Background: It has been proved that cyclooxygenase-2 (COX-2) is a key factor in lung cancer oncogenesis. COX-2 can be induced by a number of cytokines and growth factors and can be regulated by the JAK/STAT signaling pathway. Inhibiting the expression of COX-2 can prevent the development of lung cancer. The aim fo this study is to investigate whether the epidermal growth factor (EGF) can stimulate the signal transducers and activators of transcription 5 (STAT5) as well as to discover the effects of the STAT5 signaling pathway on the COX-2 in human lung adenocarcinoma A549 cells.
Methods: The phenomenon of STAT5 activation stimulated by the EGF was assayed through immunofluorescence and Western blot. The adenovirus containing the wild-type (WT)-STAT5 (AdWT-STAT5) plasmid, dominant-negative (DN)-STAT5 (Ad-CMV5Stat5aΔ740) plasmid, and STAT5 siRNA were transfected into A549 cells. The latter two groups were stimulated using EGF. Reverse transcriptase polymerase chain reaction was used to detect the mRNA expression of COX-2.
Results: STAT5 was not activated in A549 cells in vitro. EGF stimulation significantly increased the level of the p-STAT5 protein and induces the shuttling of p-STAT5 from the cytoplasm into the nucleus. STAT5 activation was crucial for the COX-2 expression induced by the EGF. STAT5 was required for COX-2 expression, but can mediated the effects of the COX-2 expression through pathways that were independent of transcriptional activation.
Conclusions: COX-2 expression is dependent on STAT5 phosphorylation. A second pathway does not require STAT5 phosphorylation.
背景与目的 已有的研究表明COX-2在肺癌发生发展过程中起关键作用，它被一些细胞因子和生长因子所诱导产生，并受到JAK/STAT等信号通路的调控，抑制COX-2的表达能阻止肺癌的发展。本研究旨在探讨表皮生长因子（epidermal growth factor, EGF）在人肺腺癌A549细胞中对STAT5激活效应，以及STAT5信号通路对COX-2调控机制。方法 应用免疫荧光法及Western印迹法检测人肺腺癌A549细胞中EGF对STAT5的激活现象。分别用野生型STAT5（AdWT STAT5），STAT5显性负突变体（AdCMV5 Stat5a△740）以及STAT5 siRNA转染A549细胞，并用EGF对后两组转染细胞加以刺激，使STAT5及p-STAT5的表达发生变化，再用RT-PCR检测A549细胞中的COX-2 mRNA表达。结果 在体外A549细胞中STAT5无激活；EGF可以诱导STAT5的激活，促使磷酸化的STAT5穿梭入核；STAT5的激活是EGF诱导COX-2上调表达的必要条件；非磷酸化的STAT5可能通过非转录激活的途径参与了COX-2表达的调控。结论 在A549细胞中STAT5可以通过磷酸化和非磷酸化两种途径来实现对COX-2的调控。