Fig. 5

DARS2 was upregulated in HCC and promoted cell cycle progression and inhibited apoptosis in HCC. a Immunohistochemistry for DARS2 in HCC tissues and para-tumor tissues. DARS2 was upregulated in HCC and expressed in the cytoplasm. Images at 100X and 400X magnification are shown. b DARS2 mRNA expression was examined in 80 pairs of HCC tissues and para-tumor tissues. DARS2 relative expression was calculated by 2^-ΔΔCT, normalized to β-actin expression. DARS2 was upregulated in HCC tissues compared with para-tumor tissues (P < 0.0001). c DARS2 protein expression was also higher in HCC tissues than in para-tumor tissues. DARS2 protein expression, detected by western blotting, in six pairs of tissues is displayed. d DARS2 mRNA and protein expression in the HCC cell lines Hep3B (P < 0.0001), HepG2 (P < 0.0001), SK-Hep-1 (P < 0.0001) and Huh7 (P = 0.0021) compared with the normal liver cell line L02. Hep3B cells expressed the highest level of DARS2. e Left panel: An ROC curve was drawn based on DARS2 mRNA expression in 80 patients. Youden’s index = sensitivity% + specificity%-1. The cutoff value, sensitivity and specificity were determined by the highest Youden’s index, which is shown in the figure. Right panel: A Kaplan-Meier analysis was used to determine the correlation between DARS2 expression and survival. Follow-up visits occurred for 40 months after the patients were initially diagnosed with HCC. Patients with higher DARS2 expression had a shorter survival time. f Left panel: Apoptosis was analyzed by FCM in Hep3B cells transfected with DARS2 siRNA for 48 h. The apoptosis of cells transfected with DARS2 siRNA was higher than those transfected with a negative control. Middle panel: Statistical analysis of the FCM apoptosis assay. The apoptotic rate increased when Hep3B cells were transfected with DARS2 siRNA for 48 h. Right panel: Biomarkers for apoptosis, including BAX, BCL-2 and cleaved PARP-1, were detected by western blot analyses. DARS2 knockdown induced BAX and cleaved PARP-1 expression and reduced BCL-2 expression. g Left panel: The cell cycle was analyzed by FCM in Hep3B cells transfected with DARS2 siRNA for 48 h. DARS2 knockdown inhibited cell cycle progression. Middle panel: Statistical analysis of the FCM cell cycle assay. DARS2 knockdown induced an arrest prior to S phase. The proportion of cells in G1 phase increased (P = 0.0151), and the proportion of cells in S phase decreased (P = 0.0072). Right panel: Cyclin D1 protein decreased after 48 h of DARS2 knockdown, as detected by western blot. The efficiency of DARS2 knockdown was verified by a western blot analysis of DARS2. h The whole pathway of this research. HBV stimulated DNMTs for inducing the hypermethylation of AP1 binding site on NFAT5 promoter (−54 bp~ − 62 bp), for inhibiting NFAT5 transcription. On the other hand, HBV activated MAPK signaling pathway by suppressing miR-30e-5p, in order to inhibit NFAT5 indirectly. All in all, HBV is able to upregulated DARS2 by inhibiting NFAT5. DARS2 is an oncogene, associated with HCC cell apoptosis and cell cycle progression