Exploring OCLN and TOP2A as Molecular Markers for Cancer Stem Cell Profiling in MDAMB-231 Cells

Abstract

Background: Cancer stem cells (CSCs) are a subpopulation of tumor cells with the ability to self-renew, recurrence, and therapeutic resistance. Characterizing their molecular profile is critical for early detection and targeted treatment. While gene expression studies are informative, protein-level analysis is essential due to potential post-transcriptional regulation in CSCs. Our previous in silico analysis identified OCLN and TOP2A as downregulated secretome biomarkers in CSCs. OCLN, a tight junction protein and TOP2A, a key enzyme in DNA replication, may influence CSC malignancy. However, the correlation between gene expression and protein levels of OCLN and TOP2A in CSCs remains unclear. This study aimed to perform a comparative molecular analysis of OCLN and TOP2A gene expression and protein levels in CSC-enriched and non-CSC populations from the MDAMB-231 breast cancer model.

Methods: Quantitative PCR (qPCR) was used to assess mRNA expression, while enzyme-linked immunosorbent assay (ELISA) measured protein concentrations. Comparisons were made between CSC-enriched (CD24⁻/CD44⁺) and non-CSC (CD24⁺/CD44⁺) subpopulations.

Results: OCLN mRNA levels were comparable between CSC (1.281 ± 0.801) and non-CSC (1.157 ± 0.582) groups, yet protein concentration was markedly reduced in CSCs (0.014 ± 0.004 ng/mg vs. 0.224 ± 0.001 ng/mg). TOP2A mRNA expression was modestly higher in CSCs (1.074 ± 0.193) than in non-CSCs (0.811 ± 0.271), accompanied by an increase in protein concentration (4.172 ± 0.662 ng/mg vs. 3.675 ± 0.133 ng/mg).

Conclusion: The observed discrepancies between mRNA and protein levels, particularly for OCLN, suggest post-transcriptional regulation or protein instability in CSCs. These findings highlight the complexity of CSC molecular regulation and support the potential of OCLN and TOP2A as molecular profiling candidates in breast cancer stem cell research.