Yao Hongqiang, Sun Lihong, Luo Shuang, Ji Xiuhong
Bing Du Xue Bao. 2016 May;32(3):283-91.
This study aims to explore the tumorigenic mechanism of the target cells following JSRV interaction with its receptor. We transfected mouse lung epithelial cells (TC-1) and mouse lung epithelial cells stably expressing sheep Hyal-2(TC-1-Hyal2)with JSRV-Env eukaryotic expression vector, measured the changes in the mRNA and protein expression of AKT(serine/threonine kinase)and ERK(extracellular signal-regulated kinase)in cellular signal transduction pathways, and analyzed the role of sheep Hyal-2in JSRV-Env-induced transformation of TC-1cells.First,TC-1and TC-1-Hyal2 cells were cultured in vitro and were each divided into pEGFP-C1-env transfection group,pEGFP-C1 transfection group, and untransfected group. The expression of key enzymes was determined by PCR and Western blotting. qPCR showed that, for both cell lines, compared with untransfected cells, the expression of AKT and ERK1/2mRNA was significantly increased in the pEGFP-C1-env transfected cells(P<0.05).Western blotting showed that, relative to untransfected cells, transfection with pEGFP-C1-env significantly increased p-Akt (S473)protein expression in both cell lines(P<0.05).Moreover, p-Akt (T308)and p-Erk1/2protein expression was increased significantly in the pEGFP-C1-env transfected TC-1cells(P<0.05),and very significantly in the pEGFP-C1-env transfected TC-1-Hyal2cells(P<0.01).Cells of each type transfected with the empty vector pEGFP-C1 and the untransfected cells did not show significant differences in their mRNA and protein levels of AKT and ERK(P >0.05).Thus, the expression of JSRV-Env in the cell lines TC-1and TC-1-Hyal2 activated the cellular signal transduction pathways Ras-Raf-MAPK and PI3K-Akt.The expression of AKT and ERK was significantly increased in pEGFP-C1-env transfected TC-1and TC-1-Hyal2 cells, but a greater increase was seen in the TC-1-Hyal2 cells.We speculate that Hyal2 plays a catalytic role in JSRV-Env-induced transformation of TC-1cells.
本研究旨在探讨绵羊肺腺瘤病毒（JSRV）与其受体相互作用后靶细胞的致瘤机制。我们用JSRV-Env真核表达载体转染小鼠肺上皮细胞（TC-1）和稳定表达绵羊Hyal-2的小鼠肺上皮细胞（TC-1-Hyal2），检测细胞信号转导通路中AKT（丝氨酸/苏氨酸激酶）和ERK（细胞外信号调节激酶）的mRNA和蛋白表达变化，并分析绵羊Hyal-2在JSRV-Env诱导的TC-1细胞转化中的作用。首先，将TC-1和TC-1-Hyal2细胞在体外培养，并分别分为pEGFP-C1-env转染组、pEGFP-C1转染组和未转染组。通过PCR和蛋白质印迹法测定关键酶的表达。定量PCR显示，对于这两种细胞系，与未转染细胞相比，pEGFP-C1-env转染细胞中AKT和ERK1/2mRNA的表达显著增加（P<0.05）。蛋白质印迹法显示，相对于未转染细胞，用pEGFP-C1-env转染在两种细胞系中均显著增加了p-Akt（S473）蛋白表达（P<0.05）。此外，在pEGFP-C1-env转染的TC-1细胞中p-Akt（T308）和p-Erk1/2蛋白表达显著增加（P<0.05），而在pEGFP-C1-env转染的TC-1-Hyal2细胞中增加非常显著（P<0.01）。用空载体pEGFP-C1转染的各类型细胞和未转染细胞在AKT和ERK的mRNA和蛋白水平上均未显示出显著差异（P>0.05）。因此，JSRV-Env在细胞系TC-1和TC-1-Hyal2中的表达激活了细胞信号转导通路Ras-Raf-MAPK和PI3K-Akt。在pEGFP-C1-env转染的TC-1和TC-1-Hyal2细胞中AKT和ERK的表达显著增加，但在TC-1-Hyal2细胞中增加幅度更大。我们推测Hyal2在JSRV-Env诱导的TC-1细胞转化中起催化作用。
