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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (4): 1-7    DOI: 10.13523/j.cb.20190401
研究报告     
S100A6通过巨噬细胞促结直肠癌细胞增殖的作用及机制 *
陈露,黄茂,彭棋,赵佳丽,谢佳卿,林璐,户丽君,黄逸云,胡琴,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
S100A6 Promotes Cell Proliferation of Colorectal Cancer via Upregulating IL-6 Expression of Macrophages
Lu CHEN,Mao HUANG,Qi PENG,Jia-li ZHAO,Jia-qing XIE,Lu LIN,Li-jun HU,Yi-yun HUANG,Qin HU,Lan ZHOU()
Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing 400016, China
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摘要：

目的:探讨微环境中钙周期素S100A6 是否通过影响巨噬细胞(macrophages, Mφ)进而促进结直肠癌(colorectal cancer, CRC)细胞的增殖及其机制。方法:制备(原核表达)并鉴定带GST(glutathione S-transferase,谷胱甘肽S-转移酶)标签的人重组S100A6蛋白(recombinant GST-hS100A6,rS100A6) 和对照蛋白GST;采用台盼兰计数、CCK8和结晶紫染色检测CRC细胞系HCT116的增殖能力;用定量实时聚合酶链反应检测Mφ中IL-6 mRNA水平;用Western blot检测Mφ中IL-6的蛋白水平、HCT116细胞中JAK2和STAT3及其磷酸化水平。 结果:(1)成功制备rS100A6和GST蛋白。(2)与经rS100A6处理的Mφ(即A6-Mφ)共培养后,HCT116细胞的增殖能力增强(P<0.05);同时,HCT116细胞中的JAK2和STAT3水平无明显变化,但其磷酸化水平提高(P<0.05)。(3)A6-Mφ中,IL-6的mRNA和蛋白水平均升高(P<0.05)。(4)在HCT116与A6-Mφ的共培养体系中加入IL-6R封闭肽后,A6-Mφ促HCT116细胞的活力和增殖能力的作用被部分逆转(P<0.05)。 结论:微环境中的S100A6可通过上调巨噬细胞中IL-6的表达、进而激活HCT116细胞中IL-6/JAK2/STAT3信号通路来促进CRC细胞的增殖。
关键词： S100A6;  巨噬细胞;  结直肠癌;  IL-6/JAK2/STAT3信号通路    
Abstract:

Objective: To explore whether Calcyclin S100A6 in tumor microenvironment promotes cell proliferation of colorectal cancer(CRC) through affecting macrophages (Mφ) and its mechanism. Methods: Prokaryotic expression was used to prepare recombinant human protein GST-S100A6 (rS100A6) and GST (as control). THP-1 were induced to Mφ by PMA (Phorbol-12-myristate-13-acetate). A6-Mφ were the macrophages which were treated with rS100A6 for 24h. The proliferation of CRC HCT116 cells was detected by Trypan blue staining, CCK8 and crystal violet staining. IL-6 mRNA and protein level in A6-Mφ were tested with quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The protein levels of total JAK2 and STAT3 (t-JAK2 and t-STAT3) and the phosphorylated JAK2 and STAT3 (p-JAK2 and p-STAT3) in HCT116 cells were detected by Western blot. Results: (1) rS100A6 and GST were prepared successfully. (2) A6-Mφ promoted proliferation of HCT116 cells (P<0.05). (3) rS100A6 upregulated IL-6 expression in macrophages (P<0.05). (4) IL-6R blocking antibody partly reversed the facilitation of A6-Mφ to proliferation of HCT116 cells (P<0.05). (5) A6-Mφ increased protein levels of p-JAK2 and p-STAT3 in HCT116 cells (P<0.05), but not t-JAK2 and t-STAT3. Conclusion: S100A6 in tumor microenvironment facilitates proliferation of HCT116 cells through upregulating IL-6 expression in macrophages and activating IL-6/JAK2/STAT3 pathway in HCT116 cells.
Key words: S100A6    Macrophages    CRC    IL-6/JAK2/STAT3 pathway
收稿日期: 2018-09-06 出版日期: 2019-05-08
ZTFLH:  R73-3  
基金资助: * 重庆市渝中区基础与前沿研究科技计划资助项目(20160106)
通讯作者: 周兰     E-mail: zhoulan0111@foxmail.com
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引用本文:

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.

Lu CHEN,Mao HUANG,Qi PENG,Jia-li ZHAO,Jia-qing XIE,Lu LIN,Li-jun HU,Yi-yun HUANG,Qin HU,Lan ZHOU. S100A6 Promotes Cell Proliferation of Colorectal Cancer via Upregulating IL-6 Expression of Macrophages. China Biotechnology, 2019, 39(4): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190401    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I4/1

图1  重组蛋白的鉴定
图2  THP-1(a)被PMA诱导成为巨噬细胞(b)
图3  rS100A6处理后的巨噬细胞对HCT116细胞增殖的影响
图4  rS00A6对巨噬细胞中IL-6的mRNA和蛋白质水平的影响
图5  rS00A6诱导后的巨噬细胞对HCT116 细胞中JAK2、STAT3及其磷酸化水平的影响(Western blot)
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