Li Yaping, Sun Weixuan, Sun Dajun, Yin Dexin
Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, China.
Department of Gastrointestinal Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University, Changchun, China.
J Cell Physiol. 2019 Sep;234(9):16011-16020. doi: 10.1002/jcp.28259. Epub 2019 Feb 15.
Ras-extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling has been proposed as the crucial regulators in the development of various cancers. Histone acetylation at H3 lysine 14 (H3K14ac) is closely associated with gene expression and DNA damage. However, whether H3K14ac participates in mediating Ras-ERK1/2-induced cell proliferation and migration in uveal melanoma cells remains unknown. The purpose of this study is to investigate the effect of H3K14ac on Ras-ERK1/2 affected uveal melanoma cell phenotypes. MP65 cells were transfected with Ras and Ras , the unloaded plasmid of pEGFP-N1 served as a negative control. Protein levels of phosphorylated ERK1/2 and H3K14ac were assessed by western blot assay. Cell viability, number of colonies, migration, and the downstream genes of ERK1/2 were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide, soft-agar colony formation, transwell, and chromatin immunoprecipitation assays. HA-tag vectors of CLR3 and TIP60 and the small interfering RNAs that specific for CLR3 and MDM2 were transfected into MP65 cells to uncover the effects of CLR3, TIP60, and MDM2 on Ras-ERK1/2 mediated H3K14ac expression and MP65 cell phenotypes. We found that, Ras-ERK1/2 decreased H3K14ac expression in MP65 cells, and H3K14ac significantly suppressed Ras-ERK1/2-induced cell viability, colony formation, and migration in MP65 cells. Moreover, the transcription of CYR61, IGFBP3, WNT16B, NT5E, GDF15, and CARD16 was regulated by H3K14ac. Additionally, CLR3 silence recovered H3K14ac expression and reversed the effect of Ras-ERK1/2 on MP65 cell proliferation, migration and the mRNAs of ERK1/2 downstream genes. Besides, Ras-ERK1/2 decreased H3K14ac expression by MDM2-mediated TIP60 degradation. In conclusion, Ras-ERK1/2 promoted uveal melanoma cells growth and migration by downregulating H3K14ac via MDM2-mediated TIP60 degradation.
Ras-细胞外信号调节蛋白激酶1和2（ERK1/2）信号通路被认为是多种癌症发展的关键调节因子。组蛋白H3赖氨酸14位点（H3K14）的乙酰化与基因表达和DNA损伤密切相关。然而，H3K14ac是否参与介导Ras-ERK1/2诱导的葡萄膜黑色素瘤细胞增殖和迁移仍不清楚。本研究的目的是探讨H3K14ac对Ras-ERK1/2影响的葡萄膜黑色素瘤细胞表型的作用。用Ras和Ras转染MP65细胞，空载的pEGFP-N1质粒作为阴性对照。通过蛋白质免疫印迹法检测磷酸化ERK1/2和H3K14ac的蛋白水平。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基-2-H-四唑溴盐法、软琼脂集落形成法、Transwell法和染色质免疫沉淀法分析细胞活力、集落数量、迁移情况以及ERK1/2的下游基因。将CLR3和TIP60的HA标签载体以及针对CLR3和MDM2的小干扰RNA转染到MP65细胞中，以揭示CLR3、TIP60和MDM2对Ras-ERK1/2介导的H3K14ac表达和MP65细胞表型的影响。我们发现，Ras-ERK1/2降低了MP65细胞中H3K14ac的表达，而H3K14ac显著抑制了Ras-ERK1/2诱导的MP65细胞活力、集落形成和迁移。此外，CYR61、IGFBP3、WNT16B、NT5E、GDF15和CARD16的转录受H3K14ac调控。此外，CLR3沉默恢复了H3K14ac的表达，并逆转了Ras-ERK1/2对MP65细胞增殖、迁移及ERK1/2下游基因mRNA的影响。此外，Ras-ERK1/2通过MDM2介导的TIP60降解降低了H3K14ac的表达。总之，Ras-ERK1/2通过MDM2介导的TIP60降解下调H3K14ac，从而促进葡萄膜黑色素瘤细胞的生长和迁移。
