Fig 1: 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
Fig 2: 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)
Fig 3: Further verification of the potential therapeutic. Line chart(a) and the high-content imaging(b) illustrate decreased cell viability in A549 after transfection with si-DARS2 and si-COX5B. (c) The representative IHC staining image of low and high expression levels of DARS2 and COX5B in LUAD tumor samples. (d) The Unsupervised univariate analysis using Kaplan-Meier survival analysis of patients with lung adenocarcinoma in our hospital showed that patients with high expression of DARS2 or COX5B had shorter OS and were associated with poor prognosis.
Fig 4: The hub gene identification and prognostic validation. The Venn plot(a) showed the identification of the two hub genes. The pan-cancer dot plot identified the DARS2(b) and COX5B(c) expression profiles across all tumor samples and normal tissues. The violin plot showed the correlation between clinical stage and the expression level of DARS2(d) and COX5B(e). KM overall survival analysis of TCGA-LUAD tumor samples with high or low DARS2(f) and COX5B(g) expression. The effect of siRNA to knockdown DARS2 and COX5B mRNA levels in A549 and H358 cell lines was measured by RT–qPCR(h).
Fig 5: Effects of the DARS2 mutations on mRNA splicing in a family affected with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation. RNA and protein were extracted from fibroblasts and RT-PCR was performed to obtain cDNA. Scheme for exon 3 skipping (a). The exclusion of exon 3 was visualized by PCR with primers amplifying a fragment from exon 2 to exon 4 (inverted image). The expected sizes of 296 bp (for the full transcript with exon 3) and 229 bp (without exon 3) are shown (b). Electropherograms from Sanger sequencing confirmed lack of exon 3 in the two patients and the family members carrying the c.228-17G > C mutation (c).
Supplier Page from Abcam for Anti-DARS2 antibody