Identification and Characterization of Aptamers Targeting Ovarian Cancer Biomarker Human Epididymis Protein 4 for the Application in Urine

Abstract

Ovarian cancer is the deadliest gynecological cancer. With non-specific symptoms of the disease and the lack of effective diagnostic methods, late diagnosis remains the crucial hurdle of the poor prognosis. Therefore, development of novel diagnostic approaches are needed. The purpose of this study is to develop DNA-based aptamers as potential diagnostic probes to detect ovarian cancer biomarker Human epididymis protein 4 (HE4) in urine. HE4 is a protein overexpressed in ovarian cancer, but not in healthy or benign conditions. With high stability and diagnostic value for detection of ovarian cancer, urine HE4 appears as an attractive non-invasive biomarker. The high-affinity anti-HE4 DNA aptamers were selected through 10 cycles of High Fidelity Systematic Evolution of Ligands by EXponential enrichment (Hi-Fi SELEX), a method for aptamer selection based on digital droplet PCR. The anti-HE4 aptamers were identified using DNA sequencing and bioinformatics analysis. The candidate aptamer probes were characterized in urine for binding to HE4 protein using thermofluorimetry. Two anti-HE4 aptamers, AHE1 and AHE3, displayed binding to HE4 protein in urine, with a constant of dissociation in the nanomolar range, with Kd (AHE1) = 87 ± 9 nM and Kd (AHE3) aptamer of 127 ± 28 nM. Therefore, these aptamers could be promising tools for application in diagnostics and future development of urine tests or biosensors for ovarian cancer.

Aptamers as Potential Therapeutic Tools for Ovarian Cancer: Advancements and Challenges

“…The results revealed that AHE1 and AHE3 exhibited a favorable affinity towards the target HE4, as evidenced by a dissociation constant measured within the nanomolar range. These compelling findings strongly indicate the potential of AHE1 and AHE3 as promising diagnostic probes, which could be further harnessed in the development of advanced diagnostic tests and biosensors intended for the reliable detection of OC [84].…”

“…HE4 serum levels do not increase during pregnancy, menstruation, or in benign gynecological conditions. Additionally, HE4 is elevated at an earlier stage in the progression of the disease [84,89]. Nevertheless, the concentration of HE4 is substantially influenced by age [86].…”

“…Notably, HE4 is up regulated in OC, and its elevated levels have been observed in serous, endometrioid, and clear-cell ovarian tumors. Furthermore, HE4 has shown increased expression in patients with mucinous ovarian tumors, which distinguishes it from CA125 [84][85][86][87][88]. In contrast to CA125, HE4 offers several advantages as a biomarker.…”

Aptamers Versus Vascular Endothelial Growth Factor (VEGF): A New Battle against Ovarian Cancer

“…AHE3 and AHE1 are anti-HE4 aptamers, which bind to HE4 proteins in urine. Hence, these aptamers may be useful diagnostic tools for developing biosensors or urine tests for OC [ 74 ].…”