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    r> Fig. 5. Kaplan-Meier curves constructed using the log-rank survival test. (A) The overall survival (OS) rate experienced by patients that exhibited tumors with high hCG expression was significantly lower than that experienced by patients that exhibited tumors with low or no hCG expression (P < 0.001). (B) The OS rate experienced by patients that exhibited tumors with high LHCGR expression was significantly higher than that experienced by patients that exhibited tumors with low or no LHCGR expression (P < 0.05).
    positive Dehydroergosterol
    was scored as either (0–25%), 1 (26–50%), 2 (51–75%), or 3 (76–100%). The product of these percentage and intensity scores was then used as a final staining score [21], which indicated either 0–3 was considered no or low, 4–9 was considered high expression. The hCG and LHCGR antibody were bought from Abcam Biotechnology (Abcam, Cambridge, UK). The secondary antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
    2.3. qRT-PCR analysis of ovarian tumors
    The total RNA was extracted from sample tissues using a Trizol reagent (Invitrogen, Thermo Fisher, USA) according to the manu-facturer’s instructions, before 1 μg of the total RNA was reverse tran-scribed using a Reverse Transcription Kit (K1622,Thermo Fisher Scientific, Waltham, MA, USA). hCG, LHCGR, and 18S (endogenous control) mRNA levels were then measured by qRT-PCR, using a real-time PCR system (Roche Cobaz 480, Basel, Switzerland), SYBR Green Ⅰ Master Mix (Roche, 4887352001, Switzerland), and a LightCycle 480 instrument (Roche Cobaz 480, Switzerland). The PCR cycling program consisted of denaturation at 95 ℃ for 15 s, followed by annealing at 60 for 30 s, and extension of the primer at 72 ° ℃for 30 s, for a total of 45 cyclies. The primers used to amplify each gene comprised an hCG for-ward (5′-CTA CTG CCC CAC CAT GAC C-3′) and reverse primer (5′-ATG GAC TCG AAG CGC ACA TC-3′), an LHCGR forward (5′-GAA ATG GAT TTG AAG AAG TAC AAA G-3′) and reverse primer (5′−CCA TTG TGC ATC TTC TCC AG-3′), and an 18S rRNA gene forward (5′-GTA ACC CGT TGA ACC CCA TT-3′) and reverse primer (5′−CCA TCC AAT CGG TAG TAG CG-3′). Melting curves were used to evaluate the rate of non-specific amplification, and relative expression levels were calculated using the 2™ Ct method.
    2.4. Western blot analyses
    Frozen ovarian cancer tissues were rapidly lysed (on ice) using a homogenization buffer (1% NP-40, 50 mmol/l Tris, pH 7.5, 5 mmol/l EDTA, 1% sodium dodecyl sulphate (SDS), 1% sodium deoxycholate, 1% Triton X-100, 1 mmol/l PMSF, 10 mg/ml aprotinin, and 1 mg/ml leupeptin). Lysates were resolved via SDS-polyacrylamide gel electro-phoresis (SDS-PAGE), and transferred onto a PVDF membrane. The membranes were blocked (2 h) in 5% milk (in ddH2O) and im-munoblotted (overnight, 4 °C) with hCG (1:500 dilution) and LHCGR (1:500 dilution) antibodies. They were then washed (5 min) in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20) three times and in-cubated (2 h, room temperature) with horseradish peroxidase-con-jugated secondary antibodies (1:5000 dilution). Protein signals were finally visualized using the enhanced chemiluminescence western blotting system (Pierce Company, Woburn, MA, USA). The gray value of the protein is measured by Image J software (NIH, USA).
    2.5. Statistical analyses
    Chi-square tests were performed to evaluate whether the target protein and mRNA expression levels were correlated with patient clinical or pathological parameters, and/or patient prognoses. The Kaplan–Meier method was used to calculate patient survival curves. The Cox hazard regression method was used for univariate and multi-variate analyses. For all analyses, a P-value < 0.05 was considered to indicate statistical significance. All data were analyzed using SPSS17.0 software (SPSS Inc., Chicago, IL, USA).
    3. Results
    3.1. hCG and LHCGR expression levels in fresh tissue samples collected from patients with EOC
    evaluated by analyzing 18 fresh EOC, and five normal (noncancerous) ovarian tissue samples. The demographic characteristic of patients was exhibited in supplemental S1 and S2. The generated data revealed an almost 2-fold upregulation of both hCG mRNA and protein expression in the cancerous ovarian tissue samples (P < 0.05) (Fig. 1A, B, C), and conversely, an almost 2-fold decrease in LHCGR mRNA and protein expression in cancerous compared to that in normal ovarian tissue samples (P < 0.05) (Fig. 1A, C, E). The IHC staining of hCG and LHCGR in cancer and noncancerous were significantly different (Fig. 1F).
    3.2. hCG and LHCGR expression patterns in EOC TMAs
    42) of normal ovarian, benign ovarian tumor, and borderline ovarian cancer tissue samples (P Dehydroergosterol < 0.001) (Table 2). hCG and LHCGR ex-pression levels were also found to vary between tissue samples taken from ovarian cancers with different histological classifications (Fig. 3).