MELK在舌鳞癌中的表达及对舌鳞癌细胞系CAL-27的迁移与侵袭能力的影响
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摘要
目的母体胚胎亮氨酸拉链激酶(MELK)在多种肿瘤中高表达且与肿瘤的发生与发展密切相关,该研究旨在探讨MELK基因在舌鳞癌中的表达及其对肿瘤生物学行为的影响。方法获取人的舌鳞癌和癌旁组织标本,实时荧光定量PCR和Western blot检测舌鳞癌及癌旁组织中MELK表达。通过siRNA(si001组,si002组)转染技术构建低表达MELK的舌癌细胞系(CAL-27)模型。CCK-8实验检测转染后细胞增殖能力的改变;Transwell小室法检测细胞侵袭能力改变;Transwell小室法检测细胞迁移能力改变。结果 qRT-PCR和Western blot检测发现舌鳞癌组织中MELK基因的表达明显高于癌旁组织(均P<0.05);成功建立了MELK沉默舌鳞癌细胞系模型,沉默组较控制组和正常组MELK表达明显降低(均P<0.05);CCK-8实验测得沉默组细胞活力明显低于正常组和控制组(均P<0.05);Tran-swell实验检测沉默后CAL-27细胞迁移能力,si001组和si002组与正常组和控制组比较,CAL-27细胞迁移能力降低,差异有统计学意义(均P<0.01);Transwell小室法检测细胞侵袭能力,si001组、si002组与正常组和控制组比较,CAL-27细胞侵袭能力降低,差异有统计学意义(均P<0.01)。结论 MELK基因在舌鳞癌组织中高表达;siRNA-MELK转染能够有效地构建舌鳞癌细胞系MELK低表达模型;下调MELK的表达水平可抑制CAL-27细胞的生长增殖,降低迁移与侵袭能力。
Objective Maternal embryonic leucine zipper kinase(MELK)is overexpressed in many tumors and is closely related to the occurrence and development of tumors.This study aimed to investigate the expression of MELK in tongue squamous cell carcinoma and its effect on tumor biological behaviors.Methods The expression of MELK was evaluated at mRNA and protein levels in fresh tongue squamous cell carcinoma(TSCC)and adjacent tissues using quantitative real-time polymerase chain reaction and Western blotting.The tongue cancer cell line(CAL-27)model was constructed by siRNA(si001 group,si002 group)transfection technique to down-express MELK.CCK-8 assay was used to detect changes in cell proliferation after cell transfection;the invasive ability of cells was detected by Matrigel-Transwell chamber method;the migratory ability of cells was detected by Transwell chamber method.Results qPCR and Western blotting showed that the expression of MELK was significantly higher in TSCC tissues than in adjacent tissues(both P<0.05).The MELK gene silenced tongue cancer cell line model was successfully established.The expression of MELK was significantly decreased in CAL-27 cells as compared to that in the control group and the control group(both P<0.05).The cell viability of the siRNA-MELK group was significantly lower than that of the normal and the control groups(both P<0.05).Transwell test showed a weaker migratory ability of CAL-27 cells after transfection than in the normal and the control groups(all P<0.01).The Matrigel-Transwell chamber assay proved the lower cell invasive ability in si-MELK group(si001 group)than in the normal and the control groups(all P<0.01).Conclusion MELK is highly expressed in TSCC tissues.siRNA-MELK transfection technique can effectively construct a low-expressed model of tongue cancer cell line.Down-regulation of MELK expression can inhibit the growth and proliferation of CAL-27 cells and reduce their migratory and invasion ability.
Objective Maternal embryonic leucine zipper kinase(MELK)is overexpressed in many tumors and is closely related to the occurrence and development of tumors.This study aimed to investigate the expression of MELK in tongue squamous cell carcinoma and its effect on tumor biological behaviors.Methods The expression of MELK was evaluated at mRNA and protein levels in fresh tongue squamous cell carcinoma(TSCC)and adjacent tissues using quantitative real-time polymerase chain reaction and Western blotting.The tongue cancer cell line(CAL-27)model was constructed by siRNA(si001 group,si002 group)transfection technique to down-express MELK.CCK-8 assay was used to detect changes in cell proliferation after cell transfection;the invasive ability of cells was detected by Matrigel-Transwell chamber method;the migratory ability of cells was detected by Transwell chamber method.Results qPCR and Western blotting showed that the expression of MELK was significantly higher in TSCC tissues than in adjacent tissues(both P<0.05).The MELK gene silenced tongue cancer cell line model was successfully established.The expression of MELK was significantly decreased in CAL-27 cells as compared to that in the control group and the control group(both P<0.05).The cell viability of the siRNA-MELK group was significantly lower than that of the normal and the control groups(both P<0.05).Transwell test showed a weaker migratory ability of CAL-27 cells after transfection than in the normal and the control groups(all P<0.01).The Matrigel-Transwell chamber assay proved the lower cell invasive ability in si-MELK group(si001 group)than in the normal and the control groups(all P<0.01).Conclusion MELK is highly expressed in TSCC tissues.siRNA-MELK transfection technique can effectively construct a low-expressed model of tongue cancer cell line.Down-regulation of MELK expression can inhibit the growth and proliferation of CAL-27 cells and reduce their migratory and invasion ability.
引文
[1]Kimple A J,Welch C M,Zevallos J P,et al.Oral cavity squamous cell carcinoma---an overview[J].Oral Health Dent Manag,2014,13(3):877-882.
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[3]Burusapat C,Jarungroongruangchai W,Charoenpitakchai M.Prognostic factors of cervical node status in head and neck squamous cell carcinoma[J].World J Surg Oncol,2015,13:51.
[4]Haksever M,Inancli H M,Tuncel U,et al.The effects of tumor size,degree of differentiation,and depth of invasion on the risk of neck node metastasis in squamous cell carcinoma of the oral cavity[J].Ear Nose Throat J,2012,91(3):130-135.
[5]Feng H J,Bao Y L,Liang Z P,et al.Silencing of FANCD2enhances the radiosensitivity of metastatic cervical lymph nodederived head and neck squamous cell carcinoma HSC-4cells[J].Int J Oncol,2017,7(10)1241-1250.
[6]Davezac N,Baldin V,Blot J,et al.Human pEg3kinase associates with and phosphorylates CDC25Bphosphatase:apotential role for pEg3in cell cycle regulation[J].Oncogene,2002,21(50):7630-7641.
[7]Nakano I,Paucar A A,Bajpai R,et al.Maternal embryonic leucine zipper kinase(MELK)regulates multipotent neural progenitor proliferation[J].J Cell Biol,2005,170(3):413-427.
[8]Vulsteke V,Beullens M,Boudrez A,et al.Inhibition of spliceosome assembly by the cell cycle-regulated protein kinase MELK and involvement of splicing factor NIPP1[J].J Biol Chem,2004,279(10):8642-8647.
[9]Beullens M,Vancauwenbergh S,Morrice N,et al.Substrate specificity and activity regulation of protein kinase MELK[J].J Biol Chem,2006,280(48):40003-40011.
[10]Kig C,Beullens M,Beke L,et al.Maternal embryonic leucine zipper kinase(MELK)reduces replication stress in glioblastoma cells[J].J Biol Chem,2013,288(33):24200-24212.
[11]Kuner R,Falth M,Pressinotti N C,et al.The maternal embryonic leucine zipper kinase(MELK)is upregulated in highgrade prostate cancer[J].J Mol Med(Berl),2013,91(2):237-248.
[12]Shimo A,Tanikawa C,Nishidate T,et al.Involvement of kinesin family member 2C/mitotic centromere-associated kinesin overexpression in mammary carcinogenesis[J].Cancer Sci,2008,99(1):62-70.
[13]Marie S K,Okamoto O K,Uno M,et al.Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas[J].Int J Cancer,2008,122(4):807-815.
[14]Nakano I,Masterman-Smith M,Saigusa K,et al.Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors,including brain tumor stem cells[J].J Neurosci Res,2010,86(1):48-60.
[15]Pickard M R,Green A R,Ellis I O,et al.Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer[J].Breast Cancer Res,2009,11(4):1-8.
[16]Du T,Qu Y,Li J,et al.Maternal embryonic leucine zipper kinase enhances gastric cancer progression via the FAK/Paxillin pathway[J].Mol Cancer,2014,13:100.
[17]Krishna Rao S V,Mejia G,Roberts-Thomson K,et al.Epidemiology of oral cancer in Asia in the past decade---an update(2000-2012)[J].Asian Pac J Cancer Prev,2013,14(10):5567-5577.
[18]Mao L.Oral squamous cell carcinoma-progresses from risk assessment to treatment[J].Chin J Dent Res,2012,15(2):83-88.
[19]de Vicente J C,Rodriguez-Santamarta T,Rosado P,et al.Survival after free flap reconstruction in patients with advanced oral squamous cell carcinoma[J].J Oral Maxillofac Surg,2012,70(2):453-459.
[20]Brocklehurst P R,Baker S R,Speight P M.Oral cancer screening:what have we learnt and what is there still to achieve?[J].Future Oncol,2010,6(2):299-304.,
[21]Jiang P,Zhang D.Maternal embryonic leucine zipper kinase(MELK):a novel regulator in cell cycle control,embryonic development,and cancer[J].Int J Mol Sci,2013,14(11):21551-21560.
[22]Ganguly R,Mohyeldin A,Thiel J,et al.MELK-a conserved kinase:functions,signaling,cancer,and controversy[J].Clin Transl Med,2015,4:11
[23]Gray D,Jubb A M,Hogue D,et al.Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38is a promising therapeutic target for multiple cancers[J].Cancer Res,2005,65(21):9751-9761.
[24]Ryu B,Kim D S,Deluca A M,et al.Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression[J].PLoS One,2007,2(7):e594.
[25]Inoue H,Kato T,Olugbile S,et al.Effective growth-suppressive activity of maternal embryonic leucine-zipper kinase(MELK)inhibitor against small cell lung cancer[J].Oncotarget,2016,7(12):13621-13633
[26]Yuen A P,Ho C M,Chow T L,et al.Prospective randomized study of selective neck dissection versus observation for N0neck of early tongue carcinoma[J].Head Neck,2009,31(6):765-772.
[27]Lin M L,Park J H,Nishidate T,et al.Involvement of maternal embryonic leucine zipper kinase(MELK)in mammary carcinogenesis through interaction with Bcl-G,apro-apoptotic member of the Bcl-2family[J].Breast Cancer Res,2007,9(1):1-13.
[28]Nakano I.Maternal embryonic leucine zipper kinase(MELK)regulates multipotent neural progenitor proliferation[J].JCell Biol,2005,170(3):413-427.
[29]Yoshimoto K,Ma X,Guan Y,et al.Expression of stem cell marker and receptor kinase genes in glioblastoma tissue quantified by real-time RT-PCR[J].Brain Tumor Pathol,2011,28(4):291-296.
[30]Hebbard L W,Maurer J,Miller A,et al.Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo[J].Cancer Res,2010,70(21):8863-8873.
[31]Nakano I,Joshi K,Visnyei K,et al.Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway[J].Neuro Oncol,2011,13(6):622-634.
[32]Mathias R A,Gopal S K,Simpson R J.Contribution of cells undergoing epithelial-mesenchymal transition to the tumor microenvironment[J].J Proteomics,2013,78:545-557.
[33]Han M W,Lee J C,Kim Y M,et al.Epithelial-mesenchymal transition:clinical implications for nodal metastasis and prognosis of tongue cancer[J].Otolaryngol Head Neck Surg,2015,152(1):80-86.
[2]Schwam Z G,Judson B L.Improved prognosis for patients with oral cavity squamous cell carcinoma:Analysis of the national cancer database 1998-2006[J].Oral Oncol,2016,52:45-51.
[3]Burusapat C,Jarungroongruangchai W,Charoenpitakchai M.Prognostic factors of cervical node status in head and neck squamous cell carcinoma[J].World J Surg Oncol,2015,13:51.
[4]Haksever M,Inancli H M,Tuncel U,et al.The effects of tumor size,degree of differentiation,and depth of invasion on the risk of neck node metastasis in squamous cell carcinoma of the oral cavity[J].Ear Nose Throat J,2012,91(3):130-135.
[5]Feng H J,Bao Y L,Liang Z P,et al.Silencing of FANCD2enhances the radiosensitivity of metastatic cervical lymph nodederived head and neck squamous cell carcinoma HSC-4cells[J].Int J Oncol,2017,7(10)1241-1250.
[6]Davezac N,Baldin V,Blot J,et al.Human pEg3kinase associates with and phosphorylates CDC25Bphosphatase:apotential role for pEg3in cell cycle regulation[J].Oncogene,2002,21(50):7630-7641.
[7]Nakano I,Paucar A A,Bajpai R,et al.Maternal embryonic leucine zipper kinase(MELK)regulates multipotent neural progenitor proliferation[J].J Cell Biol,2005,170(3):413-427.
[8]Vulsteke V,Beullens M,Boudrez A,et al.Inhibition of spliceosome assembly by the cell cycle-regulated protein kinase MELK and involvement of splicing factor NIPP1[J].J Biol Chem,2004,279(10):8642-8647.
[9]Beullens M,Vancauwenbergh S,Morrice N,et al.Substrate specificity and activity regulation of protein kinase MELK[J].J Biol Chem,2006,280(48):40003-40011.
[10]Kig C,Beullens M,Beke L,et al.Maternal embryonic leucine zipper kinase(MELK)reduces replication stress in glioblastoma cells[J].J Biol Chem,2013,288(33):24200-24212.
[11]Kuner R,Falth M,Pressinotti N C,et al.The maternal embryonic leucine zipper kinase(MELK)is upregulated in highgrade prostate cancer[J].J Mol Med(Berl),2013,91(2):237-248.
[12]Shimo A,Tanikawa C,Nishidate T,et al.Involvement of kinesin family member 2C/mitotic centromere-associated kinesin overexpression in mammary carcinogenesis[J].Cancer Sci,2008,99(1):62-70.
[13]Marie S K,Okamoto O K,Uno M,et al.Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas[J].Int J Cancer,2008,122(4):807-815.
[14]Nakano I,Masterman-Smith M,Saigusa K,et al.Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors,including brain tumor stem cells[J].J Neurosci Res,2010,86(1):48-60.
[15]Pickard M R,Green A R,Ellis I O,et al.Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer[J].Breast Cancer Res,2009,11(4):1-8.
[16]Du T,Qu Y,Li J,et al.Maternal embryonic leucine zipper kinase enhances gastric cancer progression via the FAK/Paxillin pathway[J].Mol Cancer,2014,13:100.
[17]Krishna Rao S V,Mejia G,Roberts-Thomson K,et al.Epidemiology of oral cancer in Asia in the past decade---an update(2000-2012)[J].Asian Pac J Cancer Prev,2013,14(10):5567-5577.
[18]Mao L.Oral squamous cell carcinoma-progresses from risk assessment to treatment[J].Chin J Dent Res,2012,15(2):83-88.
[19]de Vicente J C,Rodriguez-Santamarta T,Rosado P,et al.Survival after free flap reconstruction in patients with advanced oral squamous cell carcinoma[J].J Oral Maxillofac Surg,2012,70(2):453-459.
[20]Brocklehurst P R,Baker S R,Speight P M.Oral cancer screening:what have we learnt and what is there still to achieve?[J].Future Oncol,2010,6(2):299-304.,
[21]Jiang P,Zhang D.Maternal embryonic leucine zipper kinase(MELK):a novel regulator in cell cycle control,embryonic development,and cancer[J].Int J Mol Sci,2013,14(11):21551-21560.
[22]Ganguly R,Mohyeldin A,Thiel J,et al.MELK-a conserved kinase:functions,signaling,cancer,and controversy[J].Clin Transl Med,2015,4:11
[23]Gray D,Jubb A M,Hogue D,et al.Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38is a promising therapeutic target for multiple cancers[J].Cancer Res,2005,65(21):9751-9761.
[24]Ryu B,Kim D S,Deluca A M,et al.Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression[J].PLoS One,2007,2(7):e594.
[25]Inoue H,Kato T,Olugbile S,et al.Effective growth-suppressive activity of maternal embryonic leucine-zipper kinase(MELK)inhibitor against small cell lung cancer[J].Oncotarget,2016,7(12):13621-13633
[26]Yuen A P,Ho C M,Chow T L,et al.Prospective randomized study of selective neck dissection versus observation for N0neck of early tongue carcinoma[J].Head Neck,2009,31(6):765-772.
[27]Lin M L,Park J H,Nishidate T,et al.Involvement of maternal embryonic leucine zipper kinase(MELK)in mammary carcinogenesis through interaction with Bcl-G,apro-apoptotic member of the Bcl-2family[J].Breast Cancer Res,2007,9(1):1-13.
[28]Nakano I.Maternal embryonic leucine zipper kinase(MELK)regulates multipotent neural progenitor proliferation[J].JCell Biol,2005,170(3):413-427.
[29]Yoshimoto K,Ma X,Guan Y,et al.Expression of stem cell marker and receptor kinase genes in glioblastoma tissue quantified by real-time RT-PCR[J].Brain Tumor Pathol,2011,28(4):291-296.
[30]Hebbard L W,Maurer J,Miller A,et al.Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo[J].Cancer Res,2010,70(21):8863-8873.
[31]Nakano I,Joshi K,Visnyei K,et al.Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway[J].Neuro Oncol,2011,13(6):622-634.
[32]Mathias R A,Gopal S K,Simpson R J.Contribution of cells undergoing epithelial-mesenchymal transition to the tumor microenvironment[J].J Proteomics,2013,78:545-557.
[33]Han M W,Lee J C,Kim Y M,et al.Epithelial-mesenchymal transition:clinical implications for nodal metastasis and prognosis of tongue cancer[J].Otolaryngol Head Neck Surg,2015,152(1):80-86.