Li Yaping, Sun Dajun, Sun Weixuan, Yin Dexin
Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, Jilin, China.
Department of Vascular Surgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China.
J Cell Physiol. 2019 Sep;234(9):16032-16042. doi: 10.1002/jcp.28261. Epub 2019 Feb 15.
Uveal melanoma (UM) is an intraocular malignant tumor characterized by rapid progression and recurrence. The current conventional treatments are unsatisfactory. Histone acetylation at H3 lysine 56 (H3K56ac) has been reported to be a tumor suppressor in breast cancer. However, whether H3K56ac prevents the occurrence and development of UM remains uninvestigated. The study aimed to explore the regulatory effect of H3K56ac on Ras-PI3K-AKT induced UM cells proliferation and migration.
The vectors of pEGFP-Ras , pEGFP-K-Ras , and pEGFP-N1 were transfected into MP46 cells, and protein levels of phosphorylated AKT and H3K56ac were examined using western blot analysis. The effect of H3K56ac on cell proliferation and migration were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, colony formation, and Transwell assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and chromatin immunoprecipitation assays were performed to determine the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) downstream genes. Further, the regulatory effects of silent mating type information regulation 2 homolog-1 (SIRT1), general control nonderepressible 5 (GCN5), and mouse double minute 2 homolog (MDM2) on Ras-PI3K-AKT affected H3K56ac expression were also investigated.
H3K56ac expression was specifically downregulated by Ras-PI3K-AKT activation pathway. H3K56ac inhibited the tumorigenic effect of Ras-PI3K-AKT on MP46 cells viability, colony formation, and migration, as well as participated in regulating the transcription of PI3K/AKT downstream genes. SIRT1 silence recovered H3K56ac expression, and reversed the tumorigenic effect of Ras-PI3K-AKT activation on MP46 cells. Downregulation of H3K56ac induced by Ras-PI3K-AKT activation was found to be associated with MDM2-mediated the degradation of GCN5.
The results demonstrated that Ras-PI3K-AKT signaling promoted UM cells proliferation and migration via downregulation of H3K56ac expression, which might be related to MDM2-mediated the degradation of GCN5.
葡萄膜黑色素瘤（UM）是一种眼内恶性肿瘤，其特点是进展迅速且易复发。目前的传统治疗方法并不令人满意。据报道，组蛋白H3赖氨酸56位点的乙酰化（H3K56ac）在乳腺癌中是一种肿瘤抑制因子。然而，H3K56ac是否能预防UM的发生和发展仍未得到研究。本研究旨在探讨H3K56ac对Ras-PI3K-AKT诱导的UM细胞增殖和迁移的调节作用。
将pEGFP-Ras、pEGFP-K-Ras和pEGFP-N1载体转染到MP46细胞中，采用蛋白质免疫印迹法检测磷酸化AKT和H3K56ac的蛋白水平。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法、集落形成实验和Transwell实验研究H3K56ac对细胞增殖和迁移的影响。进行逆转录定量聚合酶链反应（RT-qPCR）和染色质免疫沉淀实验以确定磷酸肌醇3激酶/蛋白激酶B（PI3K/AKT）下游基因。此外，还研究了沉默信息调节因子2同源物1（SIRT1）、一般控制非抑制性蛋白5（GCN5）和小鼠双微体2同源物（MDM2）对Ras-PI3K-AKT影响H3K56ac表达的调节作用。
Ras-PI3K-AKT激活途径特异性下调H3K56ac表达。H3K56ac抑制Ras-PI3K-AKT对MP46细胞活力、集落形成和迁移的致瘤作用，并参与调节PI3K/AKT下游基因的转录。SIRT1沉默恢复了H3K56ac表达，并逆转了Ras-PI3K-AKT激活对MP46细胞的致瘤作用。发现Ras-PI3K-AKT激活诱导的H3K56ac下调与MDM2介导的GCN5降解有关。
结果表明，Ras-PI3K-AKT信号通路通过下调H3K56ac表达促进UM细胞增殖和迁移，这可能与MDM2介导的GCN5降解有关。
