目的:探究体外培养胰腺癌细胞系中L1CAM与α5-整合素产生促血管生成因子对内皮细胞血管生成方面的影响。方法:将重组质粒pc DNA3.1-L1CAM和α5-整合素小干扰RNA分别转入Patu8988和Capan-2胰腺癌细胞系上调和下调靶基因的表达。Western b...目的:探究体外培养胰腺癌细胞系中L1CAM与α5-整合素产生促血管生成因子对内皮细胞血管生成方面的影响。方法:将重组质粒pc DNA3.1-L1CAM和α5-整合素小干扰RNA分别转入Patu8988和Capan-2胰腺癌细胞系上调和下调靶基因的表达。Western blot及实时定量PCR检测Patu8988及Capan-2胰腺癌细胞系中促血管生成因子[信号转导及转录激活因子3(signal transducers and activators of transcription3,STAT3)、磷酸化信号转导及转录因子3(phosphorylated signal transducers and activators of transcription 3,pSTAT3)、血管内皮生长因子-A(vascular endothelial growth factor-A,VEGF-A)、血管生成素2(angiopoietin-2,Ang-2)]表达的水平差异;血管生成实验检测改变L1CAM及α5-整合素表达水平后Patu8988和Capan-2胰腺癌细胞系上清液对体外血管生成的影响。结果:上调Patu8988及Capan-2胰腺癌细胞系中L1CAM的表达水平可促进pSTAT3、VEGF-A及Ang-2的mRNA及蛋白表达水平(P<0.01),并促进人脐静脉内皮细胞系(human umbilical vein endothelium cell,HUVEC)的完整血管形成(P<0.01)。而抑制Patu8988及Capan-2胰腺癌细胞系中的α5-整合素表达水平显著减少pSTAT3、VEGF-A及Ang-2的mRNA及蛋白表达水平(P<0.01),并明显削减了HUVEC的成管能力(P<0.01)。结论:体外培养胰腺癌细胞系中,L1CAM表达水平增加显著促进促血管生成因子表达和血管生成能力;而抑制α5-整合素的表达水平则可显著抑制促血管生成因子表达和成管能力。展开更多
Objective:L1 cell adhesion molecule(L1 CAM)exhibits oncogenic activity in tumors.However,the link between L1 CAM and the tumor microenvironment remains poorly understood in patients with esophageal squamous cell carci...Objective:L1 cell adhesion molecule(L1 CAM)exhibits oncogenic activity in tumors.However,the link between L1 CAM and the tumor microenvironment remains poorly understood in patients with esophageal squamous cell carcinoma(ESCC).In this study,we investigated how L1 CAM expression in ESCC affects the oncogenic characteristics of tumor cells and the tumor microenvironment.Methods:Human ESCC samples were collected,and the m RNA and protein levels of L1 CAM were examined by real-time PCR and immunohistochemistry.Overexpression and knockdown gene expression assays were used for mechanistic studies.The cell proliferation and cell cycle were measured with CCK-8 assays and flow cytometry.Cell migration and invasion ability were measured with Transwell assays.Multiplex bead-based assays were performed to identity the factors downstream of L1 CAM.Xenograft studies were performed in nude mice to evaluate the effects of L1 CAM on tumor growth and regulatory T cell(Treg)recruitment.Results:L1 CAM expression was significantly elevated in ESCC tissues(P<0.001)and correlated with poorer prognosis(P<0.05).Ablation of L1 CAM in ESCC cells inhibited tumor growth and migration,and increased tumor cell apoptosis(P<0.05).In the tumor microenvironment,L1 CAM expression correlated with Treg infiltration in ESCC by affecting CCL22 secretion.Mechanistically,L1 CAM facilitated CCL22 expression by activating the PI3 K/Akt/NF-κB signaling pathway.Furthermore,CCL22 promoted Treg recruitment to the tumor site;the Tregs then secreted TGF-β,which in turn promoted L1 CAM expression via Smad2/3 in a positive feedback loop.Conclusions:Our findings provide new insight into the mechanism of immune evasion mediated by L1 CAM,suggesting that targeting L1 CAM-CCL22-TGF-βcrosstalk between tumor cells and Tregs may offer a unique means to improve treatment of patients with ESCC.展开更多
文摘目的:探究体外培养胰腺癌细胞系中L1CAM与α5-整合素产生促血管生成因子对内皮细胞血管生成方面的影响。方法:将重组质粒pc DNA3.1-L1CAM和α5-整合素小干扰RNA分别转入Patu8988和Capan-2胰腺癌细胞系上调和下调靶基因的表达。Western blot及实时定量PCR检测Patu8988及Capan-2胰腺癌细胞系中促血管生成因子[信号转导及转录激活因子3(signal transducers and activators of transcription3,STAT3)、磷酸化信号转导及转录因子3(phosphorylated signal transducers and activators of transcription 3,pSTAT3)、血管内皮生长因子-A(vascular endothelial growth factor-A,VEGF-A)、血管生成素2(angiopoietin-2,Ang-2)]表达的水平差异;血管生成实验检测改变L1CAM及α5-整合素表达水平后Patu8988和Capan-2胰腺癌细胞系上清液对体外血管生成的影响。结果:上调Patu8988及Capan-2胰腺癌细胞系中L1CAM的表达水平可促进pSTAT3、VEGF-A及Ang-2的mRNA及蛋白表达水平(P<0.01),并促进人脐静脉内皮细胞系(human umbilical vein endothelium cell,HUVEC)的完整血管形成(P<0.01)。而抑制Patu8988及Capan-2胰腺癌细胞系中的α5-整合素表达水平显著减少pSTAT3、VEGF-A及Ang-2的mRNA及蛋白表达水平(P<0.01),并明显削减了HUVEC的成管能力(P<0.01)。结论:体外培养胰腺癌细胞系中,L1CAM表达水平增加显著促进促血管生成因子表达和血管生成能力;而抑制α5-整合素的表达水平则可显著抑制促血管生成因子表达和成管能力。
基金supported by grants from the National Natural Science Foundation of China(Grant No.81802857)State’s Key Project of Research and Development Plan(Grant No.2016YFC1303501)+1 种基金Henan Science and Technology Research Project(Grant No.172102310143)The Key Research Project of Henan Provincial Colleges and Universities(Grant No.19A320062)。
文摘Objective:L1 cell adhesion molecule(L1 CAM)exhibits oncogenic activity in tumors.However,the link between L1 CAM and the tumor microenvironment remains poorly understood in patients with esophageal squamous cell carcinoma(ESCC).In this study,we investigated how L1 CAM expression in ESCC affects the oncogenic characteristics of tumor cells and the tumor microenvironment.Methods:Human ESCC samples were collected,and the m RNA and protein levels of L1 CAM were examined by real-time PCR and immunohistochemistry.Overexpression and knockdown gene expression assays were used for mechanistic studies.The cell proliferation and cell cycle were measured with CCK-8 assays and flow cytometry.Cell migration and invasion ability were measured with Transwell assays.Multiplex bead-based assays were performed to identity the factors downstream of L1 CAM.Xenograft studies were performed in nude mice to evaluate the effects of L1 CAM on tumor growth and regulatory T cell(Treg)recruitment.Results:L1 CAM expression was significantly elevated in ESCC tissues(P<0.001)and correlated with poorer prognosis(P<0.05).Ablation of L1 CAM in ESCC cells inhibited tumor growth and migration,and increased tumor cell apoptosis(P<0.05).In the tumor microenvironment,L1 CAM expression correlated with Treg infiltration in ESCC by affecting CCL22 secretion.Mechanistically,L1 CAM facilitated CCL22 expression by activating the PI3 K/Akt/NF-κB signaling pathway.Furthermore,CCL22 promoted Treg recruitment to the tumor site;the Tregs then secreted TGF-β,which in turn promoted L1 CAM expression via Smad2/3 in a positive feedback loop.Conclusions:Our findings provide new insight into the mechanism of immune evasion mediated by L1 CAM,suggesting that targeting L1 CAM-CCL22-TGF-βcrosstalk between tumor cells and Tregs may offer a unique means to improve treatment of patients with ESCC.
