Clinical samples and follow-up
Tumor samples were obtained from Guangxi Medical University carcinoma Hospital, and all of them were identified by pathology. The study was endorsed by the Ethics Committee of the Guangxi Medical University. All patients received an explanation for aims of this study and a signed informed consent. They all understood that they could withdraw from the study at any time without influencing their oncological or general medical treatment. 80 cases of ovarian malignant tumor were collected from different ages of people, their ages ranged from 13 to 76 years old, and the average age was 41.1 years old. In accordance with ovarian histology classification made by World Health Organization (WHO, 1973), the 80 samples we collected including 24 cases of mucinous carcinoma, 41 cases of serous carcinoma, 2 cases of endometrial sample carcinoma, 3 cases of embryo sinus tumor within, 1 case of asexual cell tumor, 2 cases of granulosa cell tumor, 5 cases of immature teratoma and 2 cases of other metastases tumor; and according to the clinical stage FIGO (2004) standard, the samples were classified as follows, I ~ II stage: 30 cases, Ⅲ ~IV stage: 50 cases. All of patients were accepted tumor destruction reduction operation. 67 cases of epithelial neoplasm and 13 cases of non-breast epithelial neoplasm were accepted treatment of cisplatin plus paclitaxel and treatment pf cisplatin plus bleomycin together with vincristine chemotherapy, respectively. 50 cases of benign ovarian tumor were collected from different ages of people, the average age was 40.1 years old(range: 10–74 years). The 50 samples included 11 cases of mucinous tumor, 36 cases of serous tumor and 3 cases of teratoma. 30 cases of normal ovarian tissues were collected from different ages of people, the average age was 43.1 years old(range: 29–60 years). Among these 30 peoples that the normal ovarian tissues were collected, 28 peoples had uterine fibroids, 1 person had cervical carcinoma and 1 person had breast carcinoma. The normal ovaries were resected while the carcinomas were cut off (agreed by the patients). The resected ovaries were further confirmed without abnormality by pathology. All of tissue samples were collected during the operation. The primary lesion organization and metastases organization of tumor were stored in liquid nitrogen, respectively, then the samples were ready to RNA extraction and histopathological examination via 10% formalin fixed slice.All of ovarian malignant tumor patients need to followed up for 6 months to 60 months according to WHO level four evaluation standard in 1988. In this study, excepted 5 patients were lost to follow-up, all of the malignant ovarian tumor patients were followed up after treatment until December 2010, and no one was died of carcinoma. The median follow-up time was 29.25 months (range 3–60 months). Short-term curative effect was decided according to WHO standard in 1988. The judgment for tumor patients who resistance or sensitive to platinum drugs were in accordance with previous study.
Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to quantify DHFR expression in all kinds of ovarian tissues
Organization RNA were extracted using Trizol one-step methods; cDNA were synthesized with RT retrovirus kit (TOYOBO company offers, M-MLV reverse transcriptase Promega company offers).All of experimental steps were strictly followed the specifications. Primers were designed with Primer 5.0 software and synthesized by Invitrogen Company. The DHFR primers were 5’- GTCATGGTTGGTTCGCTAAACTGCA-3’(forward);5’-ATACATACTTTTTTCAGAGGGAGGG-3’ (reverse). The PCR products were separated by 1.5% agarose gel and imaged by gel imaging analysis system, and the mRNA expressions of DHFR was quantified and normalized to that of GAPDH.
Plasmid generation
The hairpin siRNA targeted by DHFR gene was designed to screen out the best silencing fragment of siRNA, and the single-stranded DNA oligonucleotides were synthesized. The primer was annealed to form double-stranded DNA oligonucleotides, named shRNA. The annealed shRNA template was connected to the linear lentiviral vector pGCSIL after enzyme digestion and recovery, after the transformation of DH-5a, positive clones were extracted for sequencing.The sequencing results were compared by BLAST, and the homology was 100%.The recombinant plasmid was named DHFR-pGCSIL. The quality ratio of three plasmid included DHFR-pGCSIL (pGCSIL), pHelper1.0 and pHelper2.0 was 4:3:2, SKOV3 cells were inoculated in 6-well plates with 3 × 105 per well. On the second day, cell adherent growth converged to 70%~80% and then turned transfection, the specific operation was proceeding according to the instruction of LipofectamineTM2000. The experiment was divided into three groups: experimental group (SKOV3 cell group carrying DHFR-pGCSIL gene, negative control group (SKOV3 cell group only carrying pGCSIL virus) and blank control group (parental SKOV3 cell group).SKOV3 cells with DHFR-pGCSIL gene were sorting by flow cytometry .
Western blot assay
The cells were lysed with the radioimmuno precipitation test lysate and centrifuged at 13 987.5 × g for 5 min ,then extract the total cell protein in the supernatant.The proteins were separated by 10% sodium dodecyl sulfate-polyphenylamide gel electrophoresis, and then electrotransferred to the nitric acid fiber membrane. The buffer solution was washed and sealed by 5% skim milk powder. The antibody was incubated overnight, and the membrane was washed.The photographic film was scanned or photographed, and the relative expression level of the target band was analyzed with the gel image processing system, with GAPDH as the internal reference.
Drug cytotoxicity assay
When the three groups of cells were cultured to achieve a confluence of about 80%, then digested them with 0.25% trypsin to make a single cell suspension.The number of cells was inoculated in 96-well plat accorrding to 8 × 103 per well, and the total volume of each well was 200 uL,which were cultured was cultured at 37 ℃ for 24 h with 5% CO2.After 24 h, the three group cells were cultured with cisplatin for 24 h, 48 h and 72 h at the mass concentration gradients of 1.25, 2.5, 5.0, 10.0, 20.0 and 40.0 ug/mL.MTT solution 20 uL was added to each hole, incubated at 37 ℃ for 4 h, and the supernatant was sucked and discarded. 150uL dimethyl sulfoxide was added to each hole, and then beaten and mixed to make the dirty fully dissolved. All solutions were transferred to another hole plate.The absorbance (D) value of each hole was measured by enzyme-linked immunoassay at 492 nm(ThermoLab Systems, Chantilly, VA, USA), the results were recorded, and the growth curve was plotted.
Apoptosis detection by flow cytometry
The three group cells number were cultured to about 80%~90%, then digested them with 0.25% trypsin to make a single cell suspension.The number of cells was inoculated in 6-well plat accorrding to 5 × 105 per well, and the total volume of each well was 2 mL,which were cultured was cultured at 37 ℃ for 24 h with 5% CO2.After 24 h, the three group cells were cultured with cisplatin for 24 h, 48 h and 72 h respectively at the mass concentration gradients of 2.5, 5.0, 10.0, 20.0ug/mL,the total volume of each well was 2 mL. The operation was conducted according to the instructions of the apoptosis kit.
Cell cycle detection by flow cytometry
Three groups Cells (1 × 106) were induced by the IC50 concentration cisplatin drug (4.4、5.5、4.9 µg/ml), after 24, 48 and 72 h, respectively, accordance with the cell cycle kit instructions, 10 000 cells in every group were analyzed using MultiCycle Software for Windows (Phoenix Flow Systems, San Diego, CA, USA), Multicycle software analysis of DNA content, G1, G2, S cell number and proportion of every period.
Cell platinum drugs concentration detection by High Performance Liquid Chromatography
Three groups Cells (1 × 107) were induced by different concentration cisplatin chemotherapy drugs (0、 2.5、5.0、 7.5 ug/mL )after 24, 48 h, respectively. According to the corresponding chromatographic conditions for determination of peak area, making the linear regression with peak area of content, the concentration of DDP in 0.1ཞ1.0 µg/mL, the regression equation Y = 1.24 × 10 − 5 x to 6.41 × 10 − 3.
Transmission electron microscopy
Three group cells were cultured to a culture flask of 75 cm2 respectively, when the cell number reached 90%~100%,using IC50 cisplatin mass concentration (4.4 ug/mL) stimulated them for 24 h, 48 h and 72 h, then 0.25% trypsin digested the cells, the cell suspension inside the centrifuge tube was centrifuged (167.7 × g, 10 min) into clusters,which were fixed overnight by 3% glutaraldehyde fixation solution ,were fixed 1 h again by 1% osmic acid fixation solution .Acetone: isoamyl acetate (1:1) dehydrated them for 10 min and alcohol dehydrated them layer by layer gradient.Epoxy resin 618 embedded and then prepared them into a 1um semi-ultrathin section.Transmission electron microscopy( OH7650 TEM, Hitachi, Japan) observed the cell's ultrastructure and took photographs,and then analyzed the ultrastructure changes of cells in every group.
Statistical analysis
The experimental results were analyzed by SPSS 18.0 statistical software and the data were expressed as means ± SD. The comparison of the three groups data was conducted by ANOVA or Non-parametric Rank Sum Test. P < 0.05 was considered statistically significant.