活化T细胞核因子5对人胃癌MGC803细胞增殖及凋亡能力的影响
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摘要
目的:探讨活化T细胞核因子5(nuclear factor 5 of activated T cells, NFAT5)对人胃癌MGC803细胞增殖及凋亡能力的影响及其可能的机制。方法:设计并合成3条靶向NFAT5基因的siRNA(siRNA2567、siRNA2714和siRNA4562)及1条与NFAT5基因无同源性的阴性对照siRNA(NC-siRNA),脂质体介导转染人胃癌MGC803细胞后,采用Real-time PCR检测分析细胞中NFAT5 mRNA表达水平的变化,进而筛选出有效抑制NFAT5基因表达的siRNA(NFAT5-siRNA)。NFAT5-siRNA转染MGC803细胞48 h后,进一步采用Real-time PCR和Western blotting验证并检测细胞中NFAT5和S100A4 mRNA及蛋白表达水平的变化,流式细胞术和CCK-8法分析抑制NFAT5表达对细胞增殖及凋亡的影响。结果:转染siRNA2567对NFAT5 mRNA的表达抑制最为明显(P<0.01),siRNA2567被验证为NFAT5-siRNA。转染NFAT5-siRNA 48 h后,NFAT5和S100A4 m RNA及蛋白的表达水平均明显降低(P<0.05);与NC-siRNA组相比较,NFAT5-siRNA组MGC803细胞的增殖率在72 h和96 h均显著降低(P<0.01);NFAT5基因沉默48 h后,MGC803细胞的凋亡率由(2.7±0.2)%上升至(7.9±0.2)%(P<0.01)。结论:NFAT5-siRNA能有效沉默人胃癌MGC803细胞中NFAT5基因表达,在抑制细胞增殖率的同时能够有效促进细胞凋亡,该作用可能通过调控S100A4表达实现。
Objective: To investigate the effects of nuclear factor 5 of activated T cells(NFAT5) on proliferation and apoptosis of human gastric cancer MGC803 cells and to explore the possible mechanisms. Methods: Three siRNAs targeting NFAT5 gene(siRNA2567, siRNA2714 and siRNA4562) and one negative control siRNA were designed and chemically synthesized before transfected into human gastric cancer cell line MGC803 by liposome. Real-time PCR was used to detect the changes of NFAT5 mRNA expression in MGC803 cells to further pick out the siRNA that most effectively inhibit the expression of NFAT5. Further, Real-time PCR and Western blotting assay were carried out to test m RNA and protein levels of NFAT5 and S100 A4 in cells 48 h after NFAT5-siRNA transfection. Then, CCK-8 assay and FCM assay were used to detect the influence of silencing NFAT5 on cell proliferation and apoptosis, respectively. Results: siRNA2567 was the most effective siRNA that significantly inhibited the expression of NFAT5 mRNA(P<0.01),and thus was validated as NFAT5-siRNA. Real-time PCR and Western blotting assay confirmed that both mRNA and protein levels of NFAT5 and S100 A4 were down-regulated in cells 48 h after NFAT5-siRNA transfection. Compared with NC-siRNA group, the proliferation ability of MGC803 cells in the NFAT5-siRNA group was significantly down-regulated at 72 h and 96 h(P<0.01). And FCM assay showed that compared with NC-siRNA group, cell apoptosis rate of NFAT5-siRNA group was significantly increased from(2.7±0.2)%to(7.9±0.2)%,(P<0.01) 48 h after NFAT5-siRNA transfection. Conclusion: NFAT5-siRNA transfection can silence NFAT5 gene expression in gastric cancer MGC803 cells effectively. NFAT5 may inhibit proliferation and promote cell apoptosis of gastric cancer cells possibly through regulating S100 A4 expression.
Objective: To investigate the effects of nuclear factor 5 of activated T cells(NFAT5) on proliferation and apoptosis of human gastric cancer MGC803 cells and to explore the possible mechanisms. Methods: Three siRNAs targeting NFAT5 gene(siRNA2567, siRNA2714 and siRNA4562) and one negative control siRNA were designed and chemically synthesized before transfected into human gastric cancer cell line MGC803 by liposome. Real-time PCR was used to detect the changes of NFAT5 mRNA expression in MGC803 cells to further pick out the siRNA that most effectively inhibit the expression of NFAT5. Further, Real-time PCR and Western blotting assay were carried out to test m RNA and protein levels of NFAT5 and S100 A4 in cells 48 h after NFAT5-siRNA transfection. Then, CCK-8 assay and FCM assay were used to detect the influence of silencing NFAT5 on cell proliferation and apoptosis, respectively. Results: siRNA2567 was the most effective siRNA that significantly inhibited the expression of NFAT5 mRNA(P<0.01),and thus was validated as NFAT5-siRNA. Real-time PCR and Western blotting assay confirmed that both mRNA and protein levels of NFAT5 and S100 A4 were down-regulated in cells 48 h after NFAT5-siRNA transfection. Compared with NC-siRNA group, the proliferation ability of MGC803 cells in the NFAT5-siRNA group was significantly down-regulated at 72 h and 96 h(P<0.01). And FCM assay showed that compared with NC-siRNA group, cell apoptosis rate of NFAT5-siRNA group was significantly increased from(2.7±0.2)%to(7.9±0.2)%,(P<0.01) 48 h after NFAT5-siRNA transfection. Conclusion: NFAT5-siRNA transfection can silence NFAT5 gene expression in gastric cancer MGC803 cells effectively. NFAT5 may inhibit proliferation and promote cell apoptosis of gastric cancer cells possibly through regulating S100 A4 expression.
引文
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[14]周欣亮,赵日旸,韩晶,等.miR-141-3p在胃癌组织和患者血浆中的表达及其临床意义[J].中国肿瘤生物治疗杂志,2017,24(10):1112-1117.DOI:10.3872/j.issn.1007-385X.2017.10.012.
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[16]廖煜,任宁川,房殿亮,等.S100A4协同HIF-1通过WNT/β-catenin通路调控胃癌细胞SGC-7901的迁移和侵袭[J].中国肿瘤生物治疗杂志,2017,24(9):955-959.DOI:10.3872/j.issn.1007-385X.2017.09.005.
[17]HUA J,CHEN D,FU H,et al.Short hairpin RNA-mediated inhibition of S100A4 promotes apoptosis and suppresses proliferation of BGC823 gastric cancer cells in vitro and in vivo[J].Cancer Lett,2010,292(1):41-47.DOI:10.1016/j.canlet.2009.11.007.
[2]SHAW J P,UTZ P J,DURAND D B,et al.Identification of a putative regulator of early T cell activation genes.Science.1988.241:202-205[J].J Immunol,2010,185(9):4972-4975.DOI:10.1126/science.3260404.
[3]PUSCHECK E E,AWONUGA A O,YANG Y,et al.Molecular biology of the stress response in the early embryo and its stem cells[J].Adv Exp Med Biol,2015,843(2):127-128.DOI:10.1007/978-1-4939-2480-64.
[4]AL-ATTAR R,ZHANG Y,STOREY K B.Osmolyte regulation by TonEBP/NFAT5 during anoxia-recovery and dehydration-rehydration stresses in the freeze-tolerant wood frog(Rana sylvatica)[J/OL].Peer J,2017,5:e2797[2018-03-19].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5251939/.DOI:10.7717/peerj.2797.
[5]SHOU J,JING J,XIE J,et al.Nuclear factor of activated T cells in cancer development and treatment[J].Cancer Lett,2015,361(2):174-184.DOI:10.1016/j.canlet.2015.03.005
[6]WANG S,ZHANG Y,LIN Y.Effect of IL-8 on integrinαvβ3 expression and cell motility via PI3K,Akt,and NF-κB-dependent pathway in human breast cells[J].Clin Oncol,2012,30(2):158-163.DOI:10.1200/jco.2012.30.
[7]MISHRA S K,SIDDIQUE H R,SALEEM M.S100A4 calciumbinding protein is key player in tumor progression and metastasis:preclinical and clinical evidence[J].Cancer Metastasis Rev,2012,31(1/2):163-172.DOI:10.1007/s10555-011-9338-4.
[8]GUO J,BIAN Y,WANG Y,et al.S100A4 influences cancer stem cell-like properties of MGC803 gastric cancer cells by regulating GDF15 expression[J].Int J Oncol,2016,49(2):559-568.DOI:10.3892/ijo.2016.3556.
[9]AMARA S,ALOTAIBI D,TIRIVEEDHI V.NFAT5/STAT3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells[J].Oncol Lett,2016,12(2):933-943.DOI:10.3892/ol.2016.4713.
[10]武多娇,储以微.免疫细胞的脂肪酸代谢与肿瘤免疫[J].中国肿瘤生物治疗杂志,2017,24(10):1045-1050.DOI:10.3872/j.issn.1007-385X.2017.10.001.
[11]FOLDYNOVá-TRANTíRKOVáS,SEKYROVáP,TMEJOVáK,et al.Breast cancer-specific mutations in CK1epsilon inhibit Wnt/beta-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration[J].Breast Cancer Res,2010,12(3):R30-36.DOI:10.1186/bcr2581.
[12]RABINOVITZ I,MERCURIO A M.The integrin alpha6beta4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures[J].JCell Biol,1997,139(7):1873-1884.DOI:10.1083/jcb.139.7.1873.
[13]KüPER C,BECK FX,NEUHOFER W.NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells[J].Front Physiol,2014,5(6):293-299.DOI:10.3389/fphys.2014.00293.
[14]周欣亮,赵日旸,韩晶,等.miR-141-3p在胃癌组织和患者血浆中的表达及其临床意义[J].中国肿瘤生物治疗杂志,2017,24(10):1112-1117.DOI:10.3872/j.issn.1007-385X.2017.10.012.
[15]CHEN M,O'CONNOR K L.Integrinα6β4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells[J].Oncogene,2005,24(32):5125-5130.DOI:10.1038/sj.onc.1208729.
[16]廖煜,任宁川,房殿亮,等.S100A4协同HIF-1通过WNT/β-catenin通路调控胃癌细胞SGC-7901的迁移和侵袭[J].中国肿瘤生物治疗杂志,2017,24(9):955-959.DOI:10.3872/j.issn.1007-385X.2017.09.005.
[17]HUA J,CHEN D,FU H,et al.Short hairpin RNA-mediated inhibition of S100A4 promotes apoptosis and suppresses proliferation of BGC823 gastric cancer cells in vitro and in vivo[J].Cancer Lett,2010,292(1):41-47.DOI:10.1016/j.canlet.2009.11.007.