Identification of Endometrial Cancer-Specific microRNA Biomarkers in Endometrial Fluid

Abstract

Abnormal uterine bleeding is a common benign gynecological complaint and is also the most common symptom of endometrial cancer (EC). Although many microRNAs have been reported in endometrial carcinoma, most of them were identified from tumor tissues obtained at surgery or from cell lines cultured in laboratories. The objective of this study was to develop a method to detect EC-specific microRNA biomarkers from liquid biopsy samples to improve the early diagnosis of EC in women. Endometrial fluid samples were collected during patient-scheduled in-office visits or in the operating room prior to surgery using the same technique performed for saline infusion sonohysterography (SIS). The total RNA was extracted from the endometrial fluid specimens, followed by quantification, reverse transcription, and real-time PCR arrays. The study was conducted in two phases: exploratory phase I and validation phase II. In total, endometrial fluid samples from 82 patients were collected and processed, with 60 matched non-cancer versus endometrial carcinoma patients used in phase I and 22 in phase II. The 14 microRNA biomarkers, out of 84 miRNA candidates, with the greatest variation in expression from phase I, were selected to enter phase II validation and statistical analysis. Among them, three microRNAs had a consistent and substantial fold-change in upregulation (miR-429, miR-183-5p, and miR-146a-5p). Furthermore, four miRNAs (miR-378c, miR-4705, miR-1321, and miR-362-3p) were uniquely detected. This research elucidated the feasibility of the collection, quantification, and detection of miRNA from endometrial fluid with a minimally invasive procedure performed during a patient in-office visit. The screening of a larger set of clinical samples was necessary to validate these early detection biomarkers for endometrial cancer.

Keywords: downregulation; endometrial biopsy; endometrial cancer (EC); endometrial fluid; exploration; liquid biopsy; microRNA; saline infusion sonohysterography (SIS); upregulation; validation.

Figure 1

A schematic presentation of the miRNA profiling workflow applied to the endometrial fluid samples collected using sonohysterography (SIS). Five main steps are outlined, including endometrial fluid sample collection and preparation, RNA isolation and quantification, reverse transcription, and cDNA synthesis, miScript miRNA real-time PCR, and data analysis. The images shown beneath the workflow scheme are real images of the results.

Figure 2

Heatmap of the fold-changes of endometrial cancer (EC)-related miRNAs. In phase I, a total of 60 patients were recruited, i.e., 30 EC patients and 30 non-cancer patients were paired. A total of 84 miRNAs were screened. In total, 25 miRNA species out of 35 identified in EC patients showed significant fold-changes, compared with non-EC patient controls. Green bars indicate fold-changes in miRNAs exhibiting upregulation. Red bars indicate fold-changes in miRNAs exhibiting downregulation.

Figure 3

The expression levels of 14 miRNAs were compared between phase I (exploration) and phase II (validation) for EC patient endometrial fluid samples. The 14 miRNAs that showed significant changes in the expression in phase I (solid brown bars) were recruited for validation in phase II (patterned blue bars). The miRNA species with asterisks indicate the consistency of the fold-change when observed in EC samples for the two phases.

Figure 4

The receiver operating characteristic curves (ROC) were plotted with a fold-change in Ct values (ΔΔΔCt) at various levels of three miRNAs, i.e., miR-183-5p, miR-429, and miR-146a-5p, identified from the EC endometrial fluid specimens to differentiate between the EC and non-EC histopathology of the endometrium.