【作者】 欧周罗； 吴凤英； 邵志敏；

【Author】 Ou Zhou-Luo Wu Feng-Ying Shao Zhi-Min Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China

【机构】 复旦大学附属肿瘤医院复旦大学乳腺癌研究所复旦大学上海医学院肿瘤学系；

【摘要】 目的:CCL2(MCP—1)、CCL5(RANTES)等CC 族趋化因子与乳腺癌的生长、侵袭和转移密切相关,趋化因子诱饵受体可能参与其调控,其中以ELR阳性CXC族和部分CC族趋化因子为靶分子的Duffy antigen receptor for chemokines(DARC)已被证实是一种司负性调节的趋化因子诱饵受体。D6是一种只结合并清除CC族趋化因子的诱饵受体．其配基包括CCL2—5、7、8、11-14、17、22等共 12种。令人感兴趣的是,这些趋化因子中的多数受DARC 与D6的双重调节,而CCL4(MIP—1)、CCL8(MCP-2)、 CCL12(MCP-4)和CCL22(MDC)则只受D6的调节。我们推测乳腺癌中的趋化因子网络可能也在一定程度上受癌细胞自身产生的D6的影响,为此本文首先调查了D6在乳腺癌细胞中的组成性表达以及细胞因子诱导后D6表达的改变。方法:共10种人乳腺癌细胞系分别按美国典型培养物保藏中心(ATCC)等推荐的培养基进行培养,即MCF-7 用MEM培养基,MDA-MB-231、MDA—MB-435、MDA— MB-435H(本室建立的肺高转移性细胞系)和MDA—MB- 468用Leibovitz’s L-15培养基,SK—BR-3用McCoy’ s 5a培养基,T47D、SK—BR-3、BCaP-37、ZR-75-1、 ZR-75-30和Bcap-37用RPMI 1640培养基加10％FCS。提取对数生长期培养细胞和手术切除的人正常脾组织的总 RNA,用RT-PCR法分析D6的GAPDH相对性基因表达。在MCF-7、MDA-MB-231、MDA—MB-435和MDA- MB-435H细胞,同时进行了以SYBR Green I为荧光染料的实时荧光定量PCR分析。此外,分别以重组人IL-1 β (2ng／ml,24h)、TNF-α(20ng／ml,24h)和IFN-γ(20ng／ ml,24h)刺激MDA—MB-231细胞,通过实时PCR分析D6 基因表达的改变。结果:D6 mRNA在所有10个人乳腺癌细胞系及作为阳性对照的人正常脾组织中均可检出,表达水平因细胞而异,其中MCF-7、ZR-75-1和SK-BR-3细胞的表达量较高。此外,以MDA-MB-231细胞为研究对象的细胞因子诱导实验结果显示,IL-1 β促进D6的基因表达,而TNF-α和IFN-γ抑制D6的基因表达。结论:本研究首次证实D6 mRNA在来源于不同患者、生物学特性各异的多种人乳腺癌细胞系中均呈组成性表达,提示CC族趋化因子的诱饵受体D6也可能参与乳腺癌中趋化因子的调控,从而影响乳腺癌的发生、发展过程。乳腺癌中D6的基因表达至少部分地受IL-1β等细胞因子的调节,其意义尚不清楚。对D6的进一步研究将有助于阐明其在乳腺癌等肿瘤中的作用,为肿瘤治疗提供新的思路。更多还原

【Abstract】 Objective: CC chemokines such as CCL2 (MCP-1), CCL5 (RANTES) involve in the growth, invasion and metastasis of breast cancer, they may be regulated by decoy receptors. We have demonstrated that Duffy antigen receptor for chemokines (DARC), as a specific decoy receptor for ELR positive CXC chemokines and some CC chemokines, is a negative regulator in breast cancer. D6 is another decoy receptor for CC chemokines, including CCL2-5, 7, 8, 11-14, 17 and 22. It is worth to be mentioned, D6 is unique regulator for 4 CC chemokines, i. e. CCL4 (MIP-1), CCL8 (MCP-2), CCL12 (MCP-4) and CCL22 (MDC). We speculate that the chemokine network may be controlled by D6 on the breast cancer cells. In this study, we investigated the constitutive gene expression and cytokine regulation of D6 in breast cancer cell lines. Methods: D6 mRNA was analyzed by semi-quantitative RT-PCR in 10 of human breast cancer cell lines with ATCC recommended medium respectively, i.e. MCF-7 in MEM, MDA-MB-231, MDA-MB-435, MDA-MB-435H (with high pulmonary metastatic potential) and MDA-MB-468 in Leibovitz’ s L-15, SK-BR-3 in McCoy’ s 5a, T47D, SK-BR-3, ZR-75-1, ZR-75-30 and BCaP-37 in RPMI 1640 medium plus 10% FCS. The total RNA from normal human spleen was used as a positive control. In MCF-7, MDA-MB-231, MDA-MB-435 and MDA-MB-435H cell lines, the gene expression of D6 was also confirmed by real-time PCR using SYBR Green I as a fluorescent dye. In addition, the dynamics of D6 mRNA in MDA-MB-231 cells was monitored after stimulation with cytokines, including IL-18 (2ng/ml, 24h), TNF- α(20ng/ ml, 24h) or IFN-γ (20ng/ml, 24h). Results: D6mRNAs can be detected in all of 10 cell lines, although their relative level was different. Especially, D6 gene expression was prominent in MCF-7, ZR-75-1 and SK-BR-3 cells. The cytokine irritation experiment in MDA-MB-231 cells reveals that IL-1 β enhanced D6 mRNA expression, but TNF- α and IFN- γ decreased D6 mRNA expression. Conclusion: Our study demonstrated for the first time that multiple human breast cancer cell lines constitutively express D6 mRNA, in spite of their different origin and biological characteristics, suggesting that this decoy receptor may also play a role in chemokine network balance in breast cancer. The production of D6 in breast cancer can be controlled by some cytokines at least partly. It is necessary to disclose the role and regulation of D6 in detail in breast cancer and other tumors in the future.更多还原