特异性蛋白1在胰腺癌中的生物学作用及对细胞因子网络的影响

Biological role of specific protein 1 in pancreatic cancer and its effect on cytokine network

目的探讨特异性蛋白1(Sp1)在胰腺癌中表达和生物学功能并初步分析Sp1调控的细胞因子网络。方法Western blotting检测不同胰腺癌细胞系(BxPC3、AsPC1、CFPAC1、PANC1、Capan1、HS-766T、SW1990和HPAF-II)的Sp1水平;慢病毒感染构建稳定干扰和过表达Sp1的胰腺癌细胞系,IncuCyte生长曲线和平板克隆形成实验检测增殖活力,划痕实验和Boyden小室实验检测迁移和侵袭能力。悬液芯片进一步研究干扰Sp1前后的细胞因子变化并分析调控的信号通路和生物学功能。结果与正常胰腺上皮细胞HPDE相比,胰腺癌细胞系中Sp1呈不同程度的表达,其中BxPC3细胞的最高,而AsPC1细胞的最低。接下来成功构建稳定干扰和过表达Sp1的胰腺癌细胞系,体外实验证明Sp1促进胰腺癌细胞体外增殖、迁移和侵袭能力。悬液芯片结果:干扰Sp1后一组炎性因子、趋化因子和基质金属蛋白酶类发生变化,其中51个上调(>2倍)、26个下调(<0.5倍)。GO分析显示,差异表达的因子与细胞生长增殖、炎症反应、免疫反应、淋巴细胞迁移和趋化反应等密切相关。信号通路分析显示差异表达基因涉及细胞因子与其受体反应、趋化因子信号通路、TNF、TGF-β、JAK/STAT和uPAR等。成功构建Sp1调控差异细胞因子的网络模式图。结论胰腺癌高表达Sp1可通过分泌大量细胞因子来激活相应信号通路,进而调控微环境,共同促进胰腺癌侵袭和转移。下一步需要深入研究Sp1在细胞因子调控网络中的核心作用机制。

Objective To investigate the expression and biological function of specific protein 1(Sp1)in pancreatic cancer,and further analyze the cytokine network regulated by Sp1.Methods Expressions of Sp1 in pancreatic cancer cell lines(BxPC3,AsPC1,CFPAC1,PANC1,Capan1,HS-766T,SW1990 and HPAF-II)were detected by Western blotting.The stable interference and overexpression vector of Sp1 in pancreatic cancer cell lines were established using lentivirus infection.Cell proliferation activity was analyzed by IncuCyte growth curve and plate clone formation experiment.Cell migration and invasion abilities were detected by scratch test and Boyden chamber.The suspension chip was used to further study the changes of cytokines before and after being interfered with Sp1,and analyze the regulated signal pathways and biological functions.Results Compared to normal pancreatic epithelial cells HPDE,Sp1 was expressed in pancreatic cancer cells at different levels,among which BxPC3 cell was the highest and AsPC1 cell was the lowest.Subsequently,pancreatic cancer cells with stable interference and overexpression of Sp1 were successfully constructed.In vitro experiments showed that Sp1 enhanced abilities of cell proliferation,migration and invasion.Results of suspension microarray found that knockdown of Sp1 changed inflammatory factors,chemokines and matrix metalloproteinases,among which 51 cytokines were up-regulated(>2 folds)and 26 cytokines were down-regulated(<0.5 folds).GO analysis showed that the differentially expressed factors were closely related to cell growth and proliferation,inflammatory response,immune response,lymphocyte migration and chemotactic response.Signaling pathways analysis showed involvement of cytokines and their receptor reactions,chemokine signaling pathways,TNF,TGF-β,JAK/STAT,uPAR,etc.Finally,the network pattern diagram of Sp1 regulating differential cytokines was successfully constructed.Conclusion High Sp1 expression can activate the corresponding signaling pathway by secreting a large number of cytokines,thereby regulating the microenvironment and jointly promoting the invasion and metastasis of pancreatic cancer.Further study is to explore the core mechanism of Sp1 in the cytokine regulatory network.