Upregulation of SNHG3 expression associated with poor prognosis and enhances malignant progression of ovarian cancer

2.1 Patient tissue samples

A total of 76 human ovarian cancer tissues and adjacent normal tissues were collected from patients who underwent surgical resection at Department of Gynecology and Obstetrics, Zhujiang Hospital, Southern Medical University from March 2009 to April 2011. Ovarian cancer tissues were confirmed as epithelial ovarian carcinoma by two independent pathologists. None of patients underwent any treatment prior to surgical resection. The study was approved by ethics board of Zhujiang Hospital, Southern Medical University the study and written informed consent was obtained from all patients.

2.2 Cell lines culture and cell transfection

Human ovarian cancer (OC) cell lines (SKOV3, OVCAR3, A2780, and ES2) and one normal ovarian cell line, HOSE were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and added with 100 U/mLpenicillin and 100 μ g/mL streptomycin. Then, cells were cultured at 37 ∘ C in a humidified atmosphere containing 5% CO 2 . The specific two siRNAs targeting SNHG3 (si-SNHG3-1: 5’-GGTGACATCAGCCAAGAAT-3’, si-SNHG3-2: 5’-GCCAAGAATCTGAATGGTA-3’) and miR negative control (miR-NC) were commercially designed and synthesized by Shanghai Gene- Pharma (Shanghai, China). The full length sequence of SNHG3 was subcloned into pcDNA3.0 plasmid by Shanghai GenePharma (Shanghai, China) to increase its expression and named pcDNA3.1-SNHG3. Cells transfection was performed using the Lipofectamine 3000 (Invitrogen).

2.3 RNA isolation and quantitative real-time PCR (qRT-PCR)

The Trizol reagent (Invitrogen) was used to extract RNA from tissues and cells. RNA was reversed to First-strand cDNA using the Reverse Transcriptase Kit (TAKALA, Dalian, China). The reaction was at 37 ∘ C for 15 min and 85 ∘ C for 5 s. SYBR Green Mixture (TAKALA, Dalian, China) was applied for qRT-PCR reaction on ABI 7500 system. The SNHG3 mRNA expression level was normalized to GAPDH and was analyzed using 2 - Δ ⁢ Δ ⁢ Ct method. SNHG3 primer sequences was SNHG3-forward, 5’-TTCAAGCGATTC TCGTGCC-3’ and SNHG3-reverse, 5’-AAGATTGTC AAACCCTCCCTGT-3’.

2.4 Cell proliferation assay

Cell Counting Kit-8 (CCK-8) kit (Dojindo, Tokyo, Japan) was used to assess cell viability after cell were transfected with si-NC and si-SNHG3-2 or pcDNA3.0-vector or pcDNA3.0-SNHG3. After cells transfection, the cells were seeded into 96 well plates at a concentration of 2000 cells/well. The CCK-8 reagent was added into the well at 24, 48, 72 and 96 h and incubated at 37 ∘ C for 2 h. The optical density (OD) value was 450 nm and cell viability was analyzed by using a microplate reader.

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Cells were transfected with si-NC and si-SNHG3- 2 or pcDNA3.0-vector or pcDNA3.0-SNHG3 and then were seeded into 6 cm dishes (500 cells/wells). After cells were cultured for 7 days in a humidified atmosphere containing 5% CO 2 , colonies were fixed by 100% methanol, and then stained using 0.4% crystal violet solution and the figures were captured by a camera.

2.6 Cell invasion assay

Transwell chamber (50 mg/L BD Matrigel 1: 8 diluent) was used in the study. The cells at a concentration of 1 × 10 5 cells were added on the upper chamber with medium without FBS. In the lower chamber, we used the medium containing 20% FBS. Cells were cultured for 24 h at 37 ∘ C in a humidified atmosphere containing 5% CO 2 . Cells in the lower chamber were fixed with 100% methanol, and were stained using 0.1% crystal violet. Cells were calculated in five random fields using an IX71 inverted microscope (Olympus).

2.7 Western blot analysis

Cells were lysed using RIPA buffer (Roche, Indianapolis, IN, USA) that was added proteinase inhibitor cocktail (Roche, Indianapolis, IN, USA). Total cell protein (45 μ g) was used to separate on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (SDS/PAGE) and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane. Then, the membrane was blocked with 5% nonfat milk for 1 h and were incubated with specific antibodies including Cyclin D1 (Cell Signaling Technology, Danvers, MA, USA), CDK1 (Cell Signaling Technology, Danvers, MA, USA), MMP9 (Abcam, Cambridge, MA, USA) and MMP3 (Abcam, Cambridge, MA, USA, GSK3 β (Abcam, Cambridge, MA, USA), β -catenin (Abcam, Cambridge, MA, USA) and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Protein blots were examined using enhanced chemiluminescence (ECL) from the manufacturer (Beyotime Institute of Biotechnology) and results were normalized to GAPDH.

2.8 Statistical analysis

Statistical analyses were performed using SPSS 20.0 software and results were shown as mean ± standard deviation (SD). Groups differences were compared by paired samples t -test or one-way analysis of variance. A 0.05 was considered statistically significant.

3.1 SNHG3 expression is higher in ovarian cancer tissues and associates with clinicopathological features

First, qRT-PCR analyses were performed to assess SNHG3 in 76 ovarian cancer tissues and adjacent normal tissues collected from patients who underwent surgical resection. As shown in Fig. 1A, the relative SNHG3 expression was higher in ovarian cancer tissues than that in the corresponding adjacent normal tissues ( 0.05). Next, the relative SNHG3 expression was analyzed in four human ovarian cancer cell lines (SKOV3, OVCAR3, A2780, and ES2) and a human normal ovarian cell line HOSE. The qRT-PCR results demonstrated that SNHG3 was significantly upregulated in all four ovarian cancer cell lines than that of normal ovarian cell line HOSE (Fig. 1B). The 76 ovarian cancer patients were divided into two groups according to the median expression of SNHG3 (2.987 fold): lower SNHG3 expression group ( 2.987, 36 cases) and higher SNHG3 expression group ( $>$ 2.987, 40 cases). Association of SNHG3 expression and clinicopathological features of patients with ovarian cancer was analyzed. Results demonstrated that higher SNHG3 expression was significantly associated with FIGO stage and lymph node metastasis (all 0.05, Table 1).

3.2 Higher SNHG3 expression associates with poor prognosis of ovarian cancer patients

Furthermore, Kaplan-Meier survival curve and log-rank test were performed to determine the association between SNHG3 expression levels with overall survival (OS) rate of ovarian cancer patients. Our results demonstrated that higher SNHG3 expression significantly associated with poor OS rate compared with lower SNHG3 expression in ovarian cancer patients (Fig. 1C). Univariate and multivariate Cox regression model was performed to determine whether SNHG3 expression and other clinicopathologic feathers were independent prognostic markers for OS in ovarian cancer patients. Our results demonstrated that SNHG3 expression (HR = 2.255, 95% CI: 0.905–4.212, 0.05) (Table 2), FIGO stage (HR = 1.912, 95% CI: 0.432–3.641, 0.05) (Table 2), lymph node metastasis (HR = 2.088, 95% CI: 0.724–3.865, 0.05) (Table 2) were independent risk factors of prognosis of OS in ovarian cancer patients.

3.3 Upregulation of SNHG3 promotes cell proliferation and invasion of ovarian cancer

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To investigate whether SNHG3 expression could affect cell proliferation and invasion of ovarian cancer, we performed lose-function and gain-function assays by transfecting two siRNA oligos targeting SNHG3 or si-negative control into SKOV3 and A2780 cells, or transfecting pcDNA3.0-vector or pcDNA3.0-SNHG3 into OVCAR3 cells (Fig. 2A and B), the siRNA-SNHG3-2 was used for following experiments due to higher silencing efficiency of SNHG3 in SKOV3 and A2780 cells. The CCK8 cell viability assay demonstrated that knockdown of SNHG3 using siRNA-SNHG3-2 inhibited cell growth rate in SKOV3 and A2780 cells, while SNHG3 over-expression by pcDNA3.0-SNHG3 enhanced cell growth rate in OVCAR3 cells (Fig. 2C and D). Cell colony forming assays showed that knockdown of SNHG3 using siRNA-SNHG3-2 reduced cell colonies number in SKOV3 and A2780 cells, while SNHG3 over-expression using pcDNA3.0-SNHG3 enhanced colonies number in OVCAR3 cells (Fig. 3A–D). To determine whether SNHG3 expression affected tumor cell invasion, we performed transwell invasion assays by knocking down SNHG3 in SKOV3 and A2780 cells or SNHG3 over-expression in OVCAR3 cells. The results demonstrated that SNHG3 knockdown suppressed cell invasion ability in SKOV3 and A2780 cells, whereas, SNHG3 over-expression enhanced cell invasion ability in OVCAR3 cells (Fig. 4A and B).

Additionally, we also explore the effects of SNHG3 expression on cell proliferation and invasion related protein CyclinD1, CDK1, MMP9 and MMP3. The results showed that CyclinD1, CDK1, MMP9 and MMP3 expression levels were significantly downregulated after SNHG3 knockdown in SKOV3 cells. However, SNHG3 over-expression enhanced their expression in OVCAR3 cells (Fig. 4C and D). Thus, these results demonstrated that SNHG3 promoted cell proliferation and invasion of ovarian cancer.

GSK3 β / β -catenin signaling pathway associated with tumor proliferation and invasion. We detected the effects of SNHG3 expression on activity of GSK3 β / β -catenin signaling pathway. After SKOV3 cells were treated with si-NC or si-SNHG3-2, the glycogen synthase kinase 3 β (GSK3 β ) was significantly increased, but β -catenin was remarkably reduced in SKOV3 cells (Fig. 5A). However, SNHG3 over-expression enhanced β -catenin, but glycogen synthase kinase 3 β (GSK3 β ) was remarkably reduced in OVCAR3 cells (Fig. 5B). These results indicated that GSK3 β / β -catenin signaling is the downstream mechanism of SNHG3 in ovarian cancer.

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