ECT2基因在人胰腺导管腺癌中的表达及对胰腺癌细胞生物学特性的影响
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摘要
目的:探讨上皮细胞转化序列2(epithelial cell transforming 2,ECT2)基因在人胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)中的表达及其对胰腺癌细胞增殖、凋亡的影响。方法:采集2018年7月至2019年3月在海军军医大学附属长海医院病理科PDAC及相应癌旁样本各35例。通过GEO数据库中人胰腺癌基因表达谱筛选差异表达基因,采用TCGA数据分析该基因在PDAC中的表达及与患者生存的相关性。以qPCR及免疫组化在人PDAC样本中验证了ECT2 mRNA及蛋白的表达。以siRNA干扰人胰腺癌细胞系PANC-1中ECT2基因的表达,CCK-8增殖实验检测肿瘤细胞增殖,流式细胞术检测对胰腺癌细胞凋亡的影响,最后WB检测凋亡相关蛋白的变化。结果:生物信息学分析筛选出胰腺癌中差异表达基因ECT2,TCGA数据库分析发现其在癌组织中高表达(t=4.005,P<0.05)并与生存显著相关(P<0.01)。mRNA(1.01±0.06 vs 4.25±0.12,t=24.09,P<0.01)及蛋白水平验证其在人PDAC样本中高表达。干扰ECT2基因后,其细胞增殖受到明显抑制(P<0.01)、他莫昔芬诱导的细胞凋亡显著增加(P<0.01),同时影响凋亡相关蛋白BAX及BCL-2的表达。结论:ECT2基因在人PDAC中高表达并与患者生存相关,其增加了胰腺癌细胞的增殖及抗凋亡能力。本结果为探讨ECT2作为胰腺癌预后判断及治疗新靶点提供了实验依据。
Objective: To investigate the expression of ECT2(epithelial Transforming sequence 2) gene in human pancreatic ductal adenocarcinoma(PDAC) and its effect on the proliferation and apoptosis of pancreatic cancer cells. Methods: Carcinoma tissues and corresponding para-carcinoma tissues from 35 PDAC patients at Changhai Hospital Affiliated to Naval Medical University from July 2018 to March 2019 were collected for this study. The differentially expressed genes in pancreatic cancer were screened out by using Gene Expression Omnibus(GEO) Database. Then, the related gene expression in PDAC and its relation with patients' survival were analyzed by The Cancer Genome Atlas(TCGA) database. QPCR and immunohistochemistry were used to verify the mRNA and protein expressions of ECT2 in human PDAC samples. To explore the effect of ECT2 on the biological behaviors of pancreatic cancer cells, si-RNA was used to silence the ECT2 gene in pancreatic cancer PANC-1 cells, and CCK-8 proliferation assay and Flow cytometry were used to detect the proliferation and apoptosis rate of PANC-1 cells after ECT2 silence. Finally,the expressions of apoptosis-related proteins were detected by WB. Results: The differentially expressed gene-ECT2, was screened out by analyzing the gene expression profiles of human pancreatic cancer in GEO database. TCGA database analysis showed that ECT2 was highly expressed in pancreatic cancer tissues(t=4.005, P<0.05) and significantly correlated with patients' survival(P<0.01). Moreover, it is also verified that ECT2 was highly expressed in PDAC tissues at mRNA(1.01±0.06 vs 4.25±0.12; t=24.09,P<0.01) and protein level. After ECT2 silence in PANC-1 cells, the proliferation rate was decreased(P<0.01), while the Tamoxifeninduced apoptosis rate was increased(P<0.01), and the expressions of apoptosis-related proteins(BAX and Bcl-2) were also affected. Conclusion: ECT2 is highly expressed in human pancreatic ductal adenocarcinoma and is related with patients' survival.ECT2 promotes the proliferation and apoptosis resistance of pancreatic cancer cells, providing the basis for exploring ECT2 as a new target for the prognostic judgment and treatment of pancreatic cancer.
Objective: To investigate the expression of ECT2(epithelial Transforming sequence 2) gene in human pancreatic ductal adenocarcinoma(PDAC) and its effect on the proliferation and apoptosis of pancreatic cancer cells. Methods: Carcinoma tissues and corresponding para-carcinoma tissues from 35 PDAC patients at Changhai Hospital Affiliated to Naval Medical University from July 2018 to March 2019 were collected for this study. The differentially expressed genes in pancreatic cancer were screened out by using Gene Expression Omnibus(GEO) Database. Then, the related gene expression in PDAC and its relation with patients' survival were analyzed by The Cancer Genome Atlas(TCGA) database. QPCR and immunohistochemistry were used to verify the mRNA and protein expressions of ECT2 in human PDAC samples. To explore the effect of ECT2 on the biological behaviors of pancreatic cancer cells, si-RNA was used to silence the ECT2 gene in pancreatic cancer PANC-1 cells, and CCK-8 proliferation assay and Flow cytometry were used to detect the proliferation and apoptosis rate of PANC-1 cells after ECT2 silence. Finally,the expressions of apoptosis-related proteins were detected by WB. Results: The differentially expressed gene-ECT2, was screened out by analyzing the gene expression profiles of human pancreatic cancer in GEO database. TCGA database analysis showed that ECT2 was highly expressed in pancreatic cancer tissues(t=4.005, P<0.05) and significantly correlated with patients' survival(P<0.01). Moreover, it is also verified that ECT2 was highly expressed in PDAC tissues at mRNA(1.01±0.06 vs 4.25±0.12; t=24.09,P<0.01) and protein level. After ECT2 silence in PANC-1 cells, the proliferation rate was decreased(P<0.01), while the Tamoxifeninduced apoptosis rate was increased(P<0.01), and the expressions of apoptosis-related proteins(BAX and Bcl-2) were also affected. Conclusion: ECT2 is highly expressed in human pancreatic ductal adenocarcinoma and is related with patients' survival.ECT2 promotes the proliferation and apoptosis resistance of pancreatic cancer cells, providing the basis for exploring ECT2 as a new target for the prognostic judgment and treatment of pancreatic cancer.
引文
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[19]SALHIA B,TRAN N L,CHAN A,et al.The guanine nucleotide exchange factors trio,Ect2,and Vav3 mediate the invasive behavior of glioblastoma[J].Am J Pathol,2008,173(6):1828-1838.DOI:10.2353/ajpath.2008.080043.
[20]SANO M,GENKAI N,YAJIMA N,et al.Expression level of ECT2proto-oncogene correlates with prognosis in glioma patients[J].Oncol Rep,2006,16(5):1093-1098.DOI:10.3892/or.16.5.1093.
[21]JUNG Y,LEE S,CHOI H S,et al.Clinical validation of colorectal cancer biomarkers identified from bioinformatics analysis of public expression data[J].Clin Cancer Res,2011,17(4):700-709.DOI:10.1158/1078-0432.CCR-10-1300.
[2]MANGGE H,NIEDRIST T,RENNER W,et al.New diagnostic and therapeutic aspects of pancreatic ductal adenocarcinoma[J].Curr Med Chem,2017,24(28):3012-3024.DOI:10.2174/0929867324666170510150124.
[3]HUFF L P,KIKUCHI D S,FAIDLEY E,et al.Polymerase-δ-interacting protein 2 activates the Rho GEF epithelial cell transforming sequence2 in vascular smooth muscle cells[J].Am J Physiol,2019,316(5):C621-C631.DOI:10.1152/ajpcell.00208.2018.
[4]SHI Y X,YIN J Y,SHEN Y,et al.Genome-scale analysis identifies NEK2,DLGAP5 and ECT2 as promising diagnostic and prognostic biomarkers in human lung cancer[J/OL].Sci Rep,2017,7(1):8072[2019-03-27].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556079/.DOI:10.1038/s41598-017-08615-5.
[5]CHEN Z G,LIU J T,ZHANG Y H.Role of epithelial cell transforming sequence 2(ECT2)in predicting prognosis of osteosarcoma[J/OL].Med Sci Monit,2017,23:3861-3868[2019-03-27].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562183/.
[6]WANG H K,LIANG J F,ZHENG H X,et al.Expression and prognostic significance of ECT2 in invasive breast cancer[J].J Clin Pathol,2018,71(5):442-445.DOI:10.1136/jclinpath-2017-204569.
[7]BADEA L,HERLEA V,DIMA S O,et al.Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes specifically overexpressed in tumor epithelia[J].Hepatogastroenterology,2008,55(88):2016-2027.DOI:10.1136/gut.2007.142497corr1.
[8]PEI H,LI L,FRIDLEY B L,et al.FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt[J].Cancer Cell,2009,16(3):259-266.DOI:10.1016/j.ccr.2009.07.016.
[9]ZHANG G,HE P,TAN H,et al.Integration of metabolomics and transcriptomics revealed a fatty acid network exerting growth inhibitory effects in human pancreatic cancer[J].Clin Cancer Res,2013,19(18):4983-4993.DOI:10.1158/1078-0432.CCR-13-0209.
[10]YANG S,HE P,WANG J,et al.A novel MIF signaling pathway drives the malignant character of pancreatic cancer by targeting NR3C2[J].Cancer Res,2016,76(13):3838-3850.DOI:10.1158/0008-5472.CAN-15-2841.
[11]BRAY F,FERLAY J,SOERJOMATARAM I,et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2018,68(6):394-424.DOI:10.3322/caac.21492.
[12]CHIARAVALLI M,RENI M,O'REILLY E M.Pancreatic ductal adenocarcinoma:State-of-the-art 2017 and new therapeutic strategies[J].Cancer Treat Rev,2017,60:32-43.DOI:10.1016/j.ctrv.2017.08.007.
[13]OETTLE H,POST S,NEUHAUS P,et al.Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer:a randomized controlled trial[J].JAMA,2007,297(3):267-277.DOI:10.1001/jama.297.3.267.
[14]FIELDS A P,JUSTILIEN V.The guanine nucleotide exchange factor(GEF)Ect2 is an oncogene in human cancer[J].Adv Enzyme Regul,2010,50(1):190-200.DOI:10.1016/j.advenzreg.2009.10.010.
[15]MIKI T,SMITH C L,LONG J E,et al.Oncogene ect2 is related to regulators of small GTP-binding proteins[J].Nature,1993,362(6419):462-465.DOI:10.1038/362462a0.
[16]KIMURA K,TSUJI T,TAKADA Y,et al.Accumulation of GTP-bound RhoA during cytokinesis and a critical role of ECT2 in this accumulation[J].J Biol Chem,2000,275(23):17233-17236.DOI:10.1074/jbc.C000212200.
[17]COOK D R,ROSSMAN K L,DER C J.Rho guanine nucleotide exchange factors:regulators of Rho GTPase activity in development and disease[J].Oncogene,2014,33(31):4021-4035.DOI:10.1038/onc.2013.362.
[18]JUSTILIEN V,FIELDS A P.Ect2 links the PKPKCι-Par6αcomplex to Rac1 activation and cellular transformation[J].Oncogene,2009,28(41):3597-3607.DOI:10.1038/onc.2009.217.
[19]SALHIA B,TRAN N L,CHAN A,et al.The guanine nucleotide exchange factors trio,Ect2,and Vav3 mediate the invasive behavior of glioblastoma[J].Am J Pathol,2008,173(6):1828-1838.DOI:10.2353/ajpath.2008.080043.
[20]SANO M,GENKAI N,YAJIMA N,et al.Expression level of ECT2proto-oncogene correlates with prognosis in glioma patients[J].Oncol Rep,2006,16(5):1093-1098.DOI:10.3892/or.16.5.1093.
[21]JUNG Y,LEE S,CHOI H S,et al.Clinical validation of colorectal cancer biomarkers identified from bioinformatics analysis of public expression data[J].Clin Cancer Res,2011,17(4):700-709.DOI:10.1158/1078-0432.CCR-10-1300.