S100A4:潜在抑制膀胱癌干细胞的靶分子
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摘要
目的应用siRNA技术沉默S100A4基因表达对膀胱癌干细胞增殖及成瘤能力的影响。方法筛选鉴定出MB49膀胱癌干细胞后,应用核素标记相对和绝对定量(i TRAQ)技术,发现MB49膀胱癌细胞与膀胱癌干细胞表达差异的蛋白质S100A4蛋白。设计并合成S100A4基因特异性的siRNA序列,转染膀胱癌干细胞,应用Western blot和q PCR检测在siRNA对S100A4的影响,体内、外实验观察siRNA抑制S100A4后对膀胱癌干细胞增殖及成瘤能力的影响。结果通过i TRAQ核素标记结合液相色谱和串联质谱分析,共鉴定出差异蛋白共65个,S100A4在基因表达水平差异最显著(差异为5.70,P<0.05)。与空白对照组、阴性对照组相比,S100A4 siRNA转染组的S100A4基因和蛋白表达降低(P<0.05)。CCK8实验和裸鼠成瘤实验发现,与空白对照组与阴性对照组相比较,siRNA干扰S100A4表达后,膀胱癌干细胞生长明显受到抑制,成瘤能力受到抑制(P<0.05)。结论膀胱癌干细胞中S100A4表达与膀胱癌的复发、转移有关,S100A4蛋白可能成为消灭膀胱癌干细胞,治疗膀胱癌的分子靶标。
Objective To observe the effect of S100A4 gene silencing mediated by small interfering RNA(siRNA) on the proliferation of bladder cancer stem cells(CSCs) and their capacity of xenograft tumor formation. Methods MB49 bladder cancer stem cells(MCSCs) were isolated and identified. The differentially expressed protein S100A4 was identified in MCSCs using isobaric tags for relative and absolute quantitation technology(i TRAQ). A siRNA targeting S100A4 was constructed and transfected into MCSCs, and its inhibitory effects on S100A4 expression in MCSCs were assessed with Western blotting and q PCR. The effects of siRNA-mediated S100A4 silencing on the proliferation and xenograft tumor formation ability of MCSCs were observed. Results Among the 65 differentially expressed proteins identified by i TRAQ combined with LC/MS/MS,S100A4 protein showed the most distinct differential expression in MCSCs. Transfection of MCSCs with S100 A siRNA significantly inhibited the expressions of S100A4 at both m RNA and protein levels, caused obvious suppression of the cell proliferation, and attenuated the xenograft tumor formation ability of the cells in nude mice. Conclusion S100A4 in MCSCs is associated with the recurrence and metastasis of bladder cancer. S100A4 may serve as a potential therapeutic target for eliminating bladder CSCs.
Objective To observe the effect of S100A4 gene silencing mediated by small interfering RNA(siRNA) on the proliferation of bladder cancer stem cells(CSCs) and their capacity of xenograft tumor formation. Methods MB49 bladder cancer stem cells(MCSCs) were isolated and identified. The differentially expressed protein S100A4 was identified in MCSCs using isobaric tags for relative and absolute quantitation technology(i TRAQ). A siRNA targeting S100A4 was constructed and transfected into MCSCs, and its inhibitory effects on S100A4 expression in MCSCs were assessed with Western blotting and q PCR. The effects of siRNA-mediated S100A4 silencing on the proliferation and xenograft tumor formation ability of MCSCs were observed. Results Among the 65 differentially expressed proteins identified by i TRAQ combined with LC/MS/MS,S100A4 protein showed the most distinct differential expression in MCSCs. Transfection of MCSCs with S100 A siRNA significantly inhibited the expressions of S100A4 at both m RNA and protein levels, caused obvious suppression of the cell proliferation, and attenuated the xenograft tumor formation ability of the cells in nude mice. Conclusion S100A4 in MCSCs is associated with the recurrence and metastasis of bladder cancer. S100A4 may serve as a potential therapeutic target for eliminating bladder CSCs.
引文
[1]Hussain SA,James ND.The systemic treatment of advanced and metastatic bladder cancer[J].Lancet Oncol,2003,4(8):489-97.
[2]Clarke MF,Dick JE,Dirks PB,et al.Cancer stem cells--perspectives on current status and future directions:AACR Workshop on cancer stem cells[J].Cancer Res,2006,66(19):9339-44.
[3]刘亚辉,朱永通,罗荣城.膀胱癌肿瘤干细胞筛选方法的研究进展[J].肿瘤防治研究,2014,41(11):1251-5.
[4]Zhu YT,Lei CY,Luo Y,et al.A modified method for isolation of bladder cancer stem cells from a MB49 murine cell line[J].BMC Urol,2013,13:57.
[5]Zhu YT,Pang SY,Luo Y,et al.A modified method by differential adhesion for enrichment of bladder cancer stem cells[J].Int Braz J Urol,2016,42(4):817-24.
[6]Zhu YT,Pang SY,Lei CY,et al.Development of a therapy against metastatic bladder cancer using an interleukin-2 surface-modified MB49 bladder cancer stem cells vaccine[J].Stem Cell Res Ther,2015,6:224.
[7]Zhu YT,Zhao Z,Fu XY,et al.The granulocyte macrophage-colony stimulating factor surface modified MB49 bladder cancer stem cells vaccine against metastatic bladder cancer[J].Stem Cell Res,2014,13(1):111-22.
[8]张红燕,郑献召,轩小燕,等.S100A4在食管鳞癌中的表达及与浸润转移的关系[J].南方医科大学学报,2010,30(7):1541-4.
[9]陈香丽,王连才,张王刚,等.S100A4和MMP9在非小细胞肺癌中的表达及与浸润、转移和预后的关系[J].南方医科大学学报,2008,28(7):1254-8.
[10]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-32.
[11]余淦,许凯,许世安,等.微小RNA-34a通过靶向CD44调节膀胱癌细胞J82周期[J].南方医科大学学报,2015,35(7):935-40.
[12]郭术俊,陶象男,汪忆梦,等.SCF/c-Kit信号促进膀胱癌T24细胞侵袭作用[J].南方医科大学学报,2014,34(4):507-10.
[13]陈柯,李炳坤,许凯,等.稳定低表达DNA甲基转移酶3b基因对膀胱癌细胞生长和凋亡的影响[J].南方医科大学学报,2015,35(11):1524-9.
[14]Hanahan D,Weinberg R A.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-74.
[15]Li F,Chen DN,He CW,et al.Identification of urinary Gc-globulin as a novel biomarker for bladder cancer by two-dimensional fluorescent differential gel electrophoresis(2D-DIGE)[J].J Proteomics,2012,77:225-36.
[16]Evans C,Noirel J,Ow SY,et al.An insight into i TRAQ:where do we stand now[J]?Anal Bioanal Chem,2012,404(4):1011-27.
[17]Rideau A,Besson D,Boissard A,et al.Two-step OFFGEL approach for effective peptide separation compatible with i TRAQ labeling[J].Proteomics,2013,13(22):3261-6.
[18]Mencia N,Selga E,Rico I,et al.Overexpression of S100A4 in human cancer cell lines resistant to methotrexate[J].BMC Cancer,2010,10:250.
[19]吴爱国,马雷,纪术峰,等.短发夹RNA沉默S100A4基因表达对乳腺癌MCF-7细胞生长和侵袭能力的影响[J].肿瘤,2009,29(8):715-20.
[20]马雷,吴爱国,纪术峰,等.短发夹RNA沉默S100A4基因对乳腺癌MCF-7细胞体外增殖和迁移力的抑制[J].肿瘤防治研究,2010,37(4):402-6.
[21]Liu J,Fu S,Xu Y,et al.RNA interference targeting inhibition of S100A4 suppresses cell growth and promotes apoptosis in human laryngeal carcinoma Hep2 cells[J].Mol Med Rep,2014,10(3):1389-94.
[22]Tsumura H,Matsumoto K,Sato Y,et al.Abnormal expression of multiple proteins predicts cancer-specific mortality in patients with high-grade non-muscle-invasive bladder cancer treated with transurethral resection[J].Mol Clin Oncol,2013,1(3):473-9.
[23]Agerbaek M,Alsner J,Marcussen N,et al.Focal S100A4 protein expression is an independent predictor of development of metastatic disease in cystectomized bladder cancer patients[J].Eur Urol,2006,50(4):777-85.
[24]孙明,赵文嫣,詹运洪,等.沉默S100A4基因对人膀胱癌T-24细胞生物学特性的影响[J].现代肿瘤医学,2015,23(22):3232-6.
[2]Clarke MF,Dick JE,Dirks PB,et al.Cancer stem cells--perspectives on current status and future directions:AACR Workshop on cancer stem cells[J].Cancer Res,2006,66(19):9339-44.
[3]刘亚辉,朱永通,罗荣城.膀胱癌肿瘤干细胞筛选方法的研究进展[J].肿瘤防治研究,2014,41(11):1251-5.
[4]Zhu YT,Lei CY,Luo Y,et al.A modified method for isolation of bladder cancer stem cells from a MB49 murine cell line[J].BMC Urol,2013,13:57.
[5]Zhu YT,Pang SY,Luo Y,et al.A modified method by differential adhesion for enrichment of bladder cancer stem cells[J].Int Braz J Urol,2016,42(4):817-24.
[6]Zhu YT,Pang SY,Lei CY,et al.Development of a therapy against metastatic bladder cancer using an interleukin-2 surface-modified MB49 bladder cancer stem cells vaccine[J].Stem Cell Res Ther,2015,6:224.
[7]Zhu YT,Zhao Z,Fu XY,et al.The granulocyte macrophage-colony stimulating factor surface modified MB49 bladder cancer stem cells vaccine against metastatic bladder cancer[J].Stem Cell Res,2014,13(1):111-22.
[8]张红燕,郑献召,轩小燕,等.S100A4在食管鳞癌中的表达及与浸润转移的关系[J].南方医科大学学报,2010,30(7):1541-4.
[9]陈香丽,王连才,张王刚,等.S100A4和MMP9在非小细胞肺癌中的表达及与浸润、转移和预后的关系[J].南方医科大学学报,2008,28(7):1254-8.
[10]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-32.
[11]余淦,许凯,许世安,等.微小RNA-34a通过靶向CD44调节膀胱癌细胞J82周期[J].南方医科大学学报,2015,35(7):935-40.
[12]郭术俊,陶象男,汪忆梦,等.SCF/c-Kit信号促进膀胱癌T24细胞侵袭作用[J].南方医科大学学报,2014,34(4):507-10.
[13]陈柯,李炳坤,许凯,等.稳定低表达DNA甲基转移酶3b基因对膀胱癌细胞生长和凋亡的影响[J].南方医科大学学报,2015,35(11):1524-9.
[14]Hanahan D,Weinberg R A.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-74.
[15]Li F,Chen DN,He CW,et al.Identification of urinary Gc-globulin as a novel biomarker for bladder cancer by two-dimensional fluorescent differential gel electrophoresis(2D-DIGE)[J].J Proteomics,2012,77:225-36.
[16]Evans C,Noirel J,Ow SY,et al.An insight into i TRAQ:where do we stand now[J]?Anal Bioanal Chem,2012,404(4):1011-27.
[17]Rideau A,Besson D,Boissard A,et al.Two-step OFFGEL approach for effective peptide separation compatible with i TRAQ labeling[J].Proteomics,2013,13(22):3261-6.
[18]Mencia N,Selga E,Rico I,et al.Overexpression of S100A4 in human cancer cell lines resistant to methotrexate[J].BMC Cancer,2010,10:250.
[19]吴爱国,马雷,纪术峰,等.短发夹RNA沉默S100A4基因表达对乳腺癌MCF-7细胞生长和侵袭能力的影响[J].肿瘤,2009,29(8):715-20.
[20]马雷,吴爱国,纪术峰,等.短发夹RNA沉默S100A4基因对乳腺癌MCF-7细胞体外增殖和迁移力的抑制[J].肿瘤防治研究,2010,37(4):402-6.
[21]Liu J,Fu S,Xu Y,et al.RNA interference targeting inhibition of S100A4 suppresses cell growth and promotes apoptosis in human laryngeal carcinoma Hep2 cells[J].Mol Med Rep,2014,10(3):1389-94.
[22]Tsumura H,Matsumoto K,Sato Y,et al.Abnormal expression of multiple proteins predicts cancer-specific mortality in patients with high-grade non-muscle-invasive bladder cancer treated with transurethral resection[J].Mol Clin Oncol,2013,1(3):473-9.
[23]Agerbaek M,Alsner J,Marcussen N,et al.Focal S100A4 protein expression is an independent predictor of development of metastatic disease in cystectomized bladder cancer patients[J].Eur Urol,2006,50(4):777-85.
[24]孙明,赵文嫣,詹运洪,等.沉默S100A4基因对人膀胱癌T-24细胞生物学特性的影响[J].现代肿瘤医学,2015,23(22):3232-6.