外源性S100A6蛋白对人结直肠癌细胞<em>S100A6</em>基因转录的影响及机制探讨

doi:10.13523/j.cb.20160103

中国生物工程杂志

2016, Vol. 36

Issue (1): 14-22 DOI: 10.13523/j.cb.20160103

研究报告

外源性S100A6蛋白对人结直肠癌细胞S100A6基因转录的影响及机制探讨

孙晖, 李雪茹, 查何, 段亮, 袁世梅, 李欢, 李爱芳, 谷月, 周兰

重庆医科大学检验医学院临床检验诊断学教育部重点实验室重庆 400016

Effect of Exogenous S100A6 Protein on The Level of S100A6 mRNA of Colorectal Carcinoma Cell Lines and Its Possible Mechanism

SUN Hui, LI Xue-ru, ZHA He, DUAN Liang, YUAN Shi-mei, LI Huan, LI Ai-fang, GU Yue, ZHOU Lan

Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China

全文: PDF HTML

摘要：

目的:探讨钙周期蛋白S100A6对人结直肠癌细胞系HCT116及SW480中 S100A6 基因转录的影响及机制。方法:首先采用原核表达方法制备重组人S100A6蛋白(recombinant human S100A6,rhS100A6) 并鉴定;rhS100A6蛋白(终浓度为10μg/ml)、重组腺病毒Adsiβ-catenin(可表达β-catenin的siRNA)处理结直肠癌细胞HCT116、SW480及正常肠上皮细胞FHC,采用半定量反转录聚合酶链反应(reverse transcription and polymerase chain reaction,RT-PCR)及定量实时聚合酶链反应(quantitative real time polymerase chain reaction,qPCR)检测 S100A6 mRNA的水平,采用RT-PCR检测细胞中E-cadherin mRNA水平,采用Western blot检测并比较HCT116及SW480细胞核中β-catenin蛋白的水平变化,采用细胞免疫荧光法检测细胞中β-catenin分布的变化。结果:成功制备rhS100A6蛋白;外源性rhS100A6蛋白上调HCT116及SW480细胞中 S100A6 mRNA水平和细胞核中的β-catenin蛋白水平并促进β-catenin发生核移位,同时下调细胞中E-cadherin mRNA水平;Adsiβ-catenin与rhS100A6联合处理组的 S100A6 mRNA水平明显低于单独rhS100A6处理组。所有差异均具有统计学意义。结论:胞外的S100A6蛋白增强结直肠癌细胞中 S100A6 基因的转录,其机制可能涉及β-catenin通路的激活。

关键词： S100A6; 结直肠癌; β-catenin

Abstract:

Objective: To investigate the effect of rhS100A6 on S100A6 mRNA of colorectal carcinoma cell lines HCT116 and SW480, and to explore its possible mechanism. Methods: Recombinant human S100A6 (rhS100A6) protien was prepared by prokaryotic expression. FHC, HCT116 and SW480 cells were treated with 10μg/ml rhS100A6 and recombinant adenoviruses carrying human β-catenin siRNA (Adsiβ-catenin), semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real time polymerase chain reaction (qPCR) was used to detect the expression of S100A6 mRNA, RT-PCR was used to detect the expression of E-cadherin mRNA,Western blot was used to detect the expression of β-catenin of nuclear protein in HCT116 and SW480 cells and immunofluorescence staining was used to detect the distribution of β-catenin. Results: rhS100A6 protein was prepared successfully and significantly increased the expression of S100A6 mRNA and nuclear β-catenin protein of HCT116 and SW480 cells, rhS100A6 treatment promoted β-catenin translocating from cytoplasm to nucleus in HCT116 and SW480 cells, at the same time, the E-cadherin mRNA level was decreased with the treatment of rhS100A6 protein. Compared with rhS100A6 treatment group, the expression level of S100A6 mRNA was decreased in co-treatment group by Adsiβ-catenin and rhS100A6. Conclusion: Extracellular rhS100A6 upregulate the experssion of S100A6 mRNA in colorectal carcinoma cell lines and its mechanism is partly involved in Wnt/β-catenin pathway.

Key words: S100A6 Colorectal carcinoma β-catenin

收稿日期: 2015-07-20 出版日期: 2016-01-11

ZTFLH:

Q819

通讯作者: 周兰 E-mail: zhoulan0111@foxmail.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	孙晖
	李雪茹
	查何
	段亮
	袁世梅
	李欢
	李爱芳
	谷月
	周兰

引用本文:

孙晖, 李雪茹, 查何, 段亮, 袁世梅, 李欢, 李爱芳, 谷月, 周兰. 外源性S100A6蛋白对人结直肠癌细胞S100A6基因转录的影响及机制探讨[J]. 中国生物工程杂志, 2016, 36(1): 14-22.

SUN Hui, LI Xue-ru, ZHA He, DUAN Liang, YUAN Shi-mei, LI Huan, LI Ai-fang, GU Yue, ZHOU Lan. Effect of Exogenous S100A6 Protein on The Level of S100A6 mRNA of Colorectal Carcinoma Cell Lines and Its Possible Mechanism. China Biotechnology, 2016, 36(1): 14-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160103 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I1/14

[1] 李道娟, 李倩, 贺宇彤. 结直肠癌流行病学趋势.肿瘤防治研究,2015,42(3):305-310. Li D J, Li Q, He Y T.Epidemiological trends of colorectal cancer. Cancer Res Prev Treat,2015,42(3):305-310.
[2] Li Z Q, Tang M, Ling B, et al. Increased expression of S100A6 promotes cell proliferation and migration in human hepatocellular carcinoma.J Mol Med,2014, 92(3):291-303.
[3] De Petris L, Orre L M, Kanter L, et al. Tumor expression of S100A6 correlates with survival of patients with stage I non-small-cell lung cance. Lung Cancer,2009, 63(3):410-417.
[4] Komatsu K, Andoh A, Ishiguro S, et al. Increased expression of S100A6 (calcyclin), a calcium-binding protein of the S100 family, in human colorectal adenocarcinomas. Clin Cancer Res,2000,6(1):172-177.
[5] Wang X H, Zhang L H, Zhong X Y, et al.. S100A6 overexpression is associated with poor prognosis and is epigenetically up-regulated in gastric cancer. Am J Pathol,2010,177(2):586-597.
[6] Vimalachandran D, Greenhalf W, Thompson C, et al. High nuclear S100A6 (calcyclin) is significantly associated with poor survival in pancreatic cancer patients. Cancer Res,2005, 65(8):3218-3225.
[7] Komatsu K, Kobune-Fujiwara Y, Andoh A, et al. Increased expression of S100A6 at the invading fronts of the primary lesion and liver metastasis in patients with colorectal adenocarcinoma. Br J Cancer, 2000, 83(6):769-774.
[8] Kuznicki J, Kordowska J, Puzianowska M, et al. Calcyclin as a marker of human epithelial cells and fibroblasts.Exp Cell Res, 1992,200(2):425-430.
[9] Jurewicz E, Góral A, Filipek A. S100A6 is secreted from Wharton's jelly mesenchymal stem cells and interacts with integrin β1.The International Journal of Biochemistry & Cell Biology,2014,55:298-303.
[10] Leclerc E, Fritz G, Weibel M, et al. S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains. J Biol Chem,2007,282(43):31317-31331.
[11] Wiesawa L, Ukasz P S, Anna F. S100A6-new facts and features.Biochemical and Biophysical Research Communications,2009,390(4):1087-1092.
[12] Polakis P. The many ways of Wnt in cancer . Current Opinion in Genetics & Development,2007,17(5):45-51.
[13] Suriano G, Vrcelj N, Senz J, et al. Beta-catenin(CTNNB1)geneam plification:a new mechanism of protein over expression in cancer .Genes Chromosomes Cancer,2005,42(3):238-246.
[14] Kilanczyk E, Graczyka A, Ostrowskab H, et al. S100A6 is transcriptionally regulated by β-catenin and interacts with a novel target, lamin A/C, in colorectal cancer cells.Cell Calcium, 2012, 51(6): 470-477.
[15] 黎玉叶, 李星星, 孙双双, 等. S100A6蛋白对细胞中β-catenin水平的影响及可能机制.中国生物工程杂志,2011,31(11) :18-23. Li Y Y, Li X X, Sun S S, et al. Effects and possible mechanism of protein S100A6 on β-catenin.China Biotechnology,2011,31(11) :18-23.
[16] 邹正渝, 陈英华, 孙双双, 等. hS100A6抑制卵巢癌细胞HO8910和HO8910PM增殖促进凋亡并上调其Wnt/β-catenin活性.第三军医大学学报, 2012,34(3):214-218. Zou Z Y, Chen Y H, Sun S S, et al. hS100A6inhibits proliferation,induces apoptosis andup-regulates Wnt/ β-catenin activity in ovarian cancer cell lines HO8910 and HO8910PM. J Third Mil Med Univ, 2012,34(3):214-218.
[17] 陈英华, 孙双双, 卫佳, 等. 外源性S100A6 对黑色素瘤 B16细胞增殖、凋亡的影响及其机制. 中国生物制品学杂志,2012,25(3):304-308. Chen Y H, Sun S S, Wei J, et al. Effect of exogenous S100A6 on proliferation and apoptosis of melanoma B16 cells and relevant mechanism. Chin J Biologicals,2012,25(3):304-308.
[18] 武睿, 段亮, 叶立伟, 等. S100A6上调人乳腺癌细胞系MCF-7中β-catenin及其机制. 基础医学与临床,2013,33(7):793-798. Wu R, Duan L, Ye L W, er al. S100A6 upregulates β-catenin and its mechanism in human breast cancer cell MCF-7. Basic&Clinical Medicine,2013,33(7):793-798.
[19] 邹正渝, 王海燕, 黎玉叶, 等. 重组hS100A6蛋白的原核表达及其对人骨肉瘤细胞系143B的生物学作用.中国生物工程杂志,2012,32(12):1-7. Zou Z Y, Wang H Y, Li Y Y, et al. Prokaryotic expression of recombinant protein hS100A6 and its biological effects on human osteosarcoma cell line 143B. China Biotechnology,2012,32(12):1-7.
[20] Kuznicki J, Filipek A. Purification and properties of a novel Ca²⁺-bindingprotein(10.5kDa) from Ehrlich-ascites-tumour cells.Biochem J, 1987, 247( 3) : 663-667.
[21] Duan L, Wu R, Zou Z Y, et al. S100A6 stimulates proliferation and migration of colorectal carcinoma cells through activation of the MAPK pathways. Int J Oncol, 2014, 44(3): 781-790.
[22] 赖天霞, 苗静琨, 何焕玲, 等. 钙结合蛋白S100A6 对Wnt/ β-catenin信号途径的影响.中华肿瘤杂志,2008,30(1):12-15. Lai T X, Miao J K, He H L,et al. Effects of Ca²⁺-binding protein S100A6 on Wnt/β-catenin signaling pathway. Chinese Journal of Oncology,2008,30(1):12-15.
[23] 时杰, 王琳, 胡丽华. Wnt信号传导通路与Cadherin/Catenin复合体的关联及其在肿瘤发生中的作用.世界华人消化杂志, 2005,13(2):250-254. Shi J, Wang L, Hu L H. The association between Wnt signaling pathway and Cadherin/Catenin complex and its effect on tumorigenesis. World Chin J Digestol, 2005, 13(2):250-254.
[24] Kuphal F,Behrens J. E-cadherin modulates Wnt-dependent transcription in colorectal cancer cells but does not alter Wnt-independent gene expression in fibroblasts. Experimental Cell Research, 2006, 312(4): 457-467.

[1]	林璐,户丽君,黄逸云,陈露,黄茂,彭棋,胡琴,周兰. S100A6通过招募和活化巨噬细胞促进血管形成^*[J]. 中国生物工程杂志, 2020, 40(5): 7-14.
[2]	陈露,黄茂,彭棋,赵佳丽,谢佳卿,林璐,户丽君,黄逸云,胡琴,周兰. S100A6通过巨噬细胞促结直肠癌细胞增殖的作用及机制 ^*[J]. 中国生物工程杂志, 2019, 39(4): 1-7.
[3]	李爱芳, 谷月, 李雪茹, 孙晖, 查何, 谢佳卿, 赵佳丽, 周兰. 促宫颈癌细胞增殖、迁移及其可能机制研究[J]. 中国生物工程杂志, 2017, 37(2): 8-14.
[4]	代爽, 赵青青, 邱峰. PEI-壳聚糖在结肠癌细胞CD133⁺梯度表达的转染研究[J]. 中国生物工程杂志, 2016, 36(6): 32-38.
[5]	蒋婷婷, 温晓霞, 陈尧. *沉默c2orf68基因对结直肠癌细胞增殖的影响*[J]. 中国生物工程杂志, 2014, 34(2): 7-13.
[6]	王鑫, 陈玲, 孙飞, 陆航. RNAi沉默CXCR7对人结肠癌细胞SW620特异性靶向抑制的实验研究[J]. 中国生物工程杂志, 2014, 34(2): 14-20.
[7]	邹正渝, 王海燕, 黎玉叶, 孙双双, 叶立伟, 武睿, 段亮, 陈娴, 罗进勇, 周兰. 重组hS100A6蛋白的原核表达及其对人骨肉瘤细胞系143B的生物学作用[J]. 中国生物工程杂志, 2012, 32(12): 1-7.
[8]	韩涛, 丁劲, 王红阳. β-catenin信号分子的生物学功能及其在肝癌中的研究进展[J]. 中国生物工程杂志, 2011, 31(9): 96-102.
[9]	黎玉叶, 李星星, 孙双双, 邹正渝, 张昀源, 段亮, 叶立伟, 武睿, 杨霞, 何通川, 周兰. S100A6蛋白对细胞中β-catenin水平的影响及可能机制[J]. 中国生物工程杂志, 2011, 31(11): 18-23.

Viewed

Full text

Abstract

Cited

Shared

Discussed