Clinical tissue samples
We collected a total of 6 paired non-cancerous (NC) and ovarian cancer tissues from patients received resection surgeries in Zhongshan hospital with the signed the consent from each patient. All the experiments in the present study were conducted with the approval of the Ethics Committee of Zhongshan hospital. The pathologic type of all samples was confirmed by two independent pathological experts. Tissues were frozen at -80°C immediately after sampling until further use.
Cell lines and cell culture
A normal cell line, human ovarian surface epithelial cell line (HOSE, also known as HOSEpiC), was purchased from ScienCell (Cat. #7310; Carlsbad, CA, USA) and cultured in Ovarian Epithelial Cell Medium (OEpiCM, Cat. #7311; ScienCell). Ovarian cancer SKOV3 (ATCC® HTB-77™), OVCAR-3 (ATCC® HTB-161™), and CAOV3 (ATCC® HTB-75™) cell lines were obtained from ATCC (Manassas, VA, USA). SKOV3 cells were cultured in McCoy's 5a Medium Modified (Catalog No. 30-2007; ATCC). CAOV3 cells were cultured in Dulbecco's Modified Eagle's Medium (Catalog No. 30-2002; ATCC). OVCAR-3 cells were cultured in RPMI-1640 Medium (Catalog No. 30-2001; ATCC). All the cells were cultured with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37℃ in 5% CO2.
Cells were transfected with scramble siRNA (negative control, si-NC; RiboBio, Guangzhou, China) or NDRG2 siRNA (si-NDRG2; RiboBio) with the help of Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Cells were collected and used for further experiments 48 h after transfection.
PCR-based analyses
Total RNA was extracted from target cells with the help of TRIzol reagent (Invitrogen). The reverse transcription of extracted RNAs into cDNA was performed with the help of Maxima First Strand cDNA Synthesis Kits (K1672; Thermo Fisher). The expression of mRNA was determined with an SYBR® Green Real-time PCR Master Mix (Sigma, St. Louis, MO, USA) using GAPDH as an endogenous control. All the results were processed and analyzed using the 2-ΔΔCt method.
Immunoblotting
Cell lysate was prepared using RIPA lysis buffer (Applygen, Beijing, China) and proteins were extracted. SDS-PAGE (10%) was used to separate the extracted proteins. After that, proteins were transferred onto PVDF membranes. Nonspecific antigen was blocked by 5% non-fat milk solution, and the membranes were washed three times using PBST. The membranes were then incubated overnight at 4°C with anti-NDRG2 (Cat# 12015-1-AP, Proteintech, Rosemont, IL, USA), anti-BAX antibody (1:1000, Cat# ab32503), anti-BCL2 antibody (1:1000, Cat# ab32124), anti-Cleaved Caspase-3 antibody (Cat# ab2302), anti-Caspase-3 antibody (1:500, Cat# ab13847), anti-cyclin B1 (1:50000, Cat# ab32053), anti-cyclin A2 (1:2000, Cat# ab181591) (Abcam, Cambridge, MA, USA) and anti-β-actin (60008-1-Ig, Proteintech). After washing with PBST, the membranes were incubated with appropriate HRP goat anti-mouse/anti-rabbit IgG (Proteintech) at room temperature. The visualization of all the blots were conducted using enhanced chemiluminescence (ECL; Thermo Fisher).
Cell viability determined by CCK-8 assay
A CCK-8 kit (Sigma-Aldrich, St. Louis, MO, USA) was employed to examine the cell viability of ovarian cancer cell lines in response to different treatment and/or transfection. Cells were placed into 96-well plates at a density of 1 × 104 cells/well. CCK-8 solution was added at 0 h and 24 h thereafter and then cells were incubated for 4 h at 37°C. The absorbance (OD value) was measured at 450 nm.
Colony formation
The colony formation ability was determined. Cells were cultured 6-well plates at a density of 1 × 102 cells/well. Fourteen days later, the colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). After that, the number of visible colonies were counted.
Cell cycle and cell apoptosis determined by Flow cytometry
Flow cytometry analyses were performed to detect the cell apoptosis and cell cycle. For cell apoptosis analysis, cells were collected, resuspended, and added with annexin V-FITC and PI. After 15 min of incubation, the cell apoptosis was analyzed.
For cell cycle analysis, cells were fixed with ethanol (70%, ice-cold) for 20 min and added with PI. After 20 min incubation, the cell cycle was analyzed.
Xenograft mice assay in vivo
Pathogen free conditions were maintained through the lifetime of twelve male BALB/c nude mice (4 weeks old). The approval of xenograft in vivo assay was obtained from Zhongshan hospital. Firstly, overexpression of NDRG2 (Lv-NDRG2 OE) was performed using MISSION® shRNA lentiviral particles (Sigma-Aldrich), which were designed to overexpress the production of NDRG2 in SKOV3 cells. The cultures that were transfected with lentiviral particles with negative control vector were used as the control group (Lv-Vector). Subsequently, Lv-NDRG2 OE or Lv-Vector transfected SKOV3 cells (1×106) were subcutaneous injected into the armpit of nude mice respectively. Caliper was adapted in measuring tumor volume following the length×width2/2 formula. The average volume of tumor was measured for 3 times every 3 days. At the termination of the experiment (the 25th day), mice were killed and the tumor was excised from each mouse to measure the average volume and weight.
Data processing and statistical analysis
All data collected from three independent experiments were processed and analyzed by GraphPad (San Diego, CA, USA). The data was represented as mean ± SD. The comparison was conducted using paired or unpaired Student’s t-test. A P value of less than 0.05 was considered as statistically significant.