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. 2019 Aug 26;9(9):413.
doi: 10.3390/biom9090413.

Serum Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) Level as a Potential Biomarker of Cholangiocarcinoma

Doungdean Tummanatsakun  1 , Tanakorn Proungvitaya  1 , Sittiruk Roytrakul  2 , Temduang Limpaiboon  1   3 , Sopit Wongkham  3   4 , Chaisiri Wongkham  3   4 , Atit Silsirivanit  3   4 , Ongart Somintara  5 , Sakkarn Sangkhamanon  6 , Siriporn Proungvitaya  7   8
Affiliations

Serum Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) Level as a Potential Biomarker of Cholangiocarcinoma

Doungdean Tummanatsakun et al. Biomolecules. .

Abstract

Diagnostic and/or prognostic biomarkers for cholangiocarcinoma (CCA) are still insufficient with poor prognosis of patients. To discover a new CCA biomarker, we constructed our secretome database of three CCA cell lines and one control cholangiocyte cell line using GeLC-MS/MS. We selected candidate proteins by five bioinformatics tools for secretome analysis. The inclusion criteria were as follows: having predicted signal peptide or being predicted as non-classically secreted protein; together with having no transmembrane helix and being previously detected in plasma and having the highest number of signal peptide cleavage sites. Eventually, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) was selected for further analysis. To validate APEX1 as a bio-marker for CCA, serum APEX1 levels of 80, 39, and 40 samples collected from CCA, benign biliary diseases (BBD), and healthy control groups, respectively, were measured using dot blot analysis. The results showed that serum APEX1 level in CCA group was significantly higher than that in BBD or healthy control group. Among CCA patients, serum APEX1 level was significantly higher in patients having metastasis than in those without metastasis. The higher level of serum APEX1 was correlated with the shorter survival time of the patients. Serum APEX1 level might be a diagnostic and prognostic biomarker for CCA.

Keywords: apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1); bioinformatics; cholangiocarcinoma; metastasis; secretome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Data analysis from mass spectrometry and selection of candidate proteins. (A) A total of 1138 protein molecules were found in cholangiocarcinomas (CCAs) and immortalized cholangiocyte secretomes. Venn diagram presents the number of proteins in each cell line secretome sample and the degree of protein overlapping. The oval dashed line indicates the total proteins in the conditioned medium (CM) of MMNK1, oval solid line is the total proteins in CM_KKU-100, oval dotted line is total proteins in CM_KKU-213, and the broken line is the total proteins in CM_KKU-214. Ninety proteins were expressed commonly in three CCA secretomes. (B) Flowchart of the selection of secretory proteins from 90 proteins overexpressed in CCA. Proteins having a signal peptide or non-classical proteins that have no transmembrane helix were identified in serum/plasma database, and the maximum number of signal peptide cleavage sites was used for stepwise selection.
Figure 2
Figure 2
Validation of the selected candidate protein, APEX1 in serum samples by western blot analysis. BBD: benign biliary diseases.
Figure 3
Figure 3
The level of serum APEX1 detected by dot blot assay. (A) The image of the dot blot assay of serum APEX1. Samples A1 to A7 were standards, sample A8 was the blank control, sample A9 was positive control, and row B was the duplicated row of A. (B) The standard curve of APEX1 levels.
Figure 4
Figure 4
The validation of selected candidate protein, APEX1. (A) The result of dot blot. (B) The validation of serum APEX1 level as the biomarker using a dot blot assay. Long horizontal line: median value; short upper and lower lines: interquartile range. *: Statistically significant difference.
Figure 4
Figure 4
The validation of selected candidate protein, APEX1. (A) The result of dot blot. (B) The validation of serum APEX1 level as the biomarker using a dot blot assay. Long horizontal line: median value; short upper and lower lines: interquartile range. *: Statistically significant difference.
Figure 5
Figure 5
Kaplan–Meier survival curves of CCA patients based on serum APEX1 levels. CCA patients were divided into high and low serum APEX1 level groups using a median serum APEX1 value (0.328) of CCA patients as a cut-off. The curves show overall survival of CCA patients having high (solid line) and low (dashed line) serum APEX1 levels. A significant difference in the survival time was observed between high and low APEX1 level groups (log-rank test p-value = 0.003).
Figure 6
Figure 6
The image of western blot. The expression of APEX1 in three CCA cell lines and control immortalized cholangiocyte MMNK1 cell line.
Figure 7
Figure 7
The effects of APEX1 gene-silencing on the CCA cell line KKU-213. (A) Western blot analysis showing suppressed APEX1 protein expression after gene silencing. β-actin was used as a control for loading protein. (B) Suppression of cell motility after APEX1 gene-silencing in wound healing model. (C) Suppression of cell migration after APEX1 gene-silencing in Transwell-migration assay. (D) Suppression of cell invasion after APEX1 gene-silencing in Matrigel-invasion assay.
Figure 8
Figure 8
The interaction map of the APEX1 and metastatic processes-associated proteins. Protein-ligand interaction was predicted by STITCH Version 5.0. Protein-protein interactions are represented in solid lines. Stronger associations are represented by thicker lines. Weak associations are represented by thin lines. APEX1 was predicted to have strong interaction with vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1alpha (HIF-1α), nuclear factor kappa B (NF-κB), transforming growth factor β (TGFβ), and vimentin (VIM).
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