showed that MTBP knockdown significantly lowered the migration and invasion ability of the cells, while MTBP overexpression markedly increased the number of migrating and invading cells(P<0.01); Western blotting results showed that MTBP knockdown increased the expression of E-cadherin protein, and MTBP overexpression decreased E-cadherin expression in the prostate cancer cells. Conclusion MTBP overexpression promotes the migration and invasion of human prostate cancer cells possibly relation to the induction of EMT.
Objective To investigate the role of MTBP in regulating the migration and invasion of human prostate cancer cells.Methods The baseline expressions of MTBP in 3 different human prostate cancer cells lines(22RV1, DU145 and Lncap) were detected using Western blotting. The cells were transfected with a small interfering RNA(siRNA) for MTBP knockdown or MTBP plasmid for MTBP overexpression, and 48 h later, the cells were examined for MTBP expression with Western blotting;the changes in the migration abilities of the cells were evaluated using wound healing assay and Transwell assay, and the cell invasiveness was assessed using Matrigel Transwell assay. The expression of E-cadherin protein, a marker of epithelial mesenchymal transition(EMT), was detected using Western blotting. Results MTBP expression was the highest in DU145 cells followed by Lncap cells, and was the lowest in 22RV1 cells, indicating a positive correlation of MTBP expression with the level of malignancy of human prostate cancer cells. Transfection of the cells with siRNA or MTBP plasmids efficiently lowered or enhanced the expressions of MTBP in human prostate cancer cells. Wound healing assay showed that inhibition of MTBP expression decreased the migration ability of the prostate cancer cells, and MTBP overexpression significantly promoted the migration of the cells(P<0.01). Transwell assay showed that MTBP knockdown significantly lowered the migration and invasion ability of the cells, while MTBP overexpression markedly increased the number of migrating and invading cells(P<0.01); Western blotting results showed that MTBP knockdown increased the expression of E-cadherin protein, and MTBP overexpression decreased E-cadherin expression in the prostate cancer cells. Conclusion MTBP overexpression promotes the migration and invasion of human prostate cancer cells possibly relation to the induction of EMT.
引文
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[2]Chen MK,Luo Y,Zhang H,et al.Investigation of optimal prostate biopsy schemes for Chinese patients with different clinical characteristics[J].Urol Int,2012,89(4):425-32.
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[6]BradyMM.Regulation of p53 andMDM2 activity byMTBP[J].Mol Cell Biol,2005,25(2):545-53.
[7]Boyd MT,Vlatkovic N,Haines DS.A novel cellular protein(MTBP)binds to MDM2 and induces a G(1)arrest that is suppressed by MDM2[J].J Biol Chem,2000,275(41):31883-90.
[8]Ammer AG,Weed SA.Cortactin branches out:Roles in regulating protrusive actin dynamics[J].Cell Motil Cytoskeleton,2008,65(9):687-707.
[9]Iwakuma TG.MDM2,an introduction[J].Mol Cancer Res,2003,1(14):993-1000.
[10]Grieb BC,Gramling MW,Arrate MP,et al.Oncogenic protein MTBPinteracts with MYC to promote tumorigenesis[J].Cancer Res,2014,74(13):3591-602.
[11]Lu S,Zhou W,Wei HY,et al.MTBP promotes the invasion and metastasis of hepatocellular carcinoma by enhancing the MDM2-Mediated degradation of E-Cadherin[J].Dig Dis Sci,2015,60(12):3681-90.
[12]Grieb BC,Chen X,Eischen CM.MTBP is overexpressed in TripleNegative breast cancer and contributes to its growth and survival[J].Mol Cancer Res,2014,12(9):1216-24.
[13]Mao Y,Tian M,Pan B,et al.Hyper expression of MTBP May be an adverse signal for the survival of some malignant tumors[J].Medicine,2018,97(35):e12021.
[14]Barretina J,Caponigro G,Stransky N,et al.The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity[J].Nature,2012,483(7391):603-7.
[15]李根英,张云艳.MTBP与癌症的研究进展[J].实用肿瘤学杂志,2016,30(6):560-2.
[16]Venkatesan T,Alaseem A,Chinnaiyan AA,et al.MDM2 overexpression modulates the angiogenesis-related gene expression profile of prostate cancer cells[J].Cells,2018,7(5):41.
[17]Wang W,Chen Z,Jin JJ,et al.MDM2 binding protein as a predictor of metastasis and a novel prognostic biomarker in patients with gastric cancer[J].Oncol Lett,2017,14(6):6409-16.
[18]Lu X,Pan XL,Wu CJ,et al.An in vivo screen identifies PYGO2 as a driver for metastatic prostate cancer[J].Cancer Res,2018,78(14):3823-33.
[19]Gupta GP,Massagué J.Cancer metastasis:building a framework[J].Cell,2006,127(4):679-95.
[20]Pandya P,Orgaz JL,Sanz-Moreno V.Modes of invasion during tumour dissemination[J].Mol Oncol,2017,11(1):5-27.
[21]Christofori G.New signals from the invasive front[J].Nature,2006,441(792):444-50.
[22]Huber MA,Kraut N,Beuq H.Molecular requirements for epithelialmesenchymal transition during tumor progression[J].Curr Opin Cell Biol,2005,17(5):548-58.
[23]Lee JM,Dedhar S,Kalluri R,et al.The epithelial-mesenchymal transition:new insights in signaling,development,and disease[J].JCell Biol,2006,172(7):973-81.
[24]Thiery JP.Epithelial-mesenchymal transitions in tumour progression[J].Nat Rev Cancer,2002,2(6):442-54.
[25]Tse JC,Kalluri R.Mechanisms of metastasis:epithelial-to-mesenchymal transition and contribution of tumor microenvironment[J].JCell Biochem,2007,101(4):816-29.
[26]Christiansen JJ,Rajasekaran AK.Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J].Cancer Res,2006,66(17):8319-26.
[27]Smith BN,Bhowmick NA.Role of EMT in metastasis and therapy resistance[J].J ClinMed,2016,5(2):17.
[28]Serrano-Gomez SJ,MaziveyiM,Alahari SK.Regulation of epithelialmesenchymal transition through epigenetic and post-translational modifications[J].Mol Cancer,2016,15(1):18.
[29]Nakaya Y,Sheng G.EMT in developmental morphogenesis[J].Cancer Lett,2013,341(1):9-15.
[30]Polyak K,Weinberg RA.Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits[J].Nat Rev Cancer,2009,9(4):265-73.
[31]张可华,宋建国.EMT与肿瘤[J].生命的化学,2008,28(5):523-6.
[32]Lee TK,Poon RT,Yuen AP,et al.Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition[J].Clin Cancer Res,2006,12(18):5369-76.
[33]Alonso SR,Tracey L,Ortiz PA,et al.A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis[J].Cancer Res,2007,67(7):3450-60.
[2]Chen MK,Luo Y,Zhang H,et al.Investigation of optimal prostate biopsy schemes for Chinese patients with different clinical characteristics[J].Urol Int,2012,89(4):425-32.
[3]Siegel RA.Cancer statistics,2018[J].CACancer J Clin,2018,68(1):7-30.
[4]Ross RW,Xie WL,Regan MM,et al.Efficacy of androgen deprivation therapy(ADT)in patients with advanced prostate[J].Cancer,2008,112(6):1247-53.
[5]Psutka SP,Frank I,Karnes RJ.Risk stratification in hormonesensitive metastatic prostate cancer:more questions than answers[J].Eur Urol,2015,68(2):205-6.
[6]BradyMM.Regulation of p53 andMDM2 activity byMTBP[J].Mol Cell Biol,2005,25(2):545-53.
[7]Boyd MT,Vlatkovic N,Haines DS.A novel cellular protein(MTBP)binds to MDM2 and induces a G(1)arrest that is suppressed by MDM2[J].J Biol Chem,2000,275(41):31883-90.
[8]Ammer AG,Weed SA.Cortactin branches out:Roles in regulating protrusive actin dynamics[J].Cell Motil Cytoskeleton,2008,65(9):687-707.
[9]Iwakuma TG.MDM2,an introduction[J].Mol Cancer Res,2003,1(14):993-1000.
[10]Grieb BC,Gramling MW,Arrate MP,et al.Oncogenic protein MTBPinteracts with MYC to promote tumorigenesis[J].Cancer Res,2014,74(13):3591-602.
[11]Lu S,Zhou W,Wei HY,et al.MTBP promotes the invasion and metastasis of hepatocellular carcinoma by enhancing the MDM2-Mediated degradation of E-Cadherin[J].Dig Dis Sci,2015,60(12):3681-90.
[12]Grieb BC,Chen X,Eischen CM.MTBP is overexpressed in TripleNegative breast cancer and contributes to its growth and survival[J].Mol Cancer Res,2014,12(9):1216-24.
[13]Mao Y,Tian M,Pan B,et al.Hyper expression of MTBP May be an adverse signal for the survival of some malignant tumors[J].Medicine,2018,97(35):e12021.
[14]Barretina J,Caponigro G,Stransky N,et al.The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity[J].Nature,2012,483(7391):603-7.
[15]李根英,张云艳.MTBP与癌症的研究进展[J].实用肿瘤学杂志,2016,30(6):560-2.
[16]Venkatesan T,Alaseem A,Chinnaiyan AA,et al.MDM2 overexpression modulates the angiogenesis-related gene expression profile of prostate cancer cells[J].Cells,2018,7(5):41.
[17]Wang W,Chen Z,Jin JJ,et al.MDM2 binding protein as a predictor of metastasis and a novel prognostic biomarker in patients with gastric cancer[J].Oncol Lett,2017,14(6):6409-16.
[18]Lu X,Pan XL,Wu CJ,et al.An in vivo screen identifies PYGO2 as a driver for metastatic prostate cancer[J].Cancer Res,2018,78(14):3823-33.
[19]Gupta GP,Massagué J.Cancer metastasis:building a framework[J].Cell,2006,127(4):679-95.
[20]Pandya P,Orgaz JL,Sanz-Moreno V.Modes of invasion during tumour dissemination[J].Mol Oncol,2017,11(1):5-27.
[21]Christofori G.New signals from the invasive front[J].Nature,2006,441(792):444-50.
[22]Huber MA,Kraut N,Beuq H.Molecular requirements for epithelialmesenchymal transition during tumor progression[J].Curr Opin Cell Biol,2005,17(5):548-58.
[23]Lee JM,Dedhar S,Kalluri R,et al.The epithelial-mesenchymal transition:new insights in signaling,development,and disease[J].JCell Biol,2006,172(7):973-81.
[24]Thiery JP.Epithelial-mesenchymal transitions in tumour progression[J].Nat Rev Cancer,2002,2(6):442-54.
[25]Tse JC,Kalluri R.Mechanisms of metastasis:epithelial-to-mesenchymal transition and contribution of tumor microenvironment[J].JCell Biochem,2007,101(4):816-29.
[26]Christiansen JJ,Rajasekaran AK.Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis[J].Cancer Res,2006,66(17):8319-26.
[27]Smith BN,Bhowmick NA.Role of EMT in metastasis and therapy resistance[J].J ClinMed,2016,5(2):17.
[28]Serrano-Gomez SJ,MaziveyiM,Alahari SK.Regulation of epithelialmesenchymal transition through epigenetic and post-translational modifications[J].Mol Cancer,2016,15(1):18.
[29]Nakaya Y,Sheng G.EMT in developmental morphogenesis[J].Cancer Lett,2013,341(1):9-15.
[30]Polyak K,Weinberg RA.Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits[J].Nat Rev Cancer,2009,9(4):265-73.
[31]张可华,宋建国.EMT与肿瘤[J].生命的化学,2008,28(5):523-6.
[32]Lee TK,Poon RT,Yuen AP,et al.Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition[J].Clin Cancer Res,2006,12(18):5369-76.
[33]Alonso SR,Tracey L,Ortiz PA,et al.A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis[J].Cancer Res,2007,67(7):3450-60.