S100A6通过巨噬细胞促结直肠癌细胞增殖的作用及机制

S100A6 Promotes Cell Proliferation of Colorectal Cancer via Upregulating IL-6 Expression of Macrophages

目的:探讨微环境中钙周期素S100A6是否通过影响巨噬细胞(macrophages,M_φ)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 GSTh S100A6,r S100A6)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M_φ中IL-6 mRNA水平;用Western blot检测M_φ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r S100A6和GST蛋白。(2)与经r S100A6处理的M_φ(即A6-M_φ)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-M_φ中,IL-6的mRNA和蛋白水平均升高(P <0. 05)。(4)在HCT116与A6-M_φ的共培养体系中加入IL-6R封闭肽后,A6-M_φ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。

Objective: To explore whether Calcyclin S100A6 in tumor microenvironment promotes cell proliferation of colorectal cancer (CRC) through affecting macrophages (M_φ) and its mechanism. Methods:Prokaryotic expression was used to prepare recombinant human protein GST-S100A6 (r S100A6) and GST (as control). THP-1 were induced to M_φ by PMA (Phorbol-12-myristate-13-acetate).A6-M_φ were the macrophages which were treated with r S100A6 for 24 h. The proliferation of CRC HCT116 cells was detected by Trypan blue staining,CCK8 and crystal violet staining. IL-6 mRNA and protein level inA6-M_φ 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-M_φ promoted proliferation of HCT116 cells (P < 0. 05).(3) r S100A6 upregulated IL-6 expression in macrophages (P<0. 05).(4) IL-6 R blocking antibody partly reversed the facilitation ofA6-M_φ 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.