抗菌肽LL-37在巨噬细胞促进结直肠癌生长中的作用及其分子机制  

Antimicrobial peptide LL-37 in macrophages promotes colorectal cancer growth

作　　者：潘星昊 权文强[2] 吴军录[2] 肖卫东 孙祖俊 李冬[2] School of Biomedical Sciences;Huaqiao University;Quanzhou;China(Pan XH, Xiao WD;Department of Clinical Laboratory, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China （Quan WQ, wu JL, Sun Z J, Li D)

机构地区：[1]华侨大学生物医学学院,泉州362021 [2]同济大学附属同济医院检验科,上海200065

出　　处：《中华肿瘤杂志》2018年第6期412-417,共6页Chinese Journal of Oncology

基　　金：国家自然科学基金（81272603,81472179）;上海市浦江人才计划（13PJ1407300）;上海市加强公共卫生体系建设三年行动计划（2015年-2017年）（15GWZK0301）

摘　　要：目的研究非肿瘤细胞分泌的抗菌肽LL-37促进结直肠癌生长的作用及分子机制。方法采用Transwell？插入式细胞培养皿模拟肿瘤微环境，共培养人巨噬细胞U937和结直肠癌细胞株SW480和HCT116。采用溴脱氧核苷尿嘧啶（BrdU）-酶联免疫吸附（ELISA）法，检测巨噬细胞对结直肠癌细胞增殖能力的影响。采用实时荧光定量PCR和Western blot法检测巨噬细胞和结直肠癌细胞中LL-37 mRNA和蛋白的表达。应用LL-37中和抗体抑制LL-37的功能活性或转染LL-37 shRNA质粒，观察巨噬细胞分泌的LL-37在促进结直肠癌细胞增殖中的作用，并以Western blot法检测结直肠癌增殖相关信号通路的激活情况。结果BrdU-ELISA法检测结果显示，共培养前后，SW480细胞的吸光度（A）值分别为1.072±0.097和5.121±0.407，差异有统计学意义（P〈0.001）；HCT116细胞的A值分别为1.229±0.073和3.495±0.228，差异有统计学意义（P〈0.001）。实时荧光定量PCR检测结果显示，共培养前后，巨噬细胞中LL-37 mRNA的相对表达量分别为2.682±0.191和6.117±0.768，差异有统计学意义（P〈0.05）；SW480细胞中LL-37 mRNA的相对表达量分别为1.012±0.069和1.112±0.110，差异无统计学意义（P〉0.05）。Western blot法检测结果显示，巨噬细胞中LL-37蛋白的表达明显增高，而结直肠癌SW480细胞中LL-37蛋白的表达没有变化。加入LL-37中和抗体后，SW480细胞的增殖速度有所减缓；而转染LL-37 shRNA质粒后的巨噬细胞与SW480细胞共培养时，SW480细胞增殖速度也明显减慢。Western blot检测结果显示，共培养时SW480细胞中非磷酸化的β-catenin表达明显增高，其下游转录基因cyclin D1、c-myc的表达也明显增高。加入LL-37中和抗体后，非磷酸化的β-catenin表达受到抑制，cyclin D1、c-myc的表达也明显降低。结论在体外模拟肿瘤微环境共培养时，巨噬细胞通过增强抗菌肽LL-37的表达和分泌促ObjectiveTo investigate the effect and molecular mechanism of antimicrobial peptide LL-37 secreted by stromal cells on the growth of colorectal cancer cells.MethodsColorectal cancer cells SW480 or HCT116 were co-cultured with human macrophages using Transwell？ maxicell inserts to mimic the tumor microenvironment. The effect of macrophages on the proliferation of colorectal cancer cells was detected by Bromodeoxyuridine and enzyme-linked immunosorbent assay （BrdU-ELISA）. The expression of LL-37 mRNA and protein in macrophages and colorectal cancer cells was evaluated by reverse transcription-real-time quantitative PCR （RT-qPCR） and Western blot. LL-37 neutralizing antibody was added to abrogate the LL-37 activation. Additionally, macrophages were transfected with LL-37 shRNA plasmids to inhibit LL-37 expression. And then, the proliferation of colorectal cancer cells was observed. Furthermore, the growth-related signaling pathways were detected by Western blot.ResultsThe BrdU-ELISA results showed that the absorbance of SW480 cells increased from 1.072±0.097 to 5.121±0.407 after co-culture （P〈0.001）, and that of HCT116 cells increased from 1.229±0.073 to 3.495±0.228 （P〈0.001）. RT-qPCR results showed that LL-37 mRNA expression in macrophages significantly increased from 2.682±0.191 to 6.117±0.768 after co-incubation （P〈0.05）, whereas that in SW480 had no significant difference. Consistently the protein expression of LL-37 in macrophages was significantly increased by Western blot, while it did not change in SW480. The proliferation rate of SW480 cells was repressed by adding LL-37 neutralizing antibody or LL-37 shRNA plasmid. Furthermore, Western blot analysis showed that the expression of non-phosphorylated （activated） β-catenin and its target genes cyclin D1 as well as c-myc were distinctly increased in co-cultured SW480 cells, which could be reversed by anti-LL-37 antibodies.ConclusionMacrophages promote the in vitro proliferation of colorectal cancer cells by enhancing the e

关 键 词：结直肠肿瘤 LL-37 共培养 WNT/Β-CATENIN信号通路 

分 类 号：R735.34[医药卫生—肿瘤]