慢病毒介导的FOXJ1过表达对胃腺癌细胞凋亡及线粒体膜电位的影响
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摘要
目的:探讨慢病毒介导的叉头框转录因子J1(FOXJ1)过表达对胃腺癌细胞凋亡及线粒体膜电位的影响。方法:人胃腺癌细胞SGC-7901感染FOXJ1过表达慢病毒(Lv-FOXJ1),Real-time PCR和Western blot法检测细胞中FOXJ1 mRNA和蛋白的表达,MTT法检测细胞存活率,细胞克隆实验检测克隆形成率,流式细胞术检测细胞凋亡率,Western blot法检测细胞中激活型Caspase-3(C-Caspase-3)、激活型Caspase-9(C-Caspase-9)和胞浆中细胞色素C(Cyt C)蛋白表达水平,JC-1法检测线粒体膜电位。以感染阴性对照慢病毒和未感染细胞为阴性对照和空白对照。结果:FOXJ1组细胞中FOXJ1 mRNA和蛋白表达水平均增高,细胞存活率、克隆形成率降低,细胞凋亡率升高,细胞中C-Caspase-3、C-Caspase-9蛋白表达水平升高,线粒体膜电位下降,胞浆中Cyt C蛋白表达水平升高(P <0. 05)。结论:FOXJ1能够通过线粒体途径诱导胃腺癌细胞凋亡。
Aim: To investigate the effects of lentivirus mediated FOXJ1 over-expression on cell apoptosis and mitochondrial membrane potential in gastric adenocarcinoma cells. Methods: Human gastric adenocarcinoma cell SGC-7901 was infected with FOXJ1 over-expression lentivirus( Lv-FOXJ1),Real-time PCR and Western blot were used to detect the expressions of FOXJ1 mRNA and protein,cell viability was detected by MTT method,cell clone assay was used to detect clone formation ability,cell apoptosis was detected by flow cytometry,Western blot was used to detect the levels of CCaspase-3 and C-Caspase-9 protein in the cells and Cyt C in the cytoplasm,JC-1 method was used to detect the mitochondrial membrane potential. The cells infected with negative control lentivirus or without infection were the negative control or blank control. Results: Compared with those of the negative control group and blank control group,the expression of FOXJ1 in FOXJ1 group increased,the cell viability and the clone formation ability decreased,the apoptosis rate increased,the levels of C-Caspase-3 and C-Caspase-9 proteins in cells increased,mitochondrial membrane potential dropped,and the level of Cyt C protein in the cytoplasm increased( P < 0. 05). Conclusion: FOXJ1 can induce apoptosis of gastric adenocarcinoma cells through the mitochondrial pathway.
Aim: To investigate the effects of lentivirus mediated FOXJ1 over-expression on cell apoptosis and mitochondrial membrane potential in gastric adenocarcinoma cells. Methods: Human gastric adenocarcinoma cell SGC-7901 was infected with FOXJ1 over-expression lentivirus( Lv-FOXJ1),Real-time PCR and Western blot were used to detect the expressions of FOXJ1 mRNA and protein,cell viability was detected by MTT method,cell clone assay was used to detect clone formation ability,cell apoptosis was detected by flow cytometry,Western blot was used to detect the levels of CCaspase-3 and C-Caspase-9 protein in the cells and Cyt C in the cytoplasm,JC-1 method was used to detect the mitochondrial membrane potential. The cells infected with negative control lentivirus or without infection were the negative control or blank control. Results: Compared with those of the negative control group and blank control group,the expression of FOXJ1 in FOXJ1 group increased,the cell viability and the clone formation ability decreased,the apoptosis rate increased,the levels of C-Caspase-3 and C-Caspase-9 proteins in cells increased,mitochondrial membrane potential dropped,and the level of Cyt C protein in the cytoplasm increased( P < 0. 05). Conclusion: FOXJ1 can induce apoptosis of gastric adenocarcinoma cells through the mitochondrial pathway.
引文
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[2]HAYAKAWA S,OKADA S,TSUMURA M,et al.A Patient with CTLA-4 haploinsufficiency presenting gastric cancer[J].J Clin Immunol,2016,36(1):28
[3]CHEN HW,HUANG XD,LI HC,et al.Expression of FOXJ1 in hepatocellular carcinoma:correlation with patients'prognosis and tumor cell proliferation[J].Mol Carcinog,2013,52(8):647
[4]DEMIRCAN B,DYER LM,GERACE M,et al.Comparative epigenomics of human and mouse mammary tumors[J].Genes Chromosomes Cancer,2009,48(1):83
[5]SIU M,WONG E,KONG D,et al.Stem cell transcription factor NANOG controls cell migration and invasion via dysregulation of E-cadherin and Fox J1 and contributes to adverse clinical outcome in ovarian cancers[J].Oncogene,2013,32(30):3500
[6]王博,薛锋,刘娟妮,等.FOXJ1基因与肿瘤相关性研究进展[J].陕西医学杂志,2017,46(10):1487
[7]WANG J,CAI XQ,XIA LM,et al.Decreased expression of FOXJ1 is a potential prognostic predictor for progression and poor survival of gastric cancer[J].Ann Surg Oncol,2015,22(2):685
[8]王静.FOXJ1在胃癌恶性生物学表型中的作用和机制[D].西安:第四军医大学,2014.
[9]LIN L,SPOOR MS,GERTH AJ,et al.Modulation of Th1activation and inflammation by the NF-kappa B repressor Foxj1[J].Science,2004,303(5660):1017
[10]JACQUET BV,SALINAS-MONDRAGON R,LIANG HA,et al.Fox J1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain[J].Development,2009,136(23):4021
[11]HUANG T,YOU Y,SPOOR MS,et al.Foxj1 is required for apical localization of ezrin in airway epithelial cells[J].JCell Sci,2003,116(24):4935
[12]刘小琪,兰丽珍.TAP,FOXJ1基因多态性与自身免疫性疾病相关性的研究进展[J].中国当代医药,2014,21(14):195
[13]曹新国,王礼文.人类免疫调控转录因子FOXP3研究进展[J].临床检验杂志,2006,24(1):63
[14]于志华,李震,王伟,等.Foxj1在脑外伤后的表达变化[J].现代生物医学进展,2012,12(7):1244
[15]LIANG B,HE XW,SHANG DH,et al.The Link between FOXJ1 expression level in bladder carcinoma and tumor recurrence[J].Oncol Lett,2018,15(2,A):1483
[16]潘伟.ERK通路蛋白,ANXA2及FOXJ1蛋白表达与胃癌患者临床分期及预后的相关性研究[J].临床和实验医学杂志,2017,16(5):454
[17]KONG F,WANG H,GUO J,et al.Hsp70 suppresses apoptosis of BRL cells by regulating the expression of Bcl-2,cytochrome C,and caspase 8/3[J].In Vitro Cell Dev Biol Anim,2016,52(5):568
[18]ZHANG J,WANG J,JIANG JY,et al.Tanshinone IIA induces cytochrome c-mediated caspase cascade apoptosis in A549 human lung cancer cells via the JNK pathway[J].Int J Oncol,2014,45(2):683
[19]KOOK S,ZHAN X,CLEGHORN WM,et al.Caspasecleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death[J].Cell Death Differ,2014,21(1,SI):172
[20]HENSHALL DC,CHEN J,SIMON RP.Involvement of Caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures[J].J Neurochem,2000,74(3):1215
[21]LARSEN BD,RAMPALLI S,BURNS LE,et al.Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA Strand breaks[J].Proc Natl Acad Sci U S A,2010,107(9):4230