Fig 1: Model of HIG2A behavior in response to stress. (A) The transcription factor E2F-1 controls the HIGD2A gene, which encodes the HIG2A protein (in orange) [12]. The model describes two “pools” of HIG2A protein. Once synthesized in the cytoplasm, the HIG2A protein is distributed at the mitochondria and the nucleus [12,17]. Currently, the function of HIG2A at the nucleus has not been described, whereas, at mitochondria, HIG2A is localized in the inner mitochondrial membrane where it participates in the formation of respiratory supercomplexes (SC), interacting with complex III (CIII) and complex IV (CIV), and promoting their stability [8,11,12]. The increase in the formation of respiratory supercomplexes has been associated with a decrease in mitochondrial reactive oxygen species (ROSmt) and an increase in mitochondrial energy generation (ATPmt) [1,2,4,6]. The distribution of HIG2A protein at the subcellular level will depend on the type of stress to which cells are exposed. (B) Mitochondrial stress by Rotenone (Complex I (CI) inhibitor) causes an increase of HIG2A at the nuclear level. Inhibition at the CI level causes an increase in superoxide anion (O2-) generation and a decrease in electron (e-) flow through the electron transport chain (ETC) and mtATP generation [67,68]. In contrast, FCCP causes a reduction at the nucleus, which could suggest (dotted arrow) a shift of HIG2A into the mitochondria to make up for the deficiency in mitochondrial membrane potential (??m) that is caused by the mitochondrial uncoupler FCCP [69], participating in SC formation, stabilizing electron flow, and maintaining mitochondrial energy generation (mtATP). In comparison, H2O2 (generalized stress) caused an increase in HIG2A at the mitochondria and cytoplasm. H2O2 causes an increase in ROS at the cellular level which may participate in signaling pathways. (C) It has been described that under hypoxic conditions, the transcription factor HIF-1a is stabilized and forms a heterodimer with the transcription factor HIF-1ß recognizing consensus sequences of the target genes that are involved in lactic acid formation [70]. At mitochondria, mtROS that are generated by complex III (CIII) can inhibit the enzyme prolyl hydroxylase (PHD), which hydroxylases (OH) the transcription factor HIF-1a, for subsequent degradation via the proteasome [71]. In addition, the decrease in oxygen (O2) levels causes an increase in the formation of SCs, which helps prevent electrons from escaping, thus decreasing mtROS generation [71,72,73]. The subcellular distribution of HIG2A by hypoxic stress that is caused by physical hypoxia of 2% O2 and by chemical hypoxia by CoCl2 (hypoxia equal to or less than 1% O2) causes the HIG2A protein to present a differential distribution. Hypoxia of 2% O2 causes an increase of HIG2A at the nucleus, which could indicate new functions that are not yet described at the nuclear level and which could be regulated by stress factors. Conversely, CoCl2 stress causes a decrease in HIG2A at the nucleus. This decrease could indicate (dotted arrow) a shift of HIG2A to the mitochondrial level to promote SC formation and stability, decrease mtROS generation, and provide more efficient electron transport through the mitochondrial transport chain.
Fig 2: HIGD2A regulate proliferation ability of HCC cells partially by modulating MAPK/ERK signaling pathway. A Volcano plots of differential gene expression in shCtrl and shHIGD2A.1 infected HepG2 cells (data from 3 biological replicates). B Reactome enrichment analysis. Top 10 significantly enriched pathway in shHIGD2A-infected group were shown. C MAPK activation and the protein levels of BCL2L1 were assessed by western blotting in HepG2 and Huh7 cells transfected with shCtrl or shHIGD2A.1 lentivirus. D, E BCL2L1 overexpression alleviated HIGD2A knockdown-mediated growth inhibition and apoptosis in HCC cells measured by CCK8 assay (D) and western blotting (E). Results shown are mean ± SEM. Two-way ANOVA with Tukey multiple comparison test was used for statistical analyses. ***P < 0.001; ns not significant
Fig 3: HIGD2A promotes stemness property of hepatocellular carcinoma. A Expression of genes associated with tumor stemness are significantly upregulated in patients with high HIGD2A mRNA expression. GSEA analysis of TCGA dataset (hepatocellular carcinoma). B Positive association of mRNAsi with HIGD2A expression. C, D The mRNA expression levels of the tumor stemness-associated markers (CD133, EpCAM, CD44, NANOG, ALDH1) in HepG2 (C) and Huh7 (D) transfected with shCtrl or shHIGD2A.1 were measured by real-time PCR. E Immunoblot analysis of the protein levels of CD133 and EpCAM in HepG2 and Huh7 transfected with shCtrl or shHIGD2A.1. Results shown are mean ± SEM. Pearson correlation was used in B and The Mann–Whitney test was used in C-D for statistical analyses. *P < 0.05
Fig 4: Depletion of HIGD2A induces mitochondrial stress. A, C Intercellular ATP level in HepG2 (A) and Huh7 (C) transfected with shCtrl or shHIGD2A.1. B, D Immunofluorescent (IF) images showing mitochondria morphology in HepG2 (B) and Huh7 (D) cells transfected with shCtrl or shHIGD2A.1. Left, representative IF images (magenta, MitoTracker; yellow, DAPI) and 3D mitochondria reconstruction of shCtrl- and shHIGD2A-infected HepG2 and Huh7 cell. Scale bar, 5 µm; Sphericity heat map, 0.326–0.915. Right, sphericity analysis of 3D reconstructed mitochondria (results are presented as mean ± SD). E Immunoblot analysis of mitochondrial fusion-related protein (Mfn1 and OPA1) in HepG2 (left) and Huh7 (right) transfected with shCtrl or shHIGD2A.1. F ATF5 mRNA level in in HepG2 (left) and Huh7 (right) transfected with shCtrl or shHIGD2A.1 measured by real-time PCR. Results shown are mean ± SEM. The Mann–Whitney test was used for statistical analyses. *P < 0.05 and **P < 0.01
Fig 5: Knockdown of HIGD2A attenuated the tumorigenicity of HCC cell lines in vivo. A–F Images of HepG2 (A), Huh7 (C) and MHCC97H (E) xenograft tumors in nude mice of shCtrl group and shHIGD2A.1 group (n = 5 mice per group), and comparison of the tumor weight between shCtrl group and shHIGD2A.1 group. Tumor volume changes of HepG2 (B), Huh7 (D) and MHCC97H (F) were monitored at indicated time points. G–I Immunohistochemical staining of ki67 in HepG2 (G), Huh7 (H) and MHCC97H (I) xenograft tumor tissues from shCtrl group and shHIGD2A.1 group, and the proportion of ki67 positive cells counted by ImageJ software. Results shown are mean ± SEM. A Mann–Whitney test was used. *P < 0.05 and **P < 0.01. Scale bar, 100 µm
Supplier Page from Abcam for Anti-HIGD2A antibody