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Bioinformatic Prediction of Signaling Pathways for Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and Its Role in Cholangiocarcinoma Cells.

ABSTRACT: Apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) is involved in the DNA damage repair pathways and associates with the metastasis of several human cancers. However, the signaling pathway of APEX1 in cholangiocarcinoma (CCA) has never been reported. In this study, to predict the signaling pathways of APEX1 and related proteins and their functions, the effects of APEX1 gene silencing on APEX1 and related protein expression in CCA cell lines were investigated using mass spectrometry and bioinformatics tools. Bioinformatic analyses predicted that APEX1 might interact with cell division cycle 42 (CDC42) and son of sevenless homolog 1 (SOS1), which are involved in tumor metastasis. RNA and protein expression levels of APEX1 and its related proteins, retrieved from the Gene Expression Profiling Interactive Analysis (GEPIA) and the Human Protein Atlas databases, revealed that their expressions were higher in CCA than in the normal group. Moreover, higher levels of APEX1 expression and its related proteins were correlated with shorter survival time. In conclusion, the signaling pathway of APEX1 in metastasis might be mediated via CDC42 and SOS1. Furthermore, expression of APEX1 and related proteins is able to predict poor survival of CCA patients.

SUBMITTER: Tummanatsakun D

PROVIDER: S-EPMC8125001 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein's catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5-2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

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Bioinformatic Prediction of Signaling Pathways for Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and Its Role in Cholangiocarcinoma Cells.

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