Study design
The present study was approved by the internal review boards of the Tokyo Medical University. The written informed consent was obtained from all the patients before the collection of specimens, according to the Declaration of Helsinki. The patient backgrounds were obtained through interviews. The blood samples were collected before operation, chemotherapy, and radiation therapy. The ovarian clear cell carcinoma and endometriosis were diagnosed based on the histological examinations.
A total of 64 subjects participated in the research conducted at the Tokyo Medical University Hospital from February 2010 to January 2019 and at the Jikei University School of Medicine between August 2008 and November 2011. 29 patients had ovarian clear cell carcinoma, 17 had endometriosis, and 18 were healthy controls. The patients were diagnosed with ovarian clear cell carcinoma, 6 with stage Ia, 5 with stage Ic1, 5 with stage IC2, 2 with stage IC3, 1 with stage IIa, 2 with stage IIb, 1 with stage IIIa1, 4 with stage IIIb, 2 with stage IIIc, and 1 with stage IVb.
Serum preparation and total RNA extraction
The blood samples were collected from the patients with ovarian clear cell carcinoma, and endometriosis, as well as from the healthy controls. We measured the CA125 levels in patients with endometriosis and ovarian cancer before surgery. The blood serum was separated by centrifugation at 400 xg for 10 min and stored at -80 °C. The total RNA was extracted from the serum using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Search for candidate miRNAs with TaqMan Array Human microRNA Cards
We used TaqMan™ Array Human MicroRNA A+B Cards Set v3.0 (Thermo Fisher Scientific, Inc.) to search for candidate miRNAs in 20 samples (16 samples of ovarian clear cell carcinoma and four healthy controls). Using a volcano plot, we identified target miRNAs with significantly different expression levels in control and ovarian clear cell carcinoma patients. Then, using an amplification plot, we narrowed down the miRNAs that were amplified in almost all targets. Finally, we determined the target miRNAs based on relative gene expression.
miRNA expression analysis by qPCR and receiver operating characteristic curves
Four miRNAs (miR-146a-5p, miR-191-5p, miR-484, and miR-574-3p) were analyzed by the TaqMan miRNA expression analysis (Thermo Fisher Scientific, Inc.) and real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The expression analyses were performed using the TaqMan Advanced miRNA assays for human miR-146-5p (478399_mir), miR-191-5p (477952_mir), miR-484 (478308_mir), miR-574-3p (478163_mir), and miR-16 (477860_mir) as an endogenous control [17]. The cDNA was synthesized using the TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.).
Quantitative PCR (qPCR) was performed with RT primers using the Universal Master Mix and specific miRNAs using the Applied Biosystems StepOnePlus ™ real-time PCR system (Thermo Fisher Scientific, Inc.). The sequence detection was performed according to the manufacturer's protocol.
The reaction mixtures were incubated at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The miRNA expression levels in subjects with ovarian clear cell carcinoma and endometriosis compared to healthy controls were calculated using the comparative 2-ΔΔCt method [18, 19]. Receiver operating characteristic (ROC) curves were generated using the miR-146a miR-191 expression profile. The graphical plots of the true and false positive rates are shown. The area under the ROC curve represents the identification accuracy.
Statistical analysis
The statistical analyses of the causal association between the clinical background, the expression level of the miRNAs, and the ROC curve analysis were performed using SPSS-27 software. The statistical significance was determined by a Kruskal-Wallis test with significance set at P < 0.05.
Analysis of miRNA 146a-5p and miRNA 191-5p MiRTarBase
Subsequent to the identification of differentially expressed miRNAs, the predicted target genes for these altered miRNAs were subjected to experimental validation using the miRNA‑target interaction database MiRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php) [20].