肝再生磷酸酶-3通过肿瘤相关巨噬细胞作用促进结肠癌侵袭转移的研究  

Liver regeneration phosphatase 3 promotes invasion and metastasis of colorectal cancer cells through tumor associated macrophages

作　　者：张韬 罗自通[1] 蓝球生 许鹤洋[1] 褚自强 苏鹏伟 褚忠华[1] Zhang Tao;Luo Zitong;Lan Qiusheng;Xu Heyang;Chu Ziqiang;Su Pengwei;Chu Zhonghua(Department of Gastrointestinal Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China)

机构地区：[1]中山大学孙逸仙纪念医院胃肠外科,广州510120

出　　处：《中华实验外科杂志》2019年第4期656-659,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然青年科学基金(81602539、81702902、81602125);国家自然科学基金面上项目(81871981);广东省省级科技计划项目(2015A050502021).

摘　　要：目的探讨肝再生磷酸酶-3(PRL-3)与肿瘤相关巨噬细胞(TAM)相互作用从而促进结肠癌细胞侵袭转移的机制。方法构建稳定转染下调PRL-3(HT29-P/HT29-NC)以及上调PRL-3的结肠癌细胞株(LoVo-P/LoVo-NC),分别与肿瘤相关巨噬细胞(TAM)共培养后,通过Western blot检测TAM中丝裂原活化蛋白激酶(MAPK)的表达。采用Transwell侵袭实验比较与TAM共培养后结肠癌细胞的侵袭能力。抑制TAM中MAPK信号通路后,通过侵袭实验检测结肠癌细胞的侵袭能力。将细胞注入小鼠皮下行小鼠成瘤实验,将瘤体切片行免疫组织化学观察MAPK蛋白的表达情况。结果Westernblot显示,与高表达PRL-3的结肠癌细胞(HT29-NC、LoVo-P)共培养后的TAM中MAPK蛋白相较于低表达组(p-JNK,p-ERK)明显升高(P<0.05),其中LoVo-P比LoVo-NC升高3倍(p-JNK)及2倍(p-ERK),HT29-NC中表达为HT29-P的3倍(p-JNK)及1.3倍(p-ERK)。而Transwell侵袭实验表明,高表达PRL-3的结肠癌细胞与TAM作用后可增强自身的侵袭能力。其中LoVo细胞[(268±22)个比(129±12)个],HT29细胞[(298±19)个比(152±14)个],P<0.05。同时,抑制TAM中的MAPK信号通路后,共培养后结肠癌细胞的EMT减弱,侵袭能力也相应下降。LoVo最终通过细胞数降至1/2,HT29降至1/3,P<0.05。另外,小鼠成瘤实验中的组织切片免疫组化结果显示,在PRL-3表达高的组织中,TAM中MAPK磷酸化蛋白增加,其中LoVo[p-JNK:82%/20%,p-ERK:83%/16%],HT29[p-JNK:81%:21%,p-ERK:76%/16%]。结论PRL-3激活肿瘤相关巨噬细胞中的MAPK信号通路增强结肠癌细胞的EMT从而促进其侵袭。Objective To investigate whether the liver regeneration phosphatase 3 (PRL-3) promotes invasion and metastasis of colorectal cancer cells (CRCs) through tumor associated macrophage (TAM). Methods We selected LoVo cells to construct PRL-3-overexpression lines (LoVo-P) and HT29 cells (HT29-P) as the knockdown groups. After co-culture with LoVo-P, LoVo-NC, HT29-NC and HT29-P respectively, we performed Western blotting to identify the activation of mitogen-activated protein kinase (MAPK) pathway in TAMs. Meanwhile, Transwell assays were carried out before and after suppressing MAPK pathway in TAMs. Besides, immunohistochemistry was used to confirm the status of MAPK in xenografts of mice injected with cells mentioned above. Results Western blot showed that the interaction between PRL-3 and TAM activated MAPK pathways in TAMs promoting EMT in CRCsand upregulated the expression of IL-6 and IL-8.LoVo-P increased by a triple expression of p-JNK and double of p-ERK than LoVo-NC (P<0.05). In terms of HT29, the levels of p-JNK and p-ERK rised to 3 folds and 1.3 folds (P<0.05). Also, transwell assays showed that cell invasion and migration were enhanced with high levels of PRL-3. In LoVo, the number of migrating cells of LoVo-P vesusLoVo-NC was [268±22 vs. 129±12], and HT29-NC vesus HT29-P was [298±19 vs. 152±14](P<0.05). Results of immunohistochemistry were consistent with those in Western blot, which showed LoVo [p-JNK: 82%/20%, p-ERK: 83%/16%] and HT29 [p-JNK: 81%: 21%, p-ERK: 76%/16%](P<0.05). Conclusion PRL-3 promotes invasion and metastasis of CRCs through EMT by initiating MAPK pathways in TAMs.

关 键 词：结肠癌 蛋白酪氨酸磷酸酶-3 肿瘤相关巨噬细胞 磷酸化 

分 类 号：R735.35[医药卫生—肿瘤]