蛋白激酶D-1调控神经内分泌肿瘤细胞上皮-间充质转化的机制

The mechanism of protein kinase D-1 regulating epithelial-mesenchymal transition of tumor cell in neuroendocrine neoplasm

目的研究蛋白激酶D-1(PKD-1)在神经内分泌肿瘤(NEN)中的表达以及PKD-1信号调控NEN细胞上皮-间充质转化(EMT)的机制,探讨其与NEN转移的相关性。方法通过免疫荧光染色和免疫组织化学染色的方法,检测人胰腺NEN组织中PKD-1以及波形蛋白(Vimentin)、白细胞共同抗原(CD45)、α-平滑肌肌动蛋白(α-SMA)的表达及分布,分析转移性肿瘤细胞中PKD-1的表达;通过体外培养BON细胞,分别激活和阻断PKD-1信号,或以表皮生长因子(EGF)处理细胞,以免疫荧光染色、蛋白质印迹法(Western blot)和反转录实时荧光定量聚合酶链反应(RT-qPCR)的方法,检测BON细胞中EMT相关标志物的表达,分析PKD-1信号对BON细胞EMT的调控机制。两样本间比较采用t检验,多样本间比较采用单因素方差分析(ANOVA)。结果NEN组织中Vimentin+转移性肿瘤细胞有在小动脉周围聚集现象,且PKD-1在NEN肿瘤细胞转移过程中呈动态变化;PKD-1信号能够促进BON细胞Vimentin的表达[(56.67±1.93)%比(35.55±2.22)%,t=7.178,P<0.01]并升高PKD-1和细胞外信号调节激酶1/2(ERK1/2)的磷酸化水平,EGF能刺激BON细胞发生间质样形态变化,并降低E-钙黏蛋白(E-cadherin)的表达(0.517±0.005比1.000±0.004,t=74.63,P<0.001)而增加Vimentin的表达(9.632±0.646比1.004±0.061,t=13.29,P<0.001)。结论NEN中PKD-1信号是调控肿瘤细胞EMT的重要机制;血管内皮细胞分泌EGF可能通过PKD-1信号诱导肿瘤细胞EMT,促进肿瘤转移。

Objective To investigate the expression of protein kinase D-1(PKD-1)in neuroendocrine tumors(NEN)and the mechanism of PKD-1 signaling regulating epithelial mesenchymal transition(EMT)in NEN cells,as well as the correlation between PKD-1 and NEN metastasis.Methods The expression and distribution of PKD-1,Vimentin,CD45 andα-smooth muscle actin(α-SMA)in human pancreatic NEN tissues were detected by immunofluorescence staining and immunohistochemical staining,to analyze the expression of PKD-1 in metastatic tumor cells.BON cells were cultured in vitro and treated with PKD-1 activator or inhibitor,or epidermal growth factor(EGF).Immunofluorescence staining,Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)were used to detect the expression of EMT-related markers in BON cells,to analyze the mechanism of PKD-1 signaling regulating EMT in BON cells.The t-test was used for comparison between two groups,and one-way analysis of variance(ANOVA)was used for comparison between multiple groups.Results Vimentin+metastatic tumor cells were observed aggregating around arterioles in NEN tissues,and PKD-1 showed dynamic expression in tumor cells during metastasis.PKD-1 signaling promoted the expression of Vimentin in BON cells[(56.67±1.93)%vs.(35.55±2.22)%,t=7.178,P<0.01],and EGF induced mesenchymal morphology in BON cells and reduced E-cadherin expression(0.517±0.005 vs.1.000±0.004,t=74.63,P<0.001)but enhanced Vimentin expression(9.632±0.646 vs.1.004±0.061,t=13.29,P<0.001).Conclusion PKD-1 signaling plays an important role in regulating EMT in NEN.EGF secreted by vascular endothelial cells may induce EMT in tumor cells via PKD-1 signaling and promote tumor metastasis.