Fig. 4

ChIP-seq revealed that NFAT5 negatively mediated DARS2 to inhibit HCC tumorigenesis. a Proportion of peak annotation detected by ChIP-seq. b Fold change of novel NFAT5 targets. Fold change was defined as the ratio of peak reads in the IP group to those in the IgG group. c Motif sequence of peaks bound by the NFAT5 protein. The motif was highly conserved. d Luciferase report assay confirm that NFAT5 bound to DARS2 promoter. Reporter: positive control; basic: negative control; promoter: DARS2 promoter; blank: control plasmid; plasmid: NFAT5 plasmid. e RT-qPCR and Western blot analyses demonstrated that NFAT5 negatively mediated DARS2 protein expression. NFAT5 overexpression and knockdown efficiency were verified by western blot analyses of NFAT5. f Western blot demonstrated HepG2.2.15 expressed higher MAP4K4, DARS2 and lower NFAT5 protein, which confirmed HBV upregulated DARS2 via miR-30e-5p/MAPK/NFAT5 signaling pathway. g Left panel: In an FCM apoptosis assay, NFAT5 knockdown in HepG2 led to an attenuation of apoptosis, while knockdown of DARS2 did not regulate apoptosis. Right panel: Statistical analysis showed that NFAT5 siRNA attenuated the apoptosis rate (P = 0.0054 vs negative control), while co-transfection of NFAT5 siRNA and DARS2 siRNA inhibited NFAT5-induced apoptosis (P = 0.0032 vs NFAT siRNA, no significant difference compared with negative control). Apoptotic rate = acute apoptotic rate (right lower quadrant) + terminal apoptotic rate (right upper quadrant). h Left panel: FCM cell cycle analysis revealed that transfection with NFAT5 siRNA into HepG2 accelerated cell cycle progression, while co-transfection of NFAT5 siRNA and DARS2 siRNA failed to accelerate cell cycle progression. Right panel: Statistical analysis showed that NFAT5 knockdown decreased the proportion of cells in G1 phase (P = 0.0059 vs negative control) and increased the proportion of cells in S phase (P = 0.0021 vs negative control). Co-transfection of NFAT5 siRNA and DARS2 siRNA failed to accelerate cell cycle progression (P = 0.0051 vs NFAT5 siRNA in G1 phase, P = 0.0019 vs NFAT5 siRNA in S phase, no significant difference compared with negative control)