The antibodies for STAT3, and phospho-STAT3 were purchased from Abcam (Shanghai, China). The antibodies for phosphatase and tensin homolog (PTEN), N-cadherin, E-cadherin, SOCS6, vimentin and α-tubulin were purchased from Proteintech (Wuhan, China). RIPA lysis buffer was purchased from CW Biotech (Beijing, China). Tocilizumab was purchased from Chugai Pharmaceutical (Shizuoka, Japan).

Paired ovarian cancer tissues and normal contralateral ovary tissues were obtained from 15 patients who underwent primary surgical resection for ovarian cancer at Shandong Cancer Hospital affiliated to Shangdong University (Shandong, China). None of the patients had received pre-operative adjuvant therapy. These samples were snap-frozen in liquid nitrogen after resection. Prior patient consent and approval from the Ethics Committee of Shandong Cancer Hospital were obtained for the use of these clinical materials for research purposes.

SKOV3 and OVCAR3 cell lines were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (CLARK), 100 U/ml penicillin sodium and 100 mg/ml streptomycin sulfate (Solarbio, Beijing, China) at 37°C in a humidified air atmosphere containing 5% CO₂. Cells were used in the logarithmic growth phase.

The inhibitor of miR-106a was purchased from RiboBio (Guangzhou, China). Transfection was performed when cells were grown to 80% confluency, using the NanoFectin Transfection Reagent (ExCell Biology, Shanghai, China) according to the manufacturer's instructions.

Total RNA and miRNA were isolated using the Ultrapure RNA kit (CWBio, Beijing, China) according to the manufacturer's protocol. cDNA was reverse transcribed from total RNA samples using the miRNA cDNA kit (CWBio). miR-106a expression was detected by quantitative real-time PCR (qRT-PCR) using the miRNA real-time PCR assay kit. The small nuclear RNA U6 was used for normalization. The relative amount of miR-106a was calculated using the cycle threshold (CT) value as the relative miRNA level. Each sample was performed in triplicate. The miR-106a and U6 primers were synthesized by Genewiz (Beijing, China). The following primers were used: miR-106a-F, AAA AGT GCT TAC AGT GCA GGT AG; and human U6-F, CTC GCT TCG GCA GCA CA.

Cells were harvested and lysed in cold RIPA lysis buffer containing 1% Halt Protease (CWBio) for 30 min. The supernatant was collected after 10 min of centrifugation at 12,000 rpm; the protein concentration of which was measured using the bicinchoninic acid method, then denatured with sample loading buffer for 5 min at 95°C and stored at −20°C for future use. Equal amounts of proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C followed by secondary antibodies for 1 h at room temperature. The bands were subsequently detected by an enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA) and analyzed by Quantity One software.

The cells were cultured in a 6-well plate. When the cells were grown to 80% confluency, the cells were transfected with 160 pmol of miR-106a, negative control (NC) or treated with tocilizumab (Chugai Pharmaceutical, Shizuoka, Japan) (10 µg/ml) for 48 h.

For the Cell Counting kit-8 (CCK-8) assay, the cells (1,000/well) were seeded into a 96-well plate in 100 ml complete DMEM supplemented with 10% FBS, and cell growth was monitored at indicated time points using the CCK-8 assay.

For the cell Matrigel Transwell invasion assays, 24-well Transwell containing polycarbonate filters with 8-mm pores (Corning Costar, Corning, NY, USA) and the inserts were precoated with 50 µl Matrigel matrix (dilution at 1:3; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol, and 200 ml cell suspension (2×10⁵) obtained from the primary step in serum-free medium was placed into the upper chamber. The lower chamber was filled with 600 ml 10% FBS-DMEM. The plates were subsequently incubated for 24 h under normal conditions. The cells, which had invaded the lower surface of the membrane, were fixed and stained with 0.1% crystal violet, The number of invading and migrating cells was calculated using a microscope at a magnification of ×200 in 5 random fields. Three independent experiments were performed.

For the wound-healing assay, the cells obtained from primary step one were seeded in a 6-well plate. When the cells grew to a confluency of 90–95%, the cell monolayer was scratched using a sterile 200-µl pipette tip. After washing and removal of the detached cells, the plates were incubated at 37°C with FBS-free DMEM and the wounds were photographed every 24 h. At least 5 different wounds were performed, and the experiments were independently repeated 3 times.

Statistical testing was conducted with the assistance of SPSS 17.0 software. All data are expressed as means ± SD. Student's t-test and one-way analysis of variance (ANOVA) were used to analyze data. Results were considered significant at a P-value <0.05.

In the present study, the levels of miR-106a were measured by quantitative real-time PCR in normal ovarian tissues and primary ovarian cancer samples. As shown in Fig. 1A, we found that miR-106a expression was significantly increased in the primary ovarian cancer tissues compared with that noted in the normal tissues (P<0.05). In addition, miR-106a expression levels were high in two ovarian cancer cell lines compared with the level in the normal ovarian tissues (Fig. 1A). We used SKOV3 cells in the cell function assay, due to higher IL-6 and miR-106a expression levels (Fig. 1B). These findings suggest that upregulation of miR-106a may play a role in ovarian cancer development.

To investigate the potential biological function of miR-106a in ovarian cancer, we modulated the miR-106a expression by transfection with the miR-106a inhibitor. Using qPCR, we found that the expression of miR-106a was significantly decreased in the cells transfected with the miR-106a inhibitor compared with that noted in the control group (NC) (Fig. 2A; P<0.05). In order to observe the impact of miR-106a on the cell proliferation of SKOV3 cells, the proliferation rates of SKOV3 cells treated with miR-106a inhibitor were determined by CCK-8 assay. As shown in Fig. 2B, growth inhibition was noted when the cells were transfected with the miR-106a inhibitor. To test the effect of miR-106a on the motility of SKOV3 cells, in vitro migration and invasion assays were performed. We assessed the effect of miR-106b on the migratory capacity of SKOV3 cells using a wound-healing assay. As shown in Fig. 2C and D, the miR-106a inhibitor had no effect on SKOV3 cell migration. In contrast, miR-106a inhibitor transfection suppressed SKOV3 cell invasion as detected by the Matrigel invasion assay (Fig. 2E and F). These observations revealed that miR-106a significantly promoted the proliferation and invasion of SKOV3 cells. To investigate the molecular mechanism by which miR-106a suppresses the growth and invasion of SKOV3 cells, we detected putative target genes of miR-106a by western blotting. Among the candidates, PTEN was found to be regulated by miR-106a (Fig. 2G). On the contrary, SOCS6, N-cadherin, E-cadherin and vimentin were not significantly affected upon miR-106a inhibitor transfection (Fig. 2G). These findings suggest that miR-106a may promote the growth and invasion of SKOV3 cells by upregulating PTEN.

Furthermore, we investigated the signaling that triggers the upregulation of miR-106a. Interleukin-6 (IL-6) is one of the important immunoregulatory cytokines present in the ovarian cancer microenvironment (11); IL6-R expression is highly expressed in ovarian cancer tissues compared with levels in normal tissues or benign diseases, and the IL-6 receptor pathway is believed to be a new therapeutic target in ovarian cancer (11,19). Tocilizumab is a humanized anti-human IL-6R antibody and competitively inhibits IL-6/IL-6R signaling (19). As shown in Fig. 3A, tocilizumab decreased the mRNA level of IL-6 and its downstream target-N-cadherin herein. Notably, we found that tocilizumab significantly enhanced the expression of miR-106a (Fig. 3B), while the expression level of miR-301a was not affected (Fig. 3C). These findings suggest that IL-6 is a regulator of miR-106a in SKOV3 cells.

To investigate the molecular mechanism by which IL-6 suppresses miR-106a expression, we detected the activation level of STAT3, a target gene of IL-6. We found that tocilizumab significantly decreased the phosphoryation level of STAT3 (Fig. 3D). Furthermore, we found that STAT3 inhibitor Stattic increased the expression level of miR-106a (Fig. 3E). Thus, the present study indicated that IL-6 is a regulator of miR-106a. IL-6 inhibits miR-106a expression by activating STAT3 in SKOV3 cells. Then, miR-106a may downregulate PTEN, and promote the growth and invasion of SKOV3 cells.

Tocilizumab has proven useful in treating IL-6-related cancers (20–25). We aimed to ascertain whether tocilizumab increases miR-106a expression, thus it may not be effective in ovarian cancers. Thus, we detected the impact of miR-106a on the cell proliferation and invasion of SKOV3 cells. As shown in Fig. 4A, tocilizumab did not inhibit the cell proliferation in SKOV3 cells. Co-treatment with the miR-106a inhibitor slowed down the cell proliferation. In addition, tocilizumab did not affect the invasion of the SKOV3 cells (Fig. 4B and C). The results suggest that tocilizumab may increase miR-106a expression, and the overexpression of miR-106a may impair the effect of tocilizumab on ovarian cancer cells.