乌司他丁抑制CCL17/CCL22-CCR4介导的小鼠乳腺癌肝转移

Ulinastatin inhibits liver metastasis from breast cancer in mice through CCL17/CCL22–CCR4 pathway

背景与目的:越来越多的研究表明趋化因子及其受体在癌症的发生、发展和转移中起关键作用。本文研究了乌司他丁(ulinastatin)对胸腺和活化调节趋化因子(thymus and activation-regulated chemokine,TARC)即CCL17/巨噬细胞来源的趋化因子(macrophage-derived chemokine,MDC)即CCL22-CC族趋化因子受体4(CC chemokine receptor 4,CCR4)信号通路介导的乳腺癌肝转移的影响及其作用机制。方法:通过小鼠乳腺脂肪垫(mfp)接种4T1乳腺癌细胞的方式构建小鼠乳腺癌模型,15 d后取小鼠乳腺肿瘤,并记录乳腺肿瘤的质量;采用免疫组织化学法检测乳腺肿瘤组织中CCR4及肝脏转移瘤中CCL22、CCL17蛋白的表达;采用慢病毒转染的方式抑制4T1乳腺癌细胞CCR4基因,采用蛋白质印迹法(Western blot)检测抑制效果,并以同样方式进行小鼠乳腺成瘤并观察肿瘤生长情况;用三种不同浓度乌司他丁处理4T1细胞荷瘤小鼠,15 d后采用免疫组织化学法检测乳腺癌组织中CCR4及肝脏组织中CCL22及CCL17的表达,采用Western blot和实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)分别检测肝脏组织中TGF-β的表达以及microRNA-34a和microRNA-31的含量,并进行TGF-β和microRNA-34a、microRNA-31、CCL22以及CCL17的相关性分析。结果:小鼠乳腺癌组织高表达CCR4,肝脏转移瘤高表达CCL22和CCL17;沉默4T1乳腺癌细胞CCR4的表达,可抑制乳腺癌成瘤性;乌司他丁抑制CCR4、TGF-β、CCL22、microRNA-31和CCL17的表达,促进microRNA-34a的表达。结论:乌司他丁具有抑制乳腺癌肝转移的作用,其具体机制可能与乌司他丁通过TGF-β-microRNA-34a-CCL22轴及microRNA-31-TGF-β-CCL17轴抑制肝脏组织中与乳腺癌肝转移密切相关的CCL17/CCL22-CCR4信号通路有关。

Background and purpose： Increasing evidences have shown the key roles of chemokines in the formation and metastasis of cancers. In this study, we investigated the molecular mechanisms of liver metastasis from breast cancer and the effects of ulinastatin on liver metastasis from breast cancer mediated by CCL17/CCL22–CCR4 pathway. Methods： A mouse xenograft model and corresponding control were established by subcutaneously inoculating 4 T1 breast cancer cells into mouse mammary fat pad. Fifteen days later, the mice were sacrificed, and the breast tumors were weighed. Immunohistochemistry was performed to detect CCR4 protein expression in breast tumors and CCL22 and CCL17 protein expressions in liver metastasis. The CCR4 gene was found to be inhibited by lentiviral transduction in 4 T1 breast cancer cells. Western blot was used to examine the inhibitory effect. The same method was used to induce breast tumorigenesis and to examine tumor growth. Three different concentrations of ulinastatin were used to treat 4 T1 tumor-bearing mice. Fifteen days later, immunohistochemistry was used to assess the expression of CCR4 in breast tumors and the expression of CCL22 and CCL17 in liver tissues. Western blot and real-time fluorescent quantitative polymerase chain reaction（RTFQ-PCR） were used to detect the expression of TGF-β, microRNA-34 a and microRNA-31. We also analyzed the correlation between TGF-β and microRNA-34 a, microRNA-31, CCL22, and CCL17. Results： Our results showed that CCR4 was highly expressed in mouse breast tumors. CCL22 and CCL17 were highly expressed in the liver metastasis. CCR4 expression was silenced in the 4 T1 breast cancer cells and the in vivo growth of breast cancer xenograft tumor was inhibited. Ulinastatin significantly inhibited CCR4, TGF-β, CCL22, microRNA-31 and CCL17, but upregulated microRNA-34 a. Conclusion： These results showed that ulinastatin can inhibit liver metastasis of breast cancer. The specific mechanism may involve ulinastatin acting on TGF-β-microRNA-34 a-CCL22 and microRNA-31-TGF-β-CCL17 axes to inhibit CCL17/CCL22-CCR4 signaling pathway in liver tissues.