ERH基因对人膀胱癌T24和5637细胞迁移、侵袭的影响
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摘要
目的研究基本同源物增强子(ERH)对人膀胱癌T24和5637细胞迁移和侵袭作用的影响。方法将人膀胱癌T24和5637细胞的ERH敲除后,采用细胞划痕实验、Transwell细胞迁移实验、Transwell细胞侵袭实验验证其迁移和侵袭功能变化,Western blot检测细胞迁移与侵袭相关蛋白的表达,并通过裸鼠尾静脉转移实验验证敲除ERH基因后在体内对膀胱癌细胞转移能力的影响。结果 (1)细胞划痕实验结果显示,敲除ERH基因后的人膀胱癌5637和T24细胞迁移率均明显降低(P <0. 05);(2)Transwell细胞迁移实验、Transwell细胞侵袭实验结果显示,敲除ERH基因后的人膀胱癌5637和T24细胞迁移细胞计数和侵袭细胞计数均明显减少(P <0. 05)。(3)Western blot实验结果显示,敲除ERH基因后的人膀胱癌5637细胞和T24细胞中E-Cadherin表达明显升高(P <0. 05),而Fibronectin、Twist、Vimentin、Snail2蛋白的表达明显降低(P <0. 05)。(4)裸鼠尾静脉转移实验显示,与ERH正常组相比,ERH敲除组小鼠肺转移灶明显减少。结论体外和体内实验均提示,ERH基因敲除影响人膀胱癌T24和5637细胞的迁移与侵袭。
Objective To study the effect of enhancer of rudimentary homolog( ERH) gene on migration and invasion in human bladder cancer T24 and 5637 cells. Methods After knocking out the ERH gene of human bladder cancer T24 and 5637 cells,Wound healing assay,Transwell cell migration assay and Transwell cell invasion assay were used to verify the migration and invasion function. Cell migration related protein was detected by Western blot. Nude mouse tail vein transfer assay was used to study the metastasis ability of bladder cancer cells in vivo. Results (1)The Wound healing assay showed that there were significant differences in the migration cell counts of human bladder cancer 5637 and T24( P < 0. 05). (2) There were significant differences in migration and invasion cell counts of Transwell assay( P < 0. 05). (3) Western blot showed that the expression of E-Cadherin in human bladder cancer 5637 cells and T24 cells was significantly increased( P < 0. 05) after knocking out ERH gene,while the expression of Fibronectin,Twist,Vimentin and Snail2 protein were significantly decreased( P < 0. 05). (4) Nude mouse tail vein transfer assay showed that lung metastases were significantly reduced in the ERH knockout group compared with the normal ERH group. Conclusions Both in vitro and in vivo experiments suggest that ERH knockout affects the migration and invasion of human bladder cancer T24 and 5637 cells.
Objective To study the effect of enhancer of rudimentary homolog( ERH) gene on migration and invasion in human bladder cancer T24 and 5637 cells. Methods After knocking out the ERH gene of human bladder cancer T24 and 5637 cells,Wound healing assay,Transwell cell migration assay and Transwell cell invasion assay were used to verify the migration and invasion function. Cell migration related protein was detected by Western blot. Nude mouse tail vein transfer assay was used to study the metastasis ability of bladder cancer cells in vivo. Results (1)The Wound healing assay showed that there were significant differences in the migration cell counts of human bladder cancer 5637 and T24( P < 0. 05). (2) There were significant differences in migration and invasion cell counts of Transwell assay( P < 0. 05). (3) Western blot showed that the expression of E-Cadherin in human bladder cancer 5637 cells and T24 cells was significantly increased( P < 0. 05) after knocking out ERH gene,while the expression of Fibronectin,Twist,Vimentin and Snail2 protein were significantly decreased( P < 0. 05). (4) Nude mouse tail vein transfer assay showed that lung metastases were significantly reduced in the ERH knockout group compared with the normal ERH group. Conclusions Both in vitro and in vivo experiments suggest that ERH knockout affects the migration and invasion of human bladder cancer T24 and 5637 cells.
引文
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[12] Tsubota SI,Phillips AC. Drosophila Enhancer of Rudimentary Homolog,ERH,Is a Binding Partner of RPS3,RPL19,and DDIT4,Suggesting a Mechanism for the Nuclear Localization of ERH[J]. Mol Biol Int,2016,2016:8371819. DOI:10. 1155/2016/8371819.
[13] Smyk A,Szuminska M,Uniewicz KA,et al. Human enhancer of rudimentary is a molecular partner of PDIP46/SKAR,a protein interacting with DNA polymerase delta and S6K1 and regulating cell growth[J]. FEBS J,2006,273(20):4728-4741. DOI:10.1111/j. 1742-4658. 2006. 05477. x.
[14] Lukasik A,Uniewicz KA,Kulis M,et al. Ciz1,a p21 cip1/Waf1-interacting zinc finger protein and DNA replication factor,is a novel molecular partner for human enhancer of rudimentary homolog[J]. FEBS J,2008,275(2):332-340. DOI:10. 1111/j.1742-4658. 2007. 06203. x.
[15] Fujimura A,Kishimoto H,Yanagisawa J,et al. Enhancer of rudimentary homolog(ERH)plays an essential role in the progression of mitosis by promoting mitotic chromosome alignment[J]. Biochem Biophys Res Commun,2012,423(3):588-592. DOI:10.1016/j. bbrc. 2012. 06. 018.
[16] Sugiyama T,Thillainadesan G,Chalamcharla VR,et al. Enhancer of Rudimentary Cooperates with Conserved RNA-Processing Factors to Promote Meiotic mRNA Decay and Facultative Heterochromatin Assembly[J]. Mol Cell,2016,61(5):747-759. DOI:10. 1016/j. molcel. 2016. 01. 029.
[17] Weng MT,Lee JH,Wei SC,et al. Evolutionarily conserved protein ERH controls CENP-E mRNA splicing and is required for the survival of KRAS mutant cancer cells[J]. Proc Natl Acad Sci U S A, 2012,109(52):E3659-E3667. DOI:10. 1073/pnas.1207673110.
[18] Weng MT,Luo J. The enigmatic ERH protein:its role in cell cycle,RNA splicing and cancer[J]. Protein Cell,2013,4(11):807-812. DOI:10. 1007/s13238-013-3056-3.
[19]赵振伶,邵焕军,郝丽娜,等. miR-369-3p通过调控VEGFC基因表达对膀胱癌细胞增殖及凋亡的影响[J].中国医师杂志,2018,20(1):92-95,99. DOI:10. 3760/cma. j. issn. 1008-1372. 2018. 01. 023.
[20]余昆,张悦,刘胜,等.膀胱部分切除术联合化疗治疗局限性肌层浸润性膀胱癌的临床分析(附53例报告)[J].中国医师杂志,2017,19(8):1188-1191,1195. DOI:10. 3760/cma. j.issn. 1008-1372. 2017. 08. 017.
[2] Zheng L,Chen K,Zhu L,et al. Low expression of DAB2IP predicts an unfavorable prognosis in human bladder carcinoma[J].Onco Targets Ther,2017,10:5719-5726. DOI:10. 2147/OTT.S146952.
[3] Dettlaff K,Stawny M,Ogrodowczyk M,et al. Formulation and characterization of EGCG for the treatment of superficial bladder cancer[J]. Int J Mol Med,2017,40(2):329-336. DOI:10.3892/ijmm. 2017. 3024.
[4] Gelsthorpe M,Pulumati M,Mc Callum C,et al. The putative cell cycle gene,enhancer of rudimentary,encodes a highly conserved protein found in plants and animals[J]. Gene,1997,186(2):189-195.
[5] Pang K,Lv Q,Ning SY,et al. ERH is up-regulated in bladder cancer and regulates the proliferation and apoptosis of T24 bladder cancer cells[J]. Int J Clin Exp Med,2017,10(11):15269-15277.
[6] Arai R,Kukimoto-Niino M,Uda-Tochio H,et al. Crystal structure of an enhancer of rudimentary homolog(ERH)at 2. 1 Angstroms resolution[J]. Protein Sci,2005,14(7):1888-1893. DOI:10.1110/ps. 051484505.
[7] Wan C,Tempel W,Liu ZJ,et al. Structure of the conserved transcriptional repressor enhancer of rudimentary homolog[J]. Biochemistry,2005,44(13):5017-5023.DOI:10. 1021/bi047785w.
[8] Jin T,Guo F,Serebriiskii IG,et al. A 1. 55 A resolution X-ray crystal structure of HEF2/ERH and insights into its transcriptional and cell-cycle interaction networks[J].Proteins,2007,68(2):427-437.DOI:10. 1002/prot. 21343.
[9] Pogge v SE,Senkel S,Ryffel GU.ERH(enhancer of rudimentary homologue),a conserved factor identical between frog and human,is a transcriptional repressor[J].Biol Chem,2001,382(9):1379-1385. DOI:10. 1515/BC. 2001.170.
[10] Kavanaugh G,Zhao R,Guo Y,et al. Enhancer of Rudimentary Homolog Affects the Replication Stress Response through Regulation of RNA Processing[J]. Mol Cell Biol,2015,35(17):2979-2990.DOI:10. 1128/MCB. 01276-14.
[11] Krzyzanowski MK,Kozlowska E,Kozlowski P. Identification and functional analysis of the erh1(+)gene encoding enhancer of rudimentary homolog from the fission yeast Schizosaccharomyces pombe[J]. PLo S One, 2012,7(11):e49059. DOI:10. 1371/journal. pone. 0049059.
[12] Tsubota SI,Phillips AC. Drosophila Enhancer of Rudimentary Homolog,ERH,Is a Binding Partner of RPS3,RPL19,and DDIT4,Suggesting a Mechanism for the Nuclear Localization of ERH[J]. Mol Biol Int,2016,2016:8371819. DOI:10. 1155/2016/8371819.
[13] Smyk A,Szuminska M,Uniewicz KA,et al. Human enhancer of rudimentary is a molecular partner of PDIP46/SKAR,a protein interacting with DNA polymerase delta and S6K1 and regulating cell growth[J]. FEBS J,2006,273(20):4728-4741. DOI:10.1111/j. 1742-4658. 2006. 05477. x.
[14] Lukasik A,Uniewicz KA,Kulis M,et al. Ciz1,a p21 cip1/Waf1-interacting zinc finger protein and DNA replication factor,is a novel molecular partner for human enhancer of rudimentary homolog[J]. FEBS J,2008,275(2):332-340. DOI:10. 1111/j.1742-4658. 2007. 06203. x.
[15] Fujimura A,Kishimoto H,Yanagisawa J,et al. Enhancer of rudimentary homolog(ERH)plays an essential role in the progression of mitosis by promoting mitotic chromosome alignment[J]. Biochem Biophys Res Commun,2012,423(3):588-592. DOI:10.1016/j. bbrc. 2012. 06. 018.
[16] Sugiyama T,Thillainadesan G,Chalamcharla VR,et al. Enhancer of Rudimentary Cooperates with Conserved RNA-Processing Factors to Promote Meiotic mRNA Decay and Facultative Heterochromatin Assembly[J]. Mol Cell,2016,61(5):747-759. DOI:10. 1016/j. molcel. 2016. 01. 029.
[17] Weng MT,Lee JH,Wei SC,et al. Evolutionarily conserved protein ERH controls CENP-E mRNA splicing and is required for the survival of KRAS mutant cancer cells[J]. Proc Natl Acad Sci U S A, 2012,109(52):E3659-E3667. DOI:10. 1073/pnas.1207673110.
[18] Weng MT,Luo J. The enigmatic ERH protein:its role in cell cycle,RNA splicing and cancer[J]. Protein Cell,2013,4(11):807-812. DOI:10. 1007/s13238-013-3056-3.
[19]赵振伶,邵焕军,郝丽娜,等. miR-369-3p通过调控VEGFC基因表达对膀胱癌细胞增殖及凋亡的影响[J].中国医师杂志,2018,20(1):92-95,99. DOI:10. 3760/cma. j. issn. 1008-1372. 2018. 01. 023.
[20]余昆,张悦,刘胜,等.膀胱部分切除术联合化疗治疗局限性肌层浸润性膀胱癌的临床分析(附53例报告)[J].中国医师杂志,2017,19(8):1188-1191,1195. DOI:10. 3760/cma. j.issn. 1008-1372. 2017. 08. 017.