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. 2019 Apr 13;9(9):2424-2438.
doi: 10.7150/thno.30941. eCollection 2019.

"V体育官网入口" EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the RIP3 promoter

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EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the RIP3 promoter

V体育平台登录 - Feng Shi et al. Theranostics. .

"VSports最新版本" Abstract

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 VSports手机版. 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. .

Keywords: Epstein-Barr virus; Fumarate V体育安卓版. ; Nasopharyngeal carcinoma; Necroptosis; Receptor-interacting protein 3. .

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Conflict of interest statement (V体育官网)

Competing Interests: The authors have declared that no competing interest exists.

"VSports注册入口" Figures

Figure 1
Figure 1
Kaplan-Meier analysis according to RIP3 promoter methylation and mRNA expression status in HNSCC patients. A, Disease-free survival (left) and overall survival (right) analysis according to RIP3 mRNA expression. HNSCC patients were divided into two groups: good prognosis (positive expression of RIP3 mRNA) and poor prognosis (negative expression of RIP3 mRNA; “-”, negative; “+”, positive). B, Disease-free survival (left) and overall survival (right) analysis according to RIP3 promoter methylation. HNSCC patients were divided into two groups: good prognosis (unmethylated RIP3 promoter) and poor prognosis (methylated RIP3 promoter). U, unmethylated; M, methylated. C, Disease-free survival (left) and overall survival (right) analysis according to the combination of RIP3 promoter methylation and mRNA expression. The combination could stratify HNSCC patients more accurately than just one factor.
Figure 2
Figure 2
RIP3 expression is negatively associated with EBV(LMP1), and restoring RIP3 expression inhibits EBV(LMP1)-mediated tumorigenesis. A, Representative IHC photos for the expression of RIP3. Compared with NP tissues, RIP3 is expressed at low levels in NPC tissues. NP: nasopharyngitis; NPC: nasopharyngeal carcinoma. B, RIP3 protein expression is down-regulated in EBV(LMP1)-positive cells detected by Western blot analysis. EBV- uninfected/infected cells: NP460hTERT/ NP460hTERT-EBV (immortalized human nasopharyngeal epithelial cell), HK1/ HK1-EBV (differentiated NPC cells), C666-1 (undifferentiated NPC cells harboring EBV). LMP1-associated cells: HK1-Vec/ HK1-LMP1 (HK1 cells were transfected with LMP1- overexpression vector), C666-1/ C666-1 shLMP1 (C666-1 cells stably transfected with shLMP1). C, RIP3 mRNA expression is down-regulated in EBV(LMP1)-positive cells detected by RT-PCR (columns = mean; bars = S.D.; n = 3; **, p < 0.01). D, Representative IHC photos for the expression of LMP1 and RIP3 in consecutive sections of NPC tissues. E, RIP3 expression is calculated according to LMP1 expression in NPC tissues. F, Tumor growth curves of C666-1 xenografts in groups indicated. Data are expressed as mean values ± S.D. (n = 5; ***p < 0.001). G, Tumor weight was measured at the end of the experiment. Data are expressed as mean values ± S.D. (n = 5; ***p < 0.001).
Figure 3
Figure 3
RIP3 is silenced by methylation in EBV(LMP1)-positive cell lines. A, Methylation levels of CpG sites in the RIP3 promoter near its TSS (+164 to +404) in NP460hTERT-EBV cells are higher than those in NP460hTERT cells as detected by BSP (left and middle). The results are statistically significant (right). Dot, the mean methylation level of a single CpG island (***, p < 0.001). B, Methylation levels of RIP3 promoter are significantly higher in HK1-EBV cells compared to HK1 cells (the same as above). C, Promoter methylation of RIP3 in EBV(LMP1)-positive/negative cell lines validated by MSP (M, methylated; U, unmethylated.). D, With 5-aza-dC (10 μM, 4 days) treatment, methylation level of RIP3 promoter is down-regulated as detected by MSP (M, methylated; U, unmethylated). E, RIP3 protein is restored in a time-dependent manner in EBV-infected cells treated with 5-aza-dC (10 μM) as detected by Western blotting. F, RIP3 protein is restored in LMP1-positive cells treated with 5-aza-dC (10 μM, 4 days) as detected by Western blotting.
Figure 4
Figure 4
EBV(LMP1) regulates TET enzymatic activity and metabolic changes. A, TET 5mC-hydroxylase activity is down-regulated in EBV-infected cells (left and middle). Knockdown of LMP1 expression in C666-1 restored TET activity (right; columns = mean; bars = S.D.; n = 3; **, p < 0.01). B, Hydroxymethylated DNA immunoprecipitation (hMeDIP) assays to determine 5-hmC levels in the RIP3 promoter. The 5-hmC level is reduced in EBV-infected cells (left and middle) and knockdown of LMP1 expression in C666-1 elevates the 5-hmC level (right; columns = mean; bars = S.D.; n = 3; **, p < 0.01). C, Fumarate accumulates in EBV-infected cells (left and middle). The level of fumarate is decreased in LMP1-knockdown cells (right; columns = mean; bars = S.D.; n = 3; **, p < 0.01). D, The level of α-KG is decreased in EBV-infected cells (left and middle) and the level is increased in LMP1-knockdown cells (right; columns = mean; bars = S.D.; n = 3; **, p < 0.01).
Figure 5
Figure 5
EBV(LMP1)-associated metabolite changes regulate TET activity and RIP3 expression. A, Western blot analysis of RIP3 expression in NP460hTERT (left) and HK1 (right) cells treated with DMF (50 μM) for the indicated times. Treatment wtih DMF decreases the protein expression of RIP3 in a time-dependent manner. B, DMF (50 μM, 4 days) treatment increases methylation levels of the RIP3 promoter as detected by MSP (M, methylated; U, unmethylated). C-D, NP460hTERT (C) and HK1 (D) cells were treated with DMSO or DMF (50 μM, 4 days). The mRNA expression of RIP3 (left), TET 5mC-hydroxylase activity levels (middle), and 5-hmC levels of RIP3 promoter (right) are decreased in the DMF-treated group (columns = mean; bars = S.D.; n = 3; **, p < 0.01). E-F, NP460hTERT-EBV (E) and HK1-EBV (F) cells were treated with DMSO or octyl-α-KG (1 mM, 24 hours). The mRNA expression of RIP3 (left), TET 5mC-hydroxylase activity levels (middle), and 5-hmC levels of RIP3 promoter (right) are increased in the α-KG -treated group (columns = means; bars = S.D.; n = 3; *, p < 0.05; **, p < 0.01).
Figure 6
Figure 6
EBV(LMP1) inhibits FH activity. A, FH activity declines in EBV-infected cells (left and middle) and the activity is increased in LMP1-knockdown cells (right; columns = mean; bars = S.D.; n = 3; **, p < 0.01). B, Endogenous FH was immunoprecipitated (IP) and probed with FH and acetyl-Lys (Ac-K) antibodies. The whole cell extract was used as input. C-D, Cells were treated with 5 μM TSA or 5 mM NAM for 16 hours. The endogenous FH was immunoprecipitated and analyzed by Western blotting (C). FH activity were also detected (D; columns = means; bars = S.D.; n = 3; **, p < 0.01).
Figure 7
Figure 7
Fumarate inhibits T/S/Z-induced necroptosis. NP460hTERT and HK1 cells were untreated or pretreated with DMF (50 μM, 4 days) followed by treatment with TNF-α (T, 100ng/ml)/ Smac mimetic (S, 5 μM)/ z-VAD-fmk (Z, 20 μM) for 24 (NP460hTERT) or 48 h (HK1). A, Cell viability was determined by MTS (columns = means; bars = S.D.; n = 3; **, p < 0.01. B, Western blot analysis of RIP3, p-MLKL and MLKL expression. C, The integrity of cellular membrane was determined by Sytox Green fluorescence staining (left). The results are statistically significant (right; columns = means; bars = S.D.; n = 3; *, p < 0.05; **, p < 0.01).
Figure 8
Figure 8
A schematic for EBV-induced epigenetic reprogramming of RIP3. The metabolic reprogramming caused by EBV(LMP1) expression inhibits necroptosis signaling through the hypermethylation of the RIP3 promoter, which sheds light on the mechanism underlying EBV-related carcinogenesis.

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