过表达CSRP1基因可促进人肺腺癌H1299细胞的增殖、迁移和侵袭
|
摘要
目的:探讨半胱氨酸和甘氨酸丰富蛋白1(cysteine and glycine rich protein 1,CSRP 1)基因过表达对人肺腺癌H1299细胞增殖、周期、迁移和侵袭的影响,及其可能的作用机制。方法:构建携带有CSRP 1基因的重组慢病毒质粒pCDH-CSRP1;将重组慢病毒Ad-CDH-CSRP1和pCDH-GFP(空载体对照组)分别感染人肺腺癌H1299细胞。实时荧光定量PCR及蛋白质印迹法检测CSRP1 mRNA及蛋白在H1299细胞中的表达水平。CCK-8法和克隆形成实验检测CSRP 1基因过表达对H1299细胞增殖能力的影响;FCM法检测CSRP 1基因过表达对H1299细胞周期的影响。Transwell小室迁移实验和划痕愈合实验检测CSRP 1基因过表达对H1299细胞纵向和横向迁移能力的影响,Transwell小室侵袭实验检测对细胞侵袭能力的影响。蛋白质印迹法检测CSRP 1基因过表达对H1299细胞中黏着斑激酶(focal adhesion kinase,FAK)和磷酸化FAK(phospho-FAK,p-FAK)蛋白表达水平的影响。结果:重组慢病毒质粒pCDH-CSRP1构建成功。转入重组慢病毒质粒pCDH-CSRP1的H1299细胞中CSRP1蛋白的表达水平较pCDH-GFP组明显提高(P<0.01)。CSRP 1基因过表达可促进H1299细胞增殖(P<0.05),G1期细胞所占百分比明显下降(P<0.01),S期细胞所占百分比明显上升(P<0.01);CSRP 1基因过表达可明显增强H1299细胞的迁移和侵袭能力(P值均<0.01)。CSRP 1基因过表达的H1299细胞中p-FAK蛋白的表达水平明显升高(P<0.01),而总FAK蛋白的表达水平无明显变化(P>0.05)。结论:CSRP 1基因过表达可增强人肺腺癌H1299细胞的增殖、迁移和侵袭能力,其机制可能与FAK信号转导通路的激活有关。
Objective: To investigate the effects of cysteine and glycine rich protein 1(CSRP 1) gene overexpression on proliferation, cell cycle, migration and invasion of human lung adenocarcinoma H1299 cells, and to explore the posssible mechanism.Methods: The recombinant lentivirus plasmids pCDH-CSRP1 carrying CSRP 1 gene and pCDHGFP(as the control) were constructed and infected into human lung adenocarcinoma H1299 cells, respectively. The expression levels of CSRP1 mRNA and protein in H1299 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The proliferation of H1299 cells with CSRP 1 gene overexpression was tested by CCK-8 assay and colony formation assay. The cell cycle distribution of H1299 cells was detected by FCM.The longitudinal and lateral migration abilities of H1299 cells were detected by Transwell chamber assay and scratch wound healing assay. The invasion ability of H1299 cells was detected by Transwell chamber assay. The expression levels of focal adhesion kinase(FAK)and phospho-FAK(p-FAK) proteins in H1299 cells were detected by Western blotting.Results: The recombinant lentivirus plasmid pCDH-CSRP1 was successfully constructed, and the expression of CSRP1 in H1299 cells transfected with pCDH-CSRP1 was significantly higher than that in H1299 cells transfected with pCDH-GFP(P < 0.01). The overexpression of CSRP 1 gene promoted the proliferation of H1299 cells(P < 0.05), and contributed to the increased proportion of G1 phase(P < 0.01) and the decreased proportion of S phase(P < 0.01) in H1299 cells.Furthermore, the overexpression of CSRP 1 gene facilitated the migration and invasion abilities of H1299 cells(both P < 0.01). Compared with the control group, the expression level of p-FAK increased significantly(P < 0.01), while the expression level of FAK remained unchanged(P > 0.05)in H1299 cells with CSRP 1 gene overexpression.Conclusion: CSRP 1 gene overexpression can promote the proliferation, migration and invasion of human lung adenocarcinoma H1299 cells, in which the FAK signaling pathway may be involved.
Objective: To investigate the effects of cysteine and glycine rich protein 1(CSRP 1) gene overexpression on proliferation, cell cycle, migration and invasion of human lung adenocarcinoma H1299 cells, and to explore the posssible mechanism.Methods: The recombinant lentivirus plasmids pCDH-CSRP1 carrying CSRP 1 gene and pCDHGFP(as the control) were constructed and infected into human lung adenocarcinoma H1299 cells, respectively. The expression levels of CSRP1 mRNA and protein in H1299 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The proliferation of H1299 cells with CSRP 1 gene overexpression was tested by CCK-8 assay and colony formation assay. The cell cycle distribution of H1299 cells was detected by FCM.The longitudinal and lateral migration abilities of H1299 cells were detected by Transwell chamber assay and scratch wound healing assay. The invasion ability of H1299 cells was detected by Transwell chamber assay. The expression levels of focal adhesion kinase(FAK)and phospho-FAK(p-FAK) proteins in H1299 cells were detected by Western blotting.Results: The recombinant lentivirus plasmid pCDH-CSRP1 was successfully constructed, and the expression of CSRP1 in H1299 cells transfected with pCDH-CSRP1 was significantly higher than that in H1299 cells transfected with pCDH-GFP(P < 0.01). The overexpression of CSRP 1 gene promoted the proliferation of H1299 cells(P < 0.05), and contributed to the increased proportion of G1 phase(P < 0.01) and the decreased proportion of S phase(P < 0.01) in H1299 cells.Furthermore, the overexpression of CSRP 1 gene facilitated the migration and invasion abilities of H1299 cells(both P < 0.01). Compared with the control group, the expression level of p-FAK increased significantly(P < 0.01), while the expression level of FAK remained unchanged(P > 0.05)in H1299 cells with CSRP 1 gene overexpression.Conclusion: CSRP 1 gene overexpression can promote the proliferation, migration and invasion of human lung adenocarcinoma H1299 cells, in which the FAK signaling pathway may be involved.
引文
[1]TORRE LA,BRAY F,SIEGEL RL,et al.Global cancer statistics,2012[J].CACancer J Clin,2015,65(2):87-108.
[2]CHEN W,ZHENG R,BAADE PD,et al.Cancer statistics in China,2015[J].CACancer J Clin,2016,66(2):115-132.
[3]HIRSCH FR,SCAGLIOTTI GV,MULSHINEJL,et al.Lung cancer:current therapies and new targeted treatments[J].Lancet,2017,389(10066):299-311.
[4]ERDEL M,WEISKIRCHEN R.Assignment1 of CSRP1 encoding the LIM domain protein CRP1,to human chromosome 1q32 by fluorescence in situ hybridization[J].Cytogenet Cell Genet,1998,83(1-2):10-11.
[5]KADRMAS JL,BECKERLE MC.The LIMdomain:from the cytoskeleton to the nucleus[J].Nat Rev Mol Cell Biol,2004,5(11):920-931.
[6]HE H,LIU XL,ZHANG HL,et al.SNVand haplotype analysis reveals new CSRP1 variants associated with growth and carcass traits[J].Gene,2013,522(2):206-213.
[7]KARAPISAKOS T,TZILAS V,TRINGIDOU R,et al.Lung cancer in patients with idiopathic pulmonary fibrosis[J].Pulm Pharmacol Ther,2017,45:1-10.
[8]HIRASAWA Y,ARAI M,IMAZEKI F,et al.Methylation status of genes upr egul a t ed by deme thy l a t ing agent 5-aza-2’-deoxycytidine in hepatocellular carcinoma[J].Oncology,2006,71(1-2):77-85.
[9]闫明霞,朱淼鑫,刘蕾,等.实验性肿瘤细胞转移动物模型的活体成像观察[J].实验动物与比较医学,2012,32(5):376-382.
[10]HANAHAN D,WEINBERG RA.Hallmarks of cancer:The next generation[J].Cell,2011,144(5):646-674.
[11]F LAMINI V,J IANG W G,CUI Y.Therapeutic role of miR-140-5p for the treatment of non-small cell lung cancer[J].Anticancer Res,2017,37(8):4319-4327.
[12]张丽,袁伟燕,石红琴,等.Prdx4蛋白表达水平的改变对人肺腺癌A549细胞增殖?凋亡和迁移的影响[J].肿瘤,2017(4):340-349.
[13]TOMASSETTI S,GURIOLI C,RYU JH,et al.The impact of lung cancer on survival of idiopathic pulmonary fibrosis[J].Chest,2015,147(1):157-164.
[14]OZAWA Y,SUDA T,NAITO T,et al.Cumulative incidence of and predictive factors for lung cancer in IPF[J].Respirology,2009,14(5):723-728.
[15]TRAN TC,SINGLETON C,FRALEY TS,et al.Cysteine-rich protein 1(CRP1)regulates actin filament bundling[J].BMC Cell Biol,2005,6:45.
[16]MURPHY DA,COURTNEIDGE SA.The'ins'and'outs'of podosomes and invadopodia:characteristics,formation and function[J].Nat Rev Mol Cell Biol,2011,12(7):413-426.
[17]CERVERO P,HIMMEL M,KRUGER M,et al.Proteomic analysis of podosome fractions from macrophages reveals similarities to spreading initiation centres[J].Eur J Cell Biol,2012,91(11-12):908-922.
[18]XU H,YUAN Y,WU W,et al.Hypoxia stimulates invasion and migration of human cervical cancer cell lines HeLa/SiHa through the Rab11 trafficking of integrin alphavbeta3/FAK/PI3Kpathway-mediated Rac1 activation[J].JBiosci,2017,42(3):491-499.
[19]STAGNO MJ,ZACHAROPOULOU N,Bochem J,et al.Istaroxime inhibits motility and down-regulates orai1 expression,SOCE and FAKphosphorylation in prostate cancer cells[J].Cell Physiol Biochem,2017,42(4):1366-1376.
[20]ZHANG XS,HU YH,GAO HY,et al.Downregulation of Notch1 inhibits the invasion and metastasis of human gastric cancer cells SGC7901and MKN74 in vitro through PTENactivation and dephosphorylation of Akt and FAK[J].Mol Med Rep,2017,16(2):2318-2324.
[21]SULZMAIER FJ,JEAN C,SCHLAEPFER DD.FAK in cancer:mechanistic findings and clinical applications[J].Nat Rev Cancer,2014,14(9):598-610.
[22]SHAN Y,CAO W,WANG T,et al.ZNF259 inhibits non-small cell lung cancer cells proliferation and invasion by FAK-AKT signaling[J].Cancer Manag Res,2017,9:879-889.
[23]GAO H,ZHONG F,XIE J,et al.PTTGpromotes invasion in human breast cancer cell line by upregulating EMMP RINvia FAK/Akt/mTO Rsignaling[J].Am J Cancer Res,2016,6(2):425-439.
[2]CHEN W,ZHENG R,BAADE PD,et al.Cancer statistics in China,2015[J].CACancer J Clin,2016,66(2):115-132.
[3]HIRSCH FR,SCAGLIOTTI GV,MULSHINEJL,et al.Lung cancer:current therapies and new targeted treatments[J].Lancet,2017,389(10066):299-311.
[4]ERDEL M,WEISKIRCHEN R.Assignment1 of CSRP1 encoding the LIM domain protein CRP1,to human chromosome 1q32 by fluorescence in situ hybridization[J].Cytogenet Cell Genet,1998,83(1-2):10-11.
[5]KADRMAS JL,BECKERLE MC.The LIMdomain:from the cytoskeleton to the nucleus[J].Nat Rev Mol Cell Biol,2004,5(11):920-931.
[6]HE H,LIU XL,ZHANG HL,et al.SNVand haplotype analysis reveals new CSRP1 variants associated with growth and carcass traits[J].Gene,2013,522(2):206-213.
[7]KARAPISAKOS T,TZILAS V,TRINGIDOU R,et al.Lung cancer in patients with idiopathic pulmonary fibrosis[J].Pulm Pharmacol Ther,2017,45:1-10.
[8]HIRASAWA Y,ARAI M,IMAZEKI F,et al.Methylation status of genes upr egul a t ed by deme thy l a t ing agent 5-aza-2’-deoxycytidine in hepatocellular carcinoma[J].Oncology,2006,71(1-2):77-85.
[9]闫明霞,朱淼鑫,刘蕾,等.实验性肿瘤细胞转移动物模型的活体成像观察[J].实验动物与比较医学,2012,32(5):376-382.
[10]HANAHAN D,WEINBERG RA.Hallmarks of cancer:The next generation[J].Cell,2011,144(5):646-674.
[11]F LAMINI V,J IANG W G,CUI Y.Therapeutic role of miR-140-5p for the treatment of non-small cell lung cancer[J].Anticancer Res,2017,37(8):4319-4327.
[12]张丽,袁伟燕,石红琴,等.Prdx4蛋白表达水平的改变对人肺腺癌A549细胞增殖?凋亡和迁移的影响[J].肿瘤,2017(4):340-349.
[13]TOMASSETTI S,GURIOLI C,RYU JH,et al.The impact of lung cancer on survival of idiopathic pulmonary fibrosis[J].Chest,2015,147(1):157-164.
[14]OZAWA Y,SUDA T,NAITO T,et al.Cumulative incidence of and predictive factors for lung cancer in IPF[J].Respirology,2009,14(5):723-728.
[15]TRAN TC,SINGLETON C,FRALEY TS,et al.Cysteine-rich protein 1(CRP1)regulates actin filament bundling[J].BMC Cell Biol,2005,6:45.
[16]MURPHY DA,COURTNEIDGE SA.The'ins'and'outs'of podosomes and invadopodia:characteristics,formation and function[J].Nat Rev Mol Cell Biol,2011,12(7):413-426.
[17]CERVERO P,HIMMEL M,KRUGER M,et al.Proteomic analysis of podosome fractions from macrophages reveals similarities to spreading initiation centres[J].Eur J Cell Biol,2012,91(11-12):908-922.
[18]XU H,YUAN Y,WU W,et al.Hypoxia stimulates invasion and migration of human cervical cancer cell lines HeLa/SiHa through the Rab11 trafficking of integrin alphavbeta3/FAK/PI3Kpathway-mediated Rac1 activation[J].JBiosci,2017,42(3):491-499.
[19]STAGNO MJ,ZACHAROPOULOU N,Bochem J,et al.Istaroxime inhibits motility and down-regulates orai1 expression,SOCE and FAKphosphorylation in prostate cancer cells[J].Cell Physiol Biochem,2017,42(4):1366-1376.
[20]ZHANG XS,HU YH,GAO HY,et al.Downregulation of Notch1 inhibits the invasion and metastasis of human gastric cancer cells SGC7901and MKN74 in vitro through PTENactivation and dephosphorylation of Akt and FAK[J].Mol Med Rep,2017,16(2):2318-2324.
[21]SULZMAIER FJ,JEAN C,SCHLAEPFER DD.FAK in cancer:mechanistic findings and clinical applications[J].Nat Rev Cancer,2014,14(9):598-610.
[22]SHAN Y,CAO W,WANG T,et al.ZNF259 inhibits non-small cell lung cancer cells proliferation and invasion by FAK-AKT signaling[J].Cancer Manag Res,2017,9:879-889.
[23]GAO H,ZHONG F,XIE J,et al.PTTGpromotes invasion in human breast cancer cell line by upregulating EMMP RINvia FAK/Akt/mTO Rsignaling[J].Am J Cancer Res,2016,6(2):425-439.