The expression of mRNA profile was analyzed in 426 OC tissues and 88 normal control tissues obtained from TCGA database. GEPIA is a new interactive website for the analysis of RNA sequence data based on TCGA and GTEx (http://gepiacancer-pku.cn/index.html). PTPRZ1 expression in OC tissues and normal control tissues was analyzed. The association of PTPRZ1 expression with the overall survival (OS) and disease free survival (DFS) of OC patients was calculated by GEPIA database.
Thirty pairs of OC tissues and normal control tissues were collected. All OC patients received no chemotherapy or radiotherapy before surgery. Pathological classification and tumor staging were conducted according to the cancer staging criteria of Union for International Cancer Control. This study protocol was approved by the ethics committee of our hospital. All patients signed the informed consent. This study was performed following the Declaration of Helsinki.
Ovarian cancer cell lines SKOV3 and A2780 were commercially acquired from the Shanghai Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). Cells were cultured in Dulbecco modified Eagle’s culture medium containing 10% fetal bovine serum (Gibco, Carlsbad, California) in an incubator with 5% carbon dioxide at 37°C. Drug-resistant cell lines SKOV3 and A2780 were constructed by treating the proliferated cell cultures using DDP (Meilun Biotech, Dalian, China) at a concentration of 8 μM for consecutive 12 weeks.
A total of 5x10^4/ml SKOV3 and A2780 cells (or SKOV3/DDP and A2780/DDP) were seeded into 6-well plate, and the transfection was performed at a cell density of about 70% by reference to the instructions for use of Lipofectamine 3000 (Invitrogen, USA). Cells were transfected by using PTPRZ1 overexpression plasmids and corresponding negative references. Above transfection reagents and corresponding negative references were designed and synthesized by GenePharma (Shanghai, China). Cells were collected for subsequent experiments after 48 h of transfection.
RNA cells or tissues to be extracted were added into TRIzol reagent (Invitrogen, CA, USA) as per the instructions for use. Post chloroform extraction, the aqueous phase was transferred into a new tube. Isopropanol was used to subside RNAs in the aqueous phase. RNA sediments were washed by using 75% ethyl alcohol and dried at room temperature. DEPC water was then added for resuspension. RNAs were subject to reverse transcription into cDNAs by using PrimeScript RT reagent Kit (TAKARA, Code No. RR036A) according to its instructions for use. For qRT-PCR, SYBR® Green Master Mix (TaKaRa) was used for the detection on Roche480 as per the instructions for use. GAPDH was used as the internal reference, and the calculation was performed with 2-△△CT method. The primer sequence is presented below: PTPRZ1 forward: GCCTGGATTGGGCTAATGGAT, PTPRZ1 reverse: CAGTGCTCCTGTATAGGACCA; GAPDH forward: GGAGCGAGATCCCTCCAAAAT, GAPDH reverse: GGCTGTTGTCATACTTCTCATGG.
Cells were seeded into the 96-well plate at 4×143 cells per well. DDP at the concentration of 0μM, 1μM, 2μM, 5μM, 10μM, 20μM and 40μM was added into each group. The plate was then incubated for 48 h at 37°C. Subsequently, the culture solution was replaced with new culture medium. With MTT (0.5 mg/ml) added, the plate was incubated with 5% CO2 at 37°C for 4h. The culture solution was then carefully removed, and 150μl DMSO was added into each well to fully dissolve the generated formazan crystals. The microplate reader was used for absorbance measurement at the wavelength of 570nm. The experiment was repeated in triplicate.
Cell apoptosis experiment
After cell transfection, with SKOV3/DDP and A2780/DDP cells collected and washed with PBS, cells were stained by using Annexin V-FITC kit (Beyotime, China) according to the instructions for use. FACSCalibur Flow Cytometer (BD Bioscience, Franklin Lakes, NJ, USA) was then utilized to analyze the cell apoptosis rate.
Transfected SKOV3/DDP and A2780/DDP cells were collected. Upon protein extraction, cell lysis solution containing protease inhibitor PMSF (Beyotime, Nantong, China) was added. On the ice, the supernatant was collected post centrifugation. The protein concentration was detected by using BCA Protein Quantitation Kit (Beyotime, Nantong, China) as per its operating instructions. For albuminous degeneration, cells were heated at 100°C after adding SDS-PAGE protein loading buffer. The transfer membrane was conducted after the loading buffer was used up. Corresponding size of PVDF membrane cut as per the molecular weight was subject to antigen blocking in 5% skim milk powder blocking buffer. The incubation was conducted by adding primary antibodies. Another incubation was then performed by using secondary antibodies for subsequent exposure.
The data were analyzed with SPSS 20.0 and GraphPad Prism 6.0 statistical software. The measurement data were shown as the mean ± standard deviation. Two sample t-test was used as the statistical method for between-group comparison. P < 0.05 was considered statistically significant.