A total of 28 ovarian carcinoma tissue samples and matched adjacent non-tumorigenic tissues samples (2 cm from cancerous tissues) were obtained from patients who underwent surgical resection of the tumor at Harbin Medical University Cancer Hospital (Harbin, China) between May 2015 and March 2016. The average age of the patients was 48 years old. The samples were stored in liquid nitrogen until total RNA or protein were extracted. The present study was approved by the Ethical Committee of Harbin Medical University Cancer Hospital and written informed consent was obtained from all patients.

All tissues were initially fixed in 10% formalin for 48 h at 4°C and embedded in paraffin. Section of 5 µm thickness were pre-incubated with 5% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology, Shanghai, China) as a blocking buffer for 2 h at room temperature, and then incubated with primary hERG1 antibody (1:200; cat. no. sc-377388; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C; After washing three times with PBS at room temperature, the sections were incubated with peroxidase-labeled anti-mouse antibody (1:50; cat. no. A0216; Beyotime Institute of Biotechnology) for 30 min at room temperature. The peroxidase activity was detected with diaminobenzidine (DAB; Beyotime Institute of Biotechnology). The stained sections were analyzed using a fluorescence Olympus BX51 microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan). The immunohistochemical staining results were scored as the sum of intensity and percentage scores, according to the staining intensity (0=negative; 1=weak/trace; 2=moderate; and 3=strong) and the percentage of positive cells (0, ≤10; 1, 11–25; 2, 26–50; 3, 51–75; and 4, 76–100%). This grading produced a final score of 0–12, and samples were separated into groups based on low (0–4) or high (6–12) scores.

BBR and DMSO were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). BBR was dissolved in 10% DMSO and 90% deionized water. The drug solution was diluted to concentrations of 5, 10, 30, 50 and 100 µM in RPMI-1640 culture medium (Beyotime Institute of Biotechnology) prior to use at room temperature. For cellular experiments, cells were incubated with different concentrations of BBR (5, 10, 30, 50 or 100 µM) for 24, 48 and 72 h at 37°C, respectively. Cells in the blank control group were treated with 1% DMSO.

OVCAR-3, SKOV3, HO-8910, A2780 and ES-2 ovarian carcinoma cell lines were obtained from the Cancer Institute of Harbin Medical University. OVCAR-3 and SKOV3 cells were maintained in RPMI-1640 medium (Beyotime Institute of Biotechnology) supplemented with 12% (v/v) FBS (Thermo Fisher Scientific, Inc., Waltham, MA, USA). HO-8910, A2780 and ES-2 ovarian carcinoma cells were maintained in DMEM (Beyotime Institute of Biotechnology) supplemented with 12% (v/v) FBS (Thermo Fisher Scientific, Inc.). All cell lines were cultured at 37°C with 5% CO₂ in a humidified incubator.

Short hairpin (sh)RNA-hERG1 plasmid (4 µg) and shRNA-control (scrambled short hairpin RNA sequence) plasmids (4 µg) (Shanghai GeneChem Co., Ltd., Shanghai, China), were used to transfect SKOV3 cells. All transfections were performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were harvested at 24 h after transfection.

Cell proliferation was determined by Cell Counting kit-8 (CCK8) assay. Briefly, SKOV3 cells were seeded in 96-well plates at 1×10⁵ cells/well and maintained for 24 h at 37°C to allow cell adhesion. Subsequently, the cells in various groups (5, 10, 30, 50 or 100 µM berberine for 24, 48 or 72 h) were incubated with 10 µl CCK8 for 2 h and the absorbance was measured at 450 nm using a microplate reader (Olympus Corporation). The proliferation of treated cells was assessed by comparison with the blank control group. Each experiment was performed in triplicate.

OVCAR-3, SKOV3, HO-8910, A2780 and ES-2 ovarian carcinoma cells and tissue specimens were homogenized in TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to isolate total RNA, according to the manufacturer's protocols. The concentration of RNA was detected with a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). The total RNA of each sample (1 µg) was reverse transcribed using random primers and a PrimeScript 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocols. qPCR was performed using a SYBR green fluorescent dye (PCR master mix; Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling conditions: Pre-denaturation at 95°C for 45 sec, followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing and extension at 55°C for 30 sec. The relative hERG1 expression levels were calculated based on the 2^−ΔΔCq method and normalized to GAPDH level in each sample (30). Each experiment was performed in triplicate. The primer sequences for qPCR were as follows: hERG1 forward, 5′-CAGCGGCTGTACTCGGGCACAG-3′ and reverse, 5′-CAGAAGTGGTCGGAGAACTC-3′; and GAPDH forward, 5′-GTCAACGGATTTGGTCGTATTG-3′ and reverse, 5′-AGTGATGGCATGGACTGTGG-3′.

Briefly, total protein was extracted from tissues or OVCAR-3, SKOV3, HO-8910, A2780 and ES-2 ovarian carcinoma cell lines with RIPA buffer (Beyotime Institute of Biotechnology) and the protein concentrations were determined using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Subsequently, proteins (50 µg/lane) were separated by SDS-PAGE (8% gel) and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). To prevent non-specific binding, the membrane was blocked with 5% skimmed milk in PBS with Tween-20 (0.25%) for 2 h at room temperature. Subsequently, the membrane was incubated with hERG1 antibody (1:1,000; cat. no. sc-377388; Santa Cruz Biotechnology, Inc.) or β-actin antibody (1:1,000; cat. no. ab8227; Abcam, Cambridge, UK) in PBS at 4°C overnight. Subsequently, the membrane was rinsed with PBST three times, which was followed by incubation with corresponding IRDye^® 800CW fluorescent secondary antibodies (1:8,000; cat. nos. anti-rabbit 926–32211 and anti-mouse 926–32210; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. Finally, the protein bands were visualized using an Odyssey infrared imaging system (LI-COR Biosciences) at 800 nm.

SKOV3 cells in serum-free RPMI-1640 medium (200 µl containing 5×10⁵ cells for BBR group, 200 µl containing 2.5×10⁵ cells for other groups) were added to the upper Transwell chambers (pore size, 8 µm; Corning Inc., Corning, NY, USA). The chambers were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) for the invasion assays but not the migration assays. The bottom chamber was filled with 700 µl RPMI-1640 medium containing 15% FBS. The cells were cultured in an incubator at 37°C with 5% CO₂ for 24 h, then the cells were fixed in 4% paraformaldehyde for 15 min. Subsequently, the cells were washed with PBS and stained with 0.1% crystal violet for 10 min at room temperature. For analysis, five fields were randomly selected and the number of stained cells was counted under a light microscope at magnification of ×200 (Nikon Corporation, Tokyo, Japan). Data are expressed as the mean number of cells per insert.

Five-week-old BALB/c-nu/nu nude mice were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The experimental protocols were approved by the Ethics Committee of Harbin Medical University (Harbin, China). A total of 72 mice (36 females, 36 males) weighing 20–25 g each, were maintained under a 12-h light/dark cycle at 20.0–26.1°C and 35–70% humidity. Food and water were freely accessible to mice. SKOV3 cells (2×10⁶ per mouse) were subcutaneously injected into the flank of each mouse. Following the outgrowth of palpable tumors, the mice were randomly divided into four groups (n=3 for each group) and fed by oral gavage with saline or BBR (20 mg/kg), twice per week. BBR was dissolved in carboxymethylcellulose sodium for use via oral gavage. Tumors volumes were calculated using the following formula: Volume=L × (W)²/2, where L is the longest diameter and W is the shorter diameter. At the end of the present study, mice were euthanized and tumor tissues were harvested for further research.

Data are presented as the mean ± standard error of the mean of triplicate experiments and were analyzed with SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). The χ² test was used to analyze the association of hERG1 expression with clinicopathologic features of ovarian cancer. Unpaired Student's t-test and one-way analysis of variance with a Student-Newman-Keuls post hoc test were used to determine the significance of differences between two groups and three or more groups, respectively. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the role of hERG1 in ovarian cancer development, the mRNA and protein expression levels of hERG1 were detected by RT-qPCR and western blotting. A total of 28 tumor tissues and matched non-tumor tissues were obtained from patients with ovarian cancer with a median age of 48 years in the present study. The association of hERG1 expression with clinicopathologic features of ovarian cancer is presented in Table I. These results demonstrate that hERG1 expression level is significantly associated with ovarian cancer distant metastasis and lymph node metastasis. The immunohistochemical assay results revealed that the protein expression levels of hERG1 were markedly higher in ovarian cancer tissues compared with non-tumor tissues (Fig. 1A). As presented in Fig. 1B and C, the mRNA and protein expression levels of hERG1 were significantly upregulated in tumor tissues compared with non-tumor tissues, as expected. Furthermore, the expression level of hERG1 in OVCAR-3, SKOV3, HO-8910, A2780 and ES-2 ovarian cancer cell lines was investigated. The results (Fig. 1D and E) revealed that hERG1 was generally expressed in these ovarian cancer cell lines and highly expressed in SKOV3, in particular. Therefore, in the present study, SKOV3 cells were used for further analysis.

Subsequently, the effects of BBR on SKOV3 cells were investigated using a CCK8 assay. SKOV3 cells were treated for 24, 48 and 72 h with BBR at concentrations ranging between 5 and 100 µM, and the results were compared with the control group treated with DMSO. The CCK8 assay results demonstrated that BBR inhibited the proliferation of SKOV3 cells in a time- and dose-dependent manner (Fig. 2A). The proliferation of cells treated for 48 h decreased more significantly compared with cells treated for 24 h (P<0.05); however, no difference was observed between cells in the 48 and 72 h groups. Simultaneously, BBR significantly reduced the mRNA and protein expression levels of hERG1 in a dose-dependent manner (Fig. 2B-D), which suggests that hERG1 may serve a role in the proliferation of SKOV3 cells. Expression levels of hERG1 decreased significantly in response to 10 µM BBR and the IC₅₀ of BBR was 9.8 µM at 48 h. Therefore, in subsequent experiments BBR was administered to SKOV3 cells for 48 h at a concentration of 10 µM.

To investigate the effects of hERG1 on tumor biological activities, expression levels of hERG1 in SKOV3 cells were downregulated using a shRNA-hERG1 plasmid. As presented in Fig. 3, the mRNA and protein expression levels of hERG1 in SKOV3 cells transfected with the shRNA-hERG1 plasmid were significantly decreased compared with in the control groups.

The effects of hERG1 on the proliferation of SKOV3 cells were detected in vitro using a CCK8 assay (Fig. 4A). The results demonstrated that knockdown of hERG1 in SKOV3 cells significantly reduced the cell proliferation activity compared with the control cells. BBR (10 µM) was considered as a positive control group. As expected, knockdown of hERG1 exhibited similar cytotoxic effects as BBR treatment. These results suggest that hERG1 may serve a role in cell proliferation.

Furthermore, to determine whether hERG1 is involved in the process of cell migration and invasion, which represent the tumor migration and invasive abilities, Transwell assays were performed following the transfection of SKOV3 cells with sh-hERG1 or scramble control. As presented in Fig. 4B-E, knockdown of hERG1 significantly reduced the migration and invasion abilities of SKOV3 cells. BBR (10 µM) was used as a positive control. The results of the present study indicate that hERG1 may be involved in the proliferation, migration and invasion processes of SKOV3 cells, and that BBR may be a potential therapeutic drug in the treatment of ovarian cancer.

To assess whether hERG1 is involved in ovarian cancer growth in vivo, nude mice were inoculated with SKOV3 cells, which were previously transfected with sh-hERG1 or shRNA-control plasmids. Following the outgrowth of palpable tumors, nude mice were randomly divided into four groups (n=3 for each group) and fed via oral gavage with saline or BBR (20 mg/kg) twice per week. As presented in Fig. 5, the size and weight of ovarian cancer mass significantly decreased following the administration of BBR by oral gavage compared with the control group. Similarly, knockdown of hERG1 also significantly decreased tumor growth of xenografts compared with the control group. During the experiments, no notable weight loss was observed in mice of the different groups. These findings indicate that hERG1 may be involved in the pathogenesis of ovarian cancer and may be considered as a potential prognostic indicator in the treatment of ovarian cancer.