DHFR silencing effects on biological behaviour of ovarian carcinoma cells in vitro

Background: Dyhidrofolate reductase (DHFR) is involved in the DNA synthesis and is expressed highly in platinum resistant ovarian carcinoma tissue . The relationship between DHFR gene and platinum drug resistance in ovarian carcinoma (OC) is still unclear. Methods: To design targeting hairpin siRNA of DHFR gene, and constructed lentiviral vector carrying DHFR gene, then though flow cytometryhigh performance liquid chromatography and electron microscope, DHFR gene expression was down-regulating to investigate the biological behaviour of DHFR in OC. Results: DHFR mRNA expression in ovarian carcinoma was higher than normal ovarian tissues, in patients with omentum metastasis was lower than that in patients without omentum metastasis, in patients with CR for treatment was lower than that of patients with SD or PD or PR, cisplatin-sensitive patients was lower than that in cisplatin-multidrug resistant cases. The median survival period of patients with DHFR mRNA expression lower than 0.331(Youden index) was 16.4 months, but of that higher than 0.331 was 44.5 months. COX multivariate analysis showed that DHFR mRNA expression was a dependent prognostic factor. The apoptosis rate of DHFR-pGCSIL-SKOV3 was obvious higher than pGCSIL-SKOV3 and SKOV3 cells at 24h and 48h(P0.05). GO/G1 stage rate of three groups at 24h48h and 72h were decreasing but the G2/M and S stage rates were raising. The cells in the cisplatin concentration (2.55.0μg/mL)at 24h and 48h,the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell were significantly higher than pGCSIL-SKOV3 and SKOV3 cells. The microfilament increased and gathered together, mitochondrial structure also change obviously under no drug. But there were rarely microfilament in three

group cells induced by IC50 cisplatin concentration(4.4 5.5 4.9µg/ml)at 24 and 48 h, while at 72 h, there are obvious increase and inordinate microfilament.
Conclusion: DHFR silencing inhibits cell growth and cisplatin resistance, there were certain contact between resistance increases with microfilament gathered and the change of the mitochondria.The results laid the foundation for us to investigate the molecular mechanisms of multidrug-resistance in tumor.

Background
Ovarian carcinoma(OC) is the highest fatality rate of malignant tumor in the female reproductive system [1], Platinum resistance in the early stages of chemotherapy greatly reduces the chemotherapy sensitivity of ovarian carcinoma patients, leads to recurrence and poor prognosis [2].Drug resistance remains a major challenge in the treatment of ovarian carcinoma [3].Therefore, to research what is the chemotherapy multi-drug resistant mechanism in epithelial ovarian carcinoma and how to improve the sensitivity of chemotherapy has a great significance to treat ovarian carcinoma effectively and improve its survival rate .Our group previously using the method of fluorescence labeling differential display PCR (FDD-PCR) screening and validating the differences of platinum resistance and sensitive ovarian epithelial carcinoma cells also found that DHFR gene expression exists differences between two group ovarian epithelial carcinoma cells. The DHFR expression was significantly raised after platinum sensitive ovarian epithelial carcinoma cells in vitro was induced into drug-resistant cell [4].Dihydrofolate reductase (DHFR) is critical gene in the metabolism of synthetic folic acid to tetrahydrofolate following absorption, catalyticing dihydrofolate back into four hydrogen folic acid. Four hydrogen folic acid is one carbon unit transfer, it is necessary for the synthesis of DNA biosynthesis precursor substance thymine DNA nucleoside to provide one carbon unit, participating in the human body important metabolism of life [5].Recently, pemetrexed is used in clinical whose drug targets is inhibition of DHFR metabolic enzymes to play a role of anti-tumor [6].Reports that DHFR gene were higher expressed in leukemia cell lines/bone sarcoma with drugresistant cell line and drug-resistant breast carcinoma cell lines, which plays a certain role in multi-resistant drug [7][8][9].During the development of antitumor drugs,DHFR who were related to folic acid metabolism is an important drug targets, may be an important direction in anti-tumor drugs research. However, so far, no studies has reported about the relationship between DHFR gene and ovarian carcinoma ,especially related to platinum resistance.

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 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 × 10 5 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 × 10 3 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 × 10 5 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

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.
Results DHFR expression was significantly higher in normal ovary and benign tumor tissues than in OC tissues(P = 0.000-0.027). Relationship between DHFR mRNA expression in ovarian carcinoma tissues and its clinical pathologic factors The DHFR mRNA expression in serous ovarian carcinoma was higher than that in mucinous and endometrioid ovarian carcinoma (P < 0.05), in platinum-resistant patients was significantly higher than that of platinum-sensitive patients(P = 0.011), the patients with complete remission(CR)was significantly lower than that of  Table 2. ROC curve analysed an Youden index defined DHFR mRNA expression quantity is 0.331, more than 0.331 is DHFR positive, and less than 0.331 is DHFR negative.
Using single factor analysis, Kaplan Meier survival curve, Long -rank test shows: The median survival time of DHFR positive was 16.4 months, 44.5 months in DHFR negative (P = 0.018), Fig. 1. Cox proportional hazards model analysis shows that DHFR mRNA expression affect the prognosis of patients with ovarian carcinoma, but not independent prognostic factors. Table 3.

MTT detect cells growth curve in three groups
MTT assay detect the growth curve of DHFR -pGCSIL SKOV3, pGCSIL SKOV3 and After 48 h, three groups have intersection in cisplatin concentration between 4.0 ~ 5.0 ug/mL, before the cisplatin concentrations intersection, apoptosis rate of DHFR-pGCSIL-SKOV3 cells was significantly lower than the other two groups (P = 0.003, 0.036 < 0.05);While after the cisplatin concentration intersection, apoptosis rate of DHFR-pGCSIL SKOV3 cells was significantly higher than pGCSIL -SKOV3 and SKOV3 cell (P = 0.009, 0.047 < 0.05).After 72 h, apoptosis rate of DHFR-pGCSIL-SKOV3 cells was obviously higher than that of other two groups in every drug concentration (P = 0.023, 0.034, 0.012, 0.034 < 0.05).There was no statistically significant difference between pGCSIL SKOV3 and SKOV3 cell groups (P > 0.05). Table 4. (3) The p53 gene mutation ;(4) Apoptosis pathway obstruction (5) Cell survival pathway activation [10], more and more studies [3] have found multi-resistant may be associated with folate metabolism enzymes.It could inhibit the growth of ovarian carcinoma cells by affecting folate metabolism, and enhance chemotherapy drug toxic effected on the ovarian carcinoma cells, thereby enhancing ovarian carcinoma chemotherapy sensitivity [11].However, tumor cells may reduce the affinity between chemotherapy drug and DHFR via increasing DHFR gene copy number and (or) quantity, then reduce mechanism such as drug uptake and speed up the discharge to induce drug resistance [12]. and normal ovarian tissue, the result shows that ovarian benign tumor tissue of DHFR mRNA relative expression is significantly higher than OC and normal tissues, indicated that DHFR expression increased may be a compensatory adaptation mechanism for being lack of folic acid to the carcinomaous start or process, mediating the more folic acid into the cell. Due to tumor cells usually have low folic acid content [13], with the development of tumor, it is hard for extracellular carrier folate reduction to be intake of intracellulardue because of lacking folate reduction or its activity decline in tumor cells, thus made DHFR expression down regulation.
However, normal cells have much more folic acid, through the competitive inhibition of drug effect on DHFR, making DHFR expression level maintained at a higher level, this has the same with Matakidou [7]. The results of the study demonstrated that in malignant tumor tissues, DHFR mRNA expression has no correlation with the clinical stage, pathological classification, lymphatic metastasis, ascitic fluid, the result is consistent with some scholars researching the other malignant tumors [8,9].
According to the results of this observation DHFR mRNA expression in serous ovarian carcinoma patients is significantly higher than other ovarian epithelial carcinoma, and DHFR mRNA expression is higher in non-omentum metastasis in OC shows that the sensitivity of chemotherapy drugs was higher than the other two kinds of cells, and the sensitivity of chemotherapy drugs may be associated with the depolymerization of actin filaments.In the process of cisplatin stimulation, mitochondria and endoplasmic reticulum in cells also change obviously, suggested mechanism of cisplatin treat tumor may be through mitochondria and endoplasmic reticulum kill cells. One research arsenic trioxide drug induced sensitive leukemia K562 cells apoptosis mainly by mitochondria, while induced resistance leukemia K562 / ADM cells apoptosis mainly by a combined way of mitochondria and endoplasmic reticulum.the results also is the same time with Banka [16]. Sun [17] found that cisplatin inducing tumor cell apoptosis and inhibit tumor growth by restoring the function of p53, Klinghoffer [18] at the same time studies results of the mechanism of cisplatin induced apoptosis was consistent with Ma Yanyun' research.Based on the above literature research, the experiment mechanism of apoptosis may be also related to the p53 pathway, but as a result of this experiment did not test the endoplasmic reticulum ,mitochondria and protein and gene expression involved in the p53 signal transduction pathways, the specific mechanism that cisplatin induced to increase its drugs sensitivity after siclencing DHFR gene is unclear.
Through the research about silencing DHFR gene, we found there was a connection between DHFR expression and platinum drug sensitivity in OC cells, after down regulated DHFR expression, its drug sensitivity increases, when at the certain concentration range, the effect of cisplatin increased along with the increase of drug concentration, after reaching a certain concentration, the incremental effect weakened or disappeared, which illuminated cisplatin concentrations have saturation effect.
The G2 / M phase retardation enhanced along with drugs effecting time increased, effecting 72 h is the most obvious. Among effective cisplatin concentration range, prolonged effect of drug appropriately could be one of the methods to reduce the development of drug resistance or reverse resistance.Silencing DHFR gene in ovarian carcinoma cells could be considered as a multi-resistant reversal mechanism, cell apoptosis, cycle are involved in the study of multi-drug resistance mechanism, how is the concrete mechanism need to be further in depth research.

Conclusions
This study found that DHFR gene has a certain relationship with cisplatin resistance, the higher DHFR gene expression, the more increased resistance, and its drug resistance may be related to gathered icrofilament and the change of the mitochondria. Cisplatin kill tumor cells by a complex mechanism, cisplatin and taxol has become a first-line chemotherapy drugs for ovarian carcinoma.Through knowing the resistance mechanism of chemotherapy drug, develop a new generation of chemotherapy drugs to reduce the cell resistance and side effects, which is great significant to the clinical treatment of malignant tumors. This study only test epithelial ovarian carcinoma cell apoptosis and cell cycle along with the DHFR gene expression level changes but not study in-depth for the resistance in the process of related proteins and gene expression, which will be the next research direction. The study was approved by the Ethics Committee of Guangxi Medical University.
Written informed consent was obtained from all the subjects before study, and written informed consent was obtained from a parent or guardian for participants under 16 years old.

Consent for publication
Not applicable.  The establishment of chromatogram standard curve by the high performance liquid chromato Use electron microscope to observe ultrastructure changes of three groups cells after IC50 c

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