摘要
目的:探讨单核细胞趋化蛋白1(MCP-1)对肺癌A549细胞迁移和侵袭的影响,并阐明其作用机制。方法:采用免疫组织化学法检测80例非小细胞肺癌(NSCLC)及癌旁正常肺组织中MCP-1蛋白表达情况。体外培养人肺癌A549细胞,MCP-1-小干扰RNA(siRNA)实验分为空白组、阴性对照组(si-NC组)、MCP-1-siRNA-1组和MCP-1-siRNA-2组;MCP-1过表达实验分为对照组、空载对照组(OE-NC组,转染MCP-1过表达空载质粒)、过表达MCP-1组(OE-MCP-1组,转染MCP-1过表达质粒)、过表达MCP-1+细胞外调节蛋白激酶(ERK)信号通路抑制剂PD98059组(OE-MCP-1+PD98059组,共转染MCP-1过表达质粒和PD98059)和PD98059组(转染PD98059)。分别将MCP-1-siRNA和质粒转染至肺癌A549细胞,Western blotting法验证各组A549细胞转染效率。细胞划痕实验和Transwell小室实验观察各组A549细胞迁移率和侵袭细胞数,Western blotting法检测各组A549细胞中磷酸化ERK(p-ERK)、总ERK(t-ERK)和上皮-间质转化(EMT)相关蛋白表达水平。结果:与癌旁组织比较,NSCLC组织中MCP-1蛋白阳性表达率明显升高(P<0.05);NSCLC组织中MCP-1蛋白表达水平与TNM分期和淋巴结转移有关联(P<0.05)。与si-NC组比较,MCP-1-siRNA-1组和MCP-1-siRNA-2组A549细胞中MCP-1蛋白表达水平均明显降低(P<0.01);与对照组和OE-NC组比较,OE-MCP-1组A549细胞中MCP-1蛋白表达水平明显升高(P<0.01)。细胞划痕实验,与si-NC组比较,MCP-1-siRNA-1组和MCP-1-siRNA-2组细胞迁移率均明显降低(P<0.01);与OE-NC组比较,OE-MCP-1组细胞迁移率明显升高(P<0.01);与OE-MCP-1组比较,OE-MCP-1+PD98059组细胞迁移率明显降低(P<0.01);与OE-MCP-1+PD98059组比较,PD98059组细胞迁移率明显降低(P<0.01)。Transwell小室实验,与si-NC组比较,MCP-1-siRNA-1组和MCP-1-siRNA-2组侵袭细胞数明显减少(P<0.01);与OE-NC组比较,OE-MCP-1组侵袭细胞数明显增加(P<0.01);与OE-MCP-1组比较,OE-MCP-1+PD98059组侵袭细胞数明显减少(P<0.01);与OE-MCP-1+PD98059组比较,PD98059组侵袭细胞数明显减少(P<0.01)。Western blotting法,与si-NC组比较,MCP-1-siRNA-1组和MCP-1-siRNA-2组A549细胞中p-ERK、波形蛋白(Vimentin)和N-钙黏蛋白(N-cadherin)表达水平均明显降低(P<0.05或P<0.01),E-钙黏蛋白(E-cadherin)表达水平均明显升高(P<0.01);与OE-NC组比较,OE-MCP-1组A549细胞中p-ERK、Vimentin和N-cadherin蛋白表达水平均明显升高(P<0.01),E-cadherin蛋白表达水平均明显降低(P<0.01);与OE-MCP-1组比较,OE-MCP-1+PD98059组A549细胞中p-ERK、Vimentin和N-cadherin蛋白表达水平均明显降低(P<0.01),E-cadherin蛋白表达水平均明显升高(P<0.05);与OE-MCP-1+PD98059组比较,PD98059组A549细胞中p-ERK、 Vimentin和N-cadherin蛋白表达水平均明显降低(P<0.05或P<0.01),E-cadherin蛋白表达水平明显升高(P<0.01)。结论:MCP-1蛋白可上调肺癌A549细胞中EMT相关蛋白表达,促进肺癌A549细胞的迁移和侵袭,其作用机制与激活ERK信号通路有关。
Objective:To discuss the effects of monocyte chemoattractant protein-1(MCP-1) on the migration and invasion of lung cancer A549 cells,and to clarify the mechanisms.Methods:Immunohistochemistry method was used to detect the expression of MCP-1 protein in 80 cases of non-small cell lung cancer(NSCLC) and adjacent normal lung tissues.The human lung cancer A549 cells were cultured in vitro.The MCP-1-small interfering RNA(siRNA) experiment was divided into blank group,negative control group(si-NC group),MCP-1-siRNA-1 group,and MCP-1-siRNA-2 group.The MCP-1 over-expression experiment was divided into control group,empty vector control group(OE-NC,transfected with MCP-1 over-expression empty vector),over-expression MCP-1 group(OE-MCP-1group,transfected with MCP-1 over-expression plasmid),over-expression MCP-1+extracellular regulated protein kinase(ERK) pathway inhibitor PD98059 group(OE-MCP-1+PD98059 group,co-transfected with MCP-1 over-expression plasmid and PD98059),and PD98059 group(transfected with PD98059).The MCP-1 siRNA and plasmids were transfected into the lung cancer A549 cells;Western blotting method was used to verify the transfection efficiencies of the cells in various groups;the migration rate and the number of invasion cells in various groups were observed by wound healing assay and Transwell chamber assay,respectively;Western blotting method was also used to detect the expression levels of phosphorylated ERK(p-ERK),total ERK(t-ERK),and epithelial-mesenchymal transition(EMT)-related proteins in the A549 cells in various groups.Results:Compared with adjacent tissue,the positive expression rate of MCP-1 protein in NSCLC tissue was significantly increased(P<0.05),and the expression level of MCP-1 protein was related to TNM stage and lymph node metastasis(P<0.05).Compared with si-NC group,the expression level of MCP-1 protein in the cells in MCP-1-siRNA-1and MCP-1-siRNA-2 groups was significantly decreased(P<0.01).Compared with control group and OE-NC group,the expression level of MCP-1 protein in the cells in OE-MCP-1 group was significantly increased(P<0.01).The wound healing assay results showed that compared with si-NC group,the migration rate of the cells in MCP-1-siRNA-1 and MCP-1-siRNA-2 groups were significantly decreased(P<0.01).Compared with OE-NC group,the migration rate of the cells in OE-MCP-1 group was significantly increased(P<0.01);compared with OE-MCP-1 group,the migration rate of the cells in OEMCP-1+PD98059 group was significantly decreased(P<0.01).Compared with OE-MCP-1+PD98059group,the migration rate of the cells in PD98059 group was significantly decreased(P<0.01).The Transwell chamber assay results showed that compared with si-NC group,the number of invasion cells in MCP-1-siRNA-1 and MCP-1-siRNA-2 groups was significantly decreased(P<0.01).Compared with OE-NC group,the number of invasion cells in OE-MCP-1 group was significantly increased(P<0.01);compared with OE-MCP-1 group,the number of invasion cells in OE-MCP-1+PD98059 group was significantly decreased(P<0.01);compared with OE-MCP-1+PD98059 group,the number of invasion cells in PD98059 group was significantly decreased(P<0.01).The Western blotting results showed that compared with si-NC group,the expression levels of p-ERK,Vimentin,and N-cadherin protein in the cells in MCP-1-siRNA-1 and MCP-1-siRNA-2 groups were significantly decreased(P<0.05 or P<0.01),and the expression level of E-cadherin proteins was significantly increased(P<0.01).Compared with OE-NC group,the expression levels of p-ERK,Vimentin,and N-cadherin proteins in the cells in OE-MCP-1 group were significantly increased(P<0.01),and the expression level of E-cadherin protein was significantly decreased(P<0.01).Compared with OE-MCP-1 group,the expression levels of p-ERK,Vimentin,and N-cadherins proteins in the OE-MCP-1+PD98059 group were significantly decreased(P<0.01),and the expression level of E-cadherin protein was significantly increased(P<0.05).Compared with OE-MCP-1+PD98059 group,the expression levels of p-ERK,Vimentin,and N-cadherin proteins in the cells in PD98059 group were significantly decreased(P<0.05 or P<0.01),and the expression level of E-cadherin protein was increased(P<0.01).Conclusion:MCP-1 protein can upregulate the expression of EMT-related proteins in the lung cancer A549 cells,and promote the migration and invasion of the lung cancer A549 cells;its mechanism may be related to the activation of the ERK signaling pathway.
作者
王远
王志娟
张明姝
王艺慧
张晴
叶丽平
WANG Yuan;WANG Zhijuan;ZHANG Mingshu;WANG Yihui;ZHANG Qing;YE Liping(Department of Pathology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121001,China;Department of Pathophysiology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121001,China;Institute of Biological Anthropology,Jinzhou Medical University,Jinzhou 121001,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2024年第3期666-675,共10页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金青年科学基金项目(82103624)
吴阶平医学基金会研究基金课题项目(320.6750.2020-06-68)。
关键词
单核细胞趋化蛋白1
细胞外信号调节激酶
癌
非小细胞肺
细胞侵袭
细胞迁移
Monocyte chemoattractant protein-1
Extracellular-signal regulated protein kinase
Cancer,non-small cell lung
Cell invasion
Cell migration
