A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

Abstract

Receptor-interacting protein kinase 3 (RIP3) is a key determinant of necroptosis and participates in ischaemia-and oxidative stress-induced necroptosis, myocardial remodelling and heart failure (HF). In this study, we tested the hypothesis that common variants in RIP3 gene were associated with the risk and prognosis of HF in the Chinese Han population. By re-sequencing and luciferase assays, we identified a common functional variant in the RIP3 promoter region. The rs3212247-T allele suppressed RIP3 promoter activity by facilitating transcription factor SOX17 binding, but not the C allele. We further recruited 2961 control participants and 3194 HF patients who underwent a mean follow-up of 19 months (6-31 months) for this study. Rs3212247 and another missense variant rs3212254 were genotyped. Although rs3212247 did not significantly associate with increased risk of HF (odds ratio = 1.00, 95% CI = 0.92-1.08, P = 0.91), it raised the risk for cardiovascular death and cardiac transplantation (hazard ratio = 1.47, 95% CI = 1.13-1.91, P = 0.004). Moreover, participants carrying the rs3212247 CC genotype had higher plasma levels of RIP3 than those carrying the TT or TC genotype (p for trend = 0.02) in New York Heart Association class III HF group. No association was found between the RIP3 missense variant rs3212254 and risk or prognosis of HF after adjustment for traditional risk factors. In conclusion, genetic variant in RIP3 promoter region is associated with increased RIP3 transcription, thus contributed to the poor prognosis of HF patients. Clinical Trial Registration: https://www.clinicaltrials.gov/ct2/show/NCT03461107?term=03461107&cond=Heart+Failure&cntry=CN&rank=1. Unique identifier: NCT03461107.

Keywords: genetics; heart failure; prognosis; receptor-interacting protein kinase 3.

Conflict of interest statement

The authors declare no competing financial interests.

© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Figures

Figure 1

rs3212247 is responsible for haplotype‐specific difference in transcriptional activity. (A), Map of single‐nucleotide polymorphisms (SNPs) in the promoter and 5'UTR of Receptor‐interacting protein kinase 3 genotyped in 200 healthy individuals. The eight SNPs (indicated with boxes), which were predicted to be possible functional at RegulomeDB, comprise a single haplotype and define the 1.8 and 0.2 kb in promoter and 5'UTR, respectively. (B), Firefly luciferase expression from constructs transfected into AC16. Both the major (Hap1) and minor (Hap2) haplotypes of the 1.8 kb in promoter region were subcloned into PGL3‐basic vector. (C‐I), Luciferase assays for rs3212247, rs3834521, rs3759625, rs3212249, rs3759630, rs3212246 rs3212250‐51 (rs3212250 and rs3212251 were combined as a haplotype as they are close enough) were conducted using AC16. Approximately 250‐bp regions encompassing corresponding variants were cloned into PGL3‐basic vector. Luciferase activity was measured 48 h after transfection and was normalized against Renilla luciferase activity. Values are mean ± SE of three independent experiments each corresponding to at least six replicates. **P < 0.01, ****P < 0.0001, NS, not significant

Figure 2

SOX17 binds to the rs3212247‐T allele and mediates allelic expression. (A), Firefly luciferase assays were performed with 250‐bp rs3212247‐T or ‐C allele constructs cotransfected into AC16 cells with SOX17 vector encoding sox17. Luciferase activity was measured 48 h after transfection and was normalized against Renilla luciferase activity. (B), AC16 cells were transfected with pcDNA3.1 and SOX17 respectively. Protein was harvested 48 h after transfection and Receptor‐interacting protein kinase 3 (RIP3) expression was normalized against GAPDH. *P < 0.05

Figure 3

Chromatin immunoprecipitation with antibody against Flag‐SOX17 in AC16 (A) and HEK293T (B) with transduction with Flag‐SOX17 construct. Quantitative PCR was used to measure the Immunoprecipitation of DNA sequence surrounding rs3212247, normalized to background (control condition with IgG, no antibody)

Figure 4

In vitro SOX17 binding to rs3212247‐T allele. Gel‐shift assay was performed with a biotin‐labelled probe containing SOX17 binding elements with nuclear extract from AC16 or absence of nuclear extract in each group (lane 1), without or with competition from unlabelled oligonucleotides containing rs3212247‐T or rs3212247‐C ins probes (lanes 2 to 3 respectively) or anti‐SOX17 antibody as indicated (lane 4)

Figure 5

In vivo plasma concentrations of Receptor‐interacting protein kinase 3 (RIP3) in control group (A) HF NYHA class II group (B) HF NYHA class III group (C) HF NYHA class IV group (D) and among these four groups (E). The data are presented as box (25th percentile, median and 75th percentile)

Figure 6

Effects of rs3212247 on the prognosis of heart failure (HF) patients. (A), Cox proportional hazards models analysis showed the association of genotypes of rs3212247 with cardiovascular deaths or cardiac transplantation (unadjusted HR = 1.47, 95% CI = 1.13‐1.91; P = 0.004). (B), Comparison of 3194 HF patients with and without β‐blocker use (adjusted HR = 0.36, 95% CI = 0.29‐0.43; P < 0.001)

Figure 7

Prospective analysis of the interaction between rs3212247 and β‐blocker use as a determinant of heart failure. Cox proportional hazards model was used for comparison of rs3212247‐TT genotype (A) rs3212247‐TC genotype (B) and rs3212247‐CC genotype (C) with and without β‐blocker use, respectively. (D, E), Comparison of different genotype when stratified by β‐blocker use

Figure 8

Effect of SOX17 on Regulation of Receptor‐interacting protein kinase 3 (RIP3) Transcription. The variant rs3212247 in RIP3 promoter region is a T‐ to‐C change and is predicted to locate in the binding site of SOX17. Rs3212247‐C allele destroys the SOX17 binding site in the promoter region of RIP3 and subsequently results in increased transcription and translation of RIP3, followed by increased necroptosis of cardiomyocyte under various external stimuli, which leads to poor prognosis of heart failure (HF)