EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the RIP3 promoter
EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the RIP3 promoter
复制标题
EBV(LMP1)诱导的代谢重编程通过RIP3启动子的高甲基化抑制坏死性凋亡
作者：
Shi Feng;Zhou Min;Shang Li;Du Qianqian;Li Yueshuo;Xie Longlong;Liu Xiaolan;Tang Min;Luo Xiangjian;Fan Jia;Zhou Jian;Gao Qiang;Qiu Shuangjian;Wu Weizhong;Zhang Xin;Bode Ann M;Cao Ya
EBV感染是公认的致癌的表观遗传驱动因素。我们先前表明EBV可以保护癌细胞免受TNF诱导的坏死性凋亡。本研究旨在探讨EBV感染的癌细胞逃避RIP 3依赖性坏死性凋亡的表观遗传机制。方法：利用TCGA数据库中的数据，评估RIP 3启动子甲基化及其表达的预后价值。采用Western blotting、real-time PCR和免疫组化方法检测LMP 1和RIP 3在鼻咽癌细胞株和鼻咽癌组织中的表达。BSP、MSP和hMeDIP检测甲基化水平。通过细胞活力测定、p-MLKL和Sytox绿色染色检测坏死性凋亡的诱导。结果如下：RIP 3启动子高甲基化分别是HNSCC患者无病生存期和总生存期较差的独立预后因素。RIP 3在NPC（HNSCC的一种亚型）中下调。EBV（LMP 1）通过RIP 3启动子的超甲基化抑制RIP 3表达。RIP 3蛋白表达与LMP 1蛋白表达呈负相关。在EBV（LMP 1）阳性细胞中恢复RIP 3表达抑制异种移植肿瘤生长。EBV（LMP 1）阳性细胞中富马酸盐的蓄积和α-KG的减少导致RIP 3沉默，原因是TMP 3失活。FH活性降低导致富马酸盐蓄积，这可能与其乙酰化有关。用富马酸盐孵育细胞保护NPC细胞免于TNF诱导的坏死性凋亡。总结：这些结果表明，EBV（LMP 1）相关的代谢物变化通过DNA甲基化抑制坏死性凋亡信号传导的途径，并揭示了EBV相关致癌的机制，这可能为癌症的诊断和治疗提供新的选择。
EBV infection is a recognized epigenetic driver of carcinogenesis. We previously showed that EBV could protect cancer cells from TNF-induced necroptosis. This study aims to explore the epigenetic mechanisms allowing cancer cells with EBV infection to escape from RIP3-dependent necroptosis. Methods: Data from the TCGA database were used to evaluate the prognostic value of RIP3 promoter methylation and its expression. Western blotting, real-time PCR, and immunochemistry were conducted to investigate the relationship between LMP1 and RIP3 in cell lines and NPC tissues. BSP, MSP and hMeDIP assays were used to examine the methylation level. Induction of necroptosis was detected by cell viability assay, p-MLKL, and Sytox Green staining. Results: RIP3 promoter hypermethylation is an independent prognostic factor of poorer disease-free and overall survival in HNSCC patients, respectively. RIP3 is down-regulated in NPC (a subtype of HNSCC). EBV(LMP1) suppresses RIP3 expression by hypermethylation of the RIP3 promoter. RIP3 protein expression was inversely correlated with LMP1 expression in NPC tissues. Restoring RIP3 expression in EBV(LMP1)-positive cells inhibits xenograft tumor growth. The accumulation of fumarate and reduction of α-KG in EBV(LMP1)-positive cells led to RIP3 silencing due to the inactivation of TETs. Decreased FH activity caused fumarate accumulation, which might be associated with its acetylation. Incubating cells with fumarate protected NPC cells from TNF-induced necroptosis. Conclusion: These results demonstrate a pathway by which EBV(LMP1)-associated metabolite changes inhibited necroptosis signaling by DNA methylation, and shed light on the mechanism underlying EBV-related carcinogenesis, which may provide new options for cancer diagnosis and therapy.