BolA family member 2 enhances cell proliferation and predicts a poor prognosis in hepatocellular carcinoma with tumor hemorrhage

Abstract

Objective: BolA family member 2 (BOLA2) is a novel gene highly associated with human hepatocellular carcinoma (HCC) progression. Tumor hemorrhage (TH) acts as a poor marker for HCC patients and is a community affair in the tumor microenvironment. In the present study, we examined a possible association between BOLA2 levels and HCC patients with TH. Methods: The mRNA and protein levels of BOLA2 were determined in two independent cohorts of HCC specimens by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) analysis, respectively. Survival curves and Cox regression models were used to evaluate the prognosis of HCC patients. The CRISPR/Cas9 system was used to knock out BOLA2 in HCC cells, and the functional role of BOLA2 in HCC cell proliferation in vitro and growth in vivo was examined. Results: BOLA2 mRNA expression is significantly higher in HCC tumour tissue than in nontumour tissue. Immunohistochemistry analysis of HCC tissues showed that BOLA2 protein was significantly correlated with TH, a more metastatic phenotype and worse HCC survival. The potential clinical relevance of BOLA2 expression and TH was validated by a Cox regression model. Furthermore, loss-of-function studies determined that BOLA2 plays critical roles in promoting iron overload, tumor growth and TH. Bioinformatics analysis from Gene Expression Profiling Interactive Analysis (GEPIA) revealed that BOLA2 is closely associated with the activation of p62-Keap1 signalling and ATG4B expression. These results were confirmed by immunohistochemistry analysis in HCC tissues. Conclusions: Our results suggest that BOLA2 plays an important role in cancer biology and is an independent predictor of prognosis in HCC.

Keywords: BolA family member 2; Hepatocellular carcinoma; Iron metabolism; Tumor hemorrhage; Tumor microenvironment.

Fig 1

High expression levels of BOLA2 mRNA in HCC predicted by the Oncomine database. BOLA2 mRNA levels in Roessler Liver2 (GEO: GSE 14520/GPL3921) and Wurmbach Liver (GEO: GSE 6764) grouped by HCC and normal liver in the Oncomine database.

Fig 2

BOLA2 expression level is significantly increased in HCC and is associated with poor clinical outcomes. (A) Differential expression of BOLA2 between tumor and adjacent normal tissues from various types of cancer. (B) BOLA2 gene expression was significantly increased in HCC (n=369) compared with corresponding normal liver tissues (n=160). Data were extracted from the GEPIA web server. (C) Kaplan-Meier curves stratified by BOLA2 mRNA expression. Overall survival and disease-free survival data were generated from the GEPIA web server. (D) BOLA2 protein expression in normal liver tissues and HCC specimens. Images were obtained from the Human Protein Atlas online database.

Fig 3

Increased expression of BOLA2 mRNA is related to a high risk of TH. (A) Representative gross specimens of HCC with/without TH. (B) BOLA2 expression in a NT, a TH-negative tumor, and a TH-positive tumor. ** P < 0.01. *** P <0.001. Abbreviations: NT, nontumor tissues. (C) Spearman's correlation analysis of tumor oxidative stress markers, including p62, Keap1, Nrf2, and ATG4B, with BOLA2 expression in the HCC profiles of the TCGA dataset.

Fig 4

(A) A summary of the immunohistochemical staining results of the training cohort. Membrane and cytoplasmic expression of BOLA2, p62, and Keap1 and the nuclear expression of NRF2 and Ki-67 were observed. (B) The samples are sorted from left to right in ascending order based on the presence of TH. The positive samples are indicated by black boxes. HCC patients with TH and lower BOLA2 expression had higher levels of NRF2, whereas patients with higher BOLA2 expression had increased ATG4B, p62 and Keap1 levels. (C) Quantification of Ki-67 in HCC tissues with or without TN. Ki-67-positive cells are numbered, and box plots show the median, 25th and 75th percentiles, and minimum and maximum values. (D and E) Kaplan-Meier curves depicting overall survival (OS) and recurrence-free survival (RFS) according to the expression levels of BOLA2 in the validation cohort (n=175).

Fig 5

Western blot analysis was used to examine the expression of BOLA2 in HCC cell lines versus immortalized human hepatocytes (L02).

Fig 6

BOLA2 knockout reduces the tumorigenicity of HCC cells in vitro and in a xenograft model. (A) BOLA2 knockout by CRISPR/Cas9 technology in Hep3B cells was confirmed by Western blot analysis. The activities of p62 and Keap1 were reduced in BOLA2-deficient Hep3B cells. (B) BOLA2 knockout slows cell proliferation. (C) Colony formation assays were performed in the three BOLA2-deficient colonies derived from Hep3B and WT cells. The values are expressed as the mean ± SD of three independent experiments. (D) Tumor growth curves in the three groups are shown on the indicated days after Hep3B cell (WT, KO-1, and KO-3) injection. The xenograft tumor volumes of each group were measured two times a week. (E) Representative images of subcutaneous tumors in nude mice injected with the indicated cells. Final tumor weights are summarized in a dot chart. The average tumor weight is expressed as the mean ± SD of 6 mice. (F) H&E staining demonstrated that the KO of BOLA2 inhibited the TH phenotype of HCC in vivo (left panel). Corresponding Prussian blue staining shows iron particles scattered in the central part of the tumor with TH. The iron scores of the xenograft tumors were calculated and are depicted in the bar chart (right panel).

Fig 7

(A) Representative immunohistochemistry staining of BOLA2, p62 and Ki-67 in transplanted tumor sections from Hep3B cells (WT, KO-1, and KO-3) injected into nude mice (×400 magnification). Scale bar: 100 μm. (B) Comparison of Ki-67 staining in the transplanted tumors. (C) STRING database analysis of the PPI network for BOLA2. Interactions between 20 hub genes are illustrated with the cut-off criterion of a combined score =0.7. Network nodes represent proteins and edges represent protein-protein associations.