摘要
目的研究人乳腺癌微环境癌相关成纤维细胞(CAF)与正常成纤维细胞(NF)miRNA表达的差异及其对CAF的生物学功能的影响。方法运用1型胶原酶消化法分别处理临床乳腺癌患者的癌组织和对应癌旁组织标本,原代分离培养CAF和NF细胞;利用免疫荧光法和Western blot法分析CAF和NF细胞成纤维细胞分泌蛋白(FSP)的表达情况,对所分离培养的细胞进行纯度和特性鉴定;利用TranswellTM实验比较CAF与NF细胞的侵袭能力;利用miRNA芯片和芯片结果显著性差异(SAM)分析法分析CAF与NF细胞miRNA表达的差异;对差异miR-205和miR-221,在原代培养的CAF和NF进行实时定量PCR(qRT-PCR)验证;利用多种生物信息学软件预测差异miRNA的靶基因;利用DAVID软件对靶基因进行信号通路富集度分析。利用ELISA检测TGF-β和IL-6信号通路的核心蛋白基质金属蛋白酶1(MMP-1)、MMP-2和MMP-9的表达差异。结果成功分离得到原代培养的CAF与NF细胞,纯度大于95%;与NF细胞相比,CAF细胞的侵袭能力明显增强;miRNA芯片分析结果显示,CAF有10个变化倍数>1.5的异常表达miRNA(P<0.05),其中3个上调表达(miR-221-5p、miR-31-3p、miR-221-3p),7个下调表达(miR-205、miR-200b、miR-200c、miR-141、miR-101、miR-342-3p、let-7g)。信号通路富集度分析发现,miRNA靶基因与细胞分化、细胞黏附、细胞迁移、细胞增殖、细胞分泌和细胞间相互作用等密切相关,其中,异常表达的miR-200b/c和miR-141等miRNA通过抑制其靶基因,影响TGF-β信号通路、IL-6信号通路,进而影响CAF的侵袭和转移。结论人乳腺癌微环境CAF的miRNA表达谱发生显著变化,CAF细胞中异常表达miRNA可能参与了NF向CAF的转化,并与CAF细胞的增殖、黏附、侵袭、转移有密切关系。
Objective To investigate the difference of miRNA expression levels of cancer-associated fibroblasts（ CAFs）and normal fibroblasts（ NFs） in human breast cancer microenvironment and its effect on the biological features of CAFs.Methods Collagenase-1 was used to digest the cancer and adjacent tissues to isolate CAFs and NFs. The isolated cells were cultured and characterized in purity and biological features. The expression of fibroblast secretory protein（ FSP） in CAFs and NFs was detected by immunofluorescence staining and Western blotting. TranswellTMassay was adopted to compare the invasion ability of CAFs and NFs. The different expressions of miRNAs in CAFs versus NFs were detected by miRNA microarray and analyzed by Significance Analysis of Microarrays（ SAM）. The differences in miR-205 and miR-221 expressions were verified by real-time quantitative PCR（ qRT-PCR）. The common target genes of the miRNAs were predicted using multi-bioinformatics tools. The pathway analysis was conducted through the Database for Annotation,Visualization and Integrated Discovery（ DAVID） v6. 7. The secreting products of TGF-β or IL-6 signaling pathway,matrix metalloproteinase（ MMP）-1,MMP-2 and MMP-9 were analyzed by ELISA. Results The primary CAFs and NFs were isolated from breast cancer patients with a purity of over 95%. Compared with NFs,the expression of FSP was obviously elevated in CAFs,and the invasion ability of CAFs was enhanced. The miRNA microarray results showed that there were 10 miRNA genes dysregulated in CAFs,including 3 up-regulated（ miR-221-5p,miR-31-3p,miR-221-3p） and 7 down-regulated genes（ miR-205,miR-200 b,miR-200 c,miR-141,miR-101,miR-342-3p,let-7g）. The common targets genes of the dysregulated miRNAs were mainly focused on HGF,chemokine signaling,insulin signaling,MAPK signaling,tight junction signaling,adherence junction signaling,EGF1 signaling,androgen-receptor signaling,Wnt and IL-7 signaling. In addition,dysregulated miR-200 b /c and miR-141 et al. affect TGF-β and IL-6
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第10期1071-1075,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81172296)
