Cao Yiwei, Luo Xiangjun, Ding Xiaoqian, Cui Shichao, Guo Caihong
Department of Respiratory Medicine, The Affiliated Hospital of Qingdao University, Qingdao, China.
Department of Respiratory Medicine, Qingdao Eighth People's Hospital, Qingdao, China.
J Cell Biochem. 2018 Aug;119(8):6935-6942. doi: 10.1002/jcb.26894. Epub 2018 Apr 25.
Lung cancer is a commonly diagnosed disease with poor prognosis. Novel therapeutic targets and deep understanding of the regulatory mechanisms in lung cancer are of great importance. We aimed to figure out the functional roles of lncRNA-activated by transforming growth factor-β (ATB) in A549 cells as well as the underlying molecular mechanisms. ATB was non-physiologically expressed in A549 cells after cell transfection. Then, cell proliferation, expressions of proteins related to proliferation and epithelial-mesenchymal transition (EMT), migration, and invasion were measured by BrdU incorporation assay, Western blot analysis, and Transwell assay, respectively. Afterwards, miR-494 expression in transfected A549 cells was determined by quantitative reverse transcription PCR. Meanwhile, effects of miR-494 overexpression on ATB-overexpressed cells were assessed. Finally, the phosphorylation levels of AKT and key kinases in the Janus-activated kinase (JAK)/signal transducer and activator of transcription-3 (STAT3) pathway were detected by Western blot analysis. ATB overexpression promoted proliferation, migration, and invasion of A549 cells. Meanwhile, EMT of A549 cells was also enhanced. ATB silence showed the opposite influence. Expression of miR-494 was negatively regulated by ATB. Following experiments showed ATB-induced alterations of proliferation, migration, invasion, and EMT were all reversed by miR-494 overexpression. Finally, we proved that ATB increased phosphorylated levels of AKT, JAK1, and STAT3, and those increases were all reversed by miR-494 overexpression. We interestingly figured out that ATB promoted proliferation, migration, invasion, and EMT through down-regulating miR-494 in A549 cells. Moreover, ATB might activate AKT and the JAK/STAT3 pathway via down-regulating miR-494.
肺癌是一种常见的诊断疾病，预后较差。新型治疗靶点以及对肺癌调控机制的深入理解至关重要。我们旨在弄清楚转化生长因子-β激活的长链非编码RNA（ATB）在A549细胞中的功能作用及其潜在分子机制。细胞转染后，ATB在A549细胞中呈非生理性表达。然后，分别通过BrdU掺入试验、蛋白质印迹分析和Transwell试验检测细胞增殖、与增殖和上皮-间质转化（EMT）相关的蛋白质表达、迁移和侵袭。之后，通过定量逆转录PCR测定转染的A549细胞中miR-494的表达。同时，评估miR-494过表达对ATB过表达细胞的影响。最后，通过蛋白质印迹分析检测AKT的磷酸化水平以及Janus激活激酶（JAK）/信号转导和转录激活因子3（STAT3）通路中的关键激酶。ATB过表达促进了A549细胞的增殖、迁移和侵袭。同时，A549细胞的EMT也增强。ATB沉默则产生相反的影响。miR-494的表达受到ATB的负调控。后续实验表明，miR-494过表达可逆转ATB诱导的增殖、迁移、侵袭和EMT改变。最后，我们证明ATB增加了AKT、JAK1和STAT3的磷酸化水平，而miR-494过表达可逆转这些增加。我们有趣地发现，ATB通过下调A549细胞中的miR-494促进增殖、迁移、侵袭和EMT。此外，ATB可能通过下调miR-494激活AKT和JAK/STAT3通路。
