Dihydrofolate Reductase And Ovarian Epithelial Carcinoma Multi-resistant Relations Research

CHAPTER I RESEARCH PROGRESS FOR OVARIAN CANCER MULTIDRUG-RESISTANTOvarian cancer is female genitals one of three major malignant tumor. Ovarian hidden in the deep of the pelvic cavity, early lesions will not be discovered, once appear when symptoms are belong to late, after treatment, the easy relapse, mortality also is always in the lethal gynecological malignancy. At present the aim of treatment for ovarian cancer early, late recurrence control for cure, prolonging the survival time and improve the quality of life of the patients. Treatment is mainly surgery, chemotherapy and radiation treatment. Most of the cases, surgery is difficult to ovarian cancer metastases thoroughly clean the resection. For those who remain the tiny transfer and planting nodules. All need chemical drugs to treat them. Therefore, the chemotherapy treatment in the process of oarian cancer have extremely great significance. Adanced oarian cancer treatments mainly by tumor cells was reduced and postoperative adjuvant chemotherapy out to give priority to. But often cause ovarian cancer chemotherapy failed the relapse, the main reason is that the tumor cells to produce more of drug-resistant (multidrug resistance, MDR). With the combination chemotherapy cisplatin primarily for ovarian cancer rate was40%-60%, because disease progression and the body’s resistance to chemotherapy produce, patients with adanced oarian cancer of the5year’s survival rates below20%. So. many drug-resistant tumor chemotherapy is one of the main factors of failure. Therefore, tumours produce mechanisms of resistance in clinical research and prevent resistance against drug resistance, resistance to try and reverse became the current ovarian cancer treatment of hot and difficult, which become improve ovarian cancer cure effect and improve ovarian cancer survival one of the important targets. CHAPTER II THE EXPRESSION OF DIHYDROFOLATE REDUCTASE GENE AND ITS CLINICAL SIGNIFICANCE IN OVARIAN CANCERObjective To investigate the expression of dihydrofolate reductase (DHFR) mRNA in ovarian cancer and to elucidate the relationship between the clinical pathologic parameters and the expression of DHFR mRNA in ovarian cancer. Method The expression of DHFR in80cases of ovarian carcinoma and50cases of benign ovarian and30cases of normal control were detected by real-time fluorescent quantitative polymerase chain reaction-and the relationship between them was further demonstrated. Results (1)The content of DHFR mRNA expressed in ovarian carcinoma, benign ovarian tumor and normal ovarian tissues was0.584±0.234,0.895±0.783and0.197±0.412. There were significant differences between them (P<0.001,P=0.027, P<0.001).(2)There was not relationship between the content of DHFR mRNA expressed and the stage and pathological grade in ovarian cancer, but DHFR mRNA expression in serous carcinoma was significantly higher than that in mucinous and endometrioid carcinoma (P<0.001).(3)The expression of DHFR mRNA in ovarian cancer was not related significantly and the amount of ascites-lymph node metastasis and distant metastasis in the patients with ovarian cancer(P>0.05), but the expression of DHFR mRNA in patients with the omentum metastasis was down-regulated compared with that without the omentum metastasis, there was difference statistics significantly (P=0.044).(4)The expression content of DHFR mRNA in patients with CR for treatment was lower than that with SD or PD or PR (P<0.001), and also the expression of DHFR mRNA in patients with cisplatinum-sensitive was down-regulated compared with those of cisplatinum multidrug-resistant (0.130±0.103vs0.341±0.701P=0.011).(5)Youden index of the ROC curve for DHFR mRNA expression was0.331, which would be used to determine the nature of the ovarian tumors. The median survival time of patients with DHFR mRNA expression up-regulated (less than0.331) was16.4months, but the median survival time of patients with DHFR mRNA expression down-regulated (higher than0.331) was44.5months, there was difference statistical significantly (P<0.001), and COX multivariate analysis also showed DHFR mRNA expression was an independent prognostic factor in ovarian cancer(P=0.018). Conclusions It was suggested that DHFR would be to play in physiology metabolism in normal cells. There was a relationship between DHFR mRNA up-regulated expression and cisplatinum multidrug-resistant in ovarian cancer, and the expression of DHFR mRNA could be used to determine whether there was cisplatinum multidrug-resistant in ovarian cancer as a potential marker. CHAPTER III THE BIOLOGICAL ACTIONS EFFECT OF DIHYDROFOLATE REDUCTASE GENE EXPREESION UPREGULATION IN EPITHELIAL OVARIAN CANCER IN VITRO EXPERIMENTSObjective To construct a lentiviral expressing vector harboring human dihydrofolate reductase (DHFR) gene. Methods The cDNA length of DHFR gene was amplified by PCR and was connected to cloned vector EZ-T. The recovered PCR fragment was obtained by digesting with restriction enzymes named HindⅢ and BamHI-HF, then blunted at the gap. Lentiviral vector system adopted pWPI system.The vector pWPI was digested with PmeI enzyme, and then was recovered fragment and phosphorylated. The phosphorylated vector was connected with DHFR gene by T4enzyme. Recombinant plasmid was confirmed by PCR and sequencing nucleotide. The recombinant retroviral vector pWPI was selected to transfect packaging cell293T to gain virus particles with infection ability,which were infected SKOV3cell. Adopting the flow cytometric to detect the cell apoptosis of DHFR-pWPI-SKOV3cells, pWPI-SKOV3cells and SKOV3cells in different cisplatin concentrations (2.5ug/ml,5.0ug/ml,10.0ug/ml.20.0ug/ml) and different time period (24h,48h and72h),and the of cell periodic changes of theres cells under IC50cisplatin concentration (6.0ug/ml), we also used highly effective liquid phase method (HPLC) to test intracellular cisplatin concentration in different induction of cisplatin concentration (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h.48h and72h),what’s more.we further adopted transmission electron microscope to observe ultrastructural changes of these kinds of cells under induction of IC50cisplatin concentration (6.0ug/ml).The aim of this study is investigate how the upregulated DHFR gene expression affects biological actions of ovarian cancer cell in vitro.Results (1)The recombinant plasmid, named DHFR-pWPI, which was constructed and identified Packaged by packaging cell293T. The homology between sequencing results and DHFR gene sequence was up to100%, and the expression of DHFR in293T cells was determined by RT-PCR.(2)Through the flow cytometric to detect apoptosis changes cells carried DHFR gene,we found that DHFR-pWPI-SKOV3cell in the stimulation of cisplatin concentration (2.5ug/ml,5.0ug/ml), its apoptosis rate at24h.48h and72h was lower than pWPI-SKOV3and SKOV3cells; DHFR-pWPI-SKOV3cells in the induction of cisplatin concentration (10.0ug/ml,20.0ug/ml), whose apoptosis rate at24h and48h was lower than pWPI-SKOV3and SKOV3cells, but at72h, its apoptosis rate was higher than SKOV3cell line.The results showed that DHFR-pWPI-SKOV3cells in the concentration less than5.0ug/ml, regardless of the time change (≥72hours).whose cisplatin drug resistance is higher than the other two kinds of cells.(3) We adopted the flow cytometric to test different time period under IC50cisplatin concentration (6.0ug/ml) for three kinds of ovarian cancer cells cycle, and the results indicated that G1stage rate of DHFR-pWPI-SKOV3, pWPI-SKOV3and SKOV3cells at24h and48h were obviously lower than those of G2and S stage rates:However, at72h, DHFR-pWPI-SKOV3cells G2+S phase rates were significantly lower than the pWPI-SKOV3and SKOV3cells.(4) This experiment used HPLC method to detect intracellular cisplatin concentration, and the results showed that the cells in the cisplatin concentration (4.0ug/ml) at24h,48h and cisplatin concentration (6.0ug/ml) at24h after the medicine induction, the intracellular cisplatin content of DHFR-pWPI-SKOV3cell were significantly lower than pWPI-SKOV3and SKOV3cells; cisplatin concentration (6.0ug/ml) at48h and cisplatin concentration (8.0ug/ml) at24h and48h. the intracellular cisplatin content of DHFR-pWPI-SKOV3cell were obviously higher than pWPI-SKOV3and SKOV3cells.(5)The experiment obeserved ultrastructural changes of three different cells induced by IC50cisplatin concentration in different time period through the electron microscope.which found that for DHFR-pWPI-SKOV3cells, the microwire gathered together at24h and48h, and the number and structure of mitochondria had obvious change, for pWPI-SKOV3cells,which had rare microfilament, the number of mitochondria decreased but structure change is not apparent, we also saw seldom microfilament and obvious mitochondrial structure changes in SKOV3cells, expand and pyknotic mitochondrial structure existed at the same time; all of hese cells appeared expansion of endoplasmic reticulum and had rare normal organelles; at72h,there are inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in pWPI-SKOV3and SKOV3cells, and there were rare microfilament in DHFR-pWPI-SKOV3cells,nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells is on the verge of death.Conclusion The lentiviral expressing vector harboring human DHFR had been constructed successfully, DHFR expression increased when the cell was drug-resistant in ovarian cancer cell, which suggested DHFR gene expression is related to cisplatin drug resistance in ovarian cancer,The results made the foundation to investigate the molecular mechanism of multidrug-resistance in tumor. CHAPTER Ⅳ THE BIOLOGICAL IMPACT OF DIHYDROFOLATE REDUCTASE GENE EXPRESSION DOWNREGULATION IN EPITHELIAL OVARIAN CANCER CELLS IN VITRO EXPERIMENTSTHE FIRST QUARTER:CONSTRUCTION AND IDENTIFICATION OF DIHYDRIFOLATE REDUCTASE GENE siRNA-EXPRESSION PLASMIDObjective To construct dyhidrofolate reductase gene(DHFR) small interfering RNA(siRNA)and its expression vector,and explore the significance of DHFR inhibition in the treatment of platinum multidrug-resistance in ovarian cancer. Methods We designed DHFR gene-targeted hairpin siRNA and then screened the best siRNA silence fragment, and inserted into pGPU6/GFP/Neo plasmid,which was identified by sequencing. Human malignant ovarian cancer SKOV3cells were transfected with the constructed vector using lipofectin transfection method. Fluorescence photographs were taken. Then adopting G418method to screen the stable cell line. Results Three groups of DHFR gene-targeted hairpin siRNA were designed,and inserted into pGPU6/GFP/Neo vector after annealing. Vectors containing siRNA were right by sequencing. Fluorescence photographs showed that SKOV3cells were transfected successfully by lipofectin transfection method,and PCR method identified its interference effect. Conclusion DHFR gene-targeted siRNA and its vector was successfully constructed. And one sequence with the highest inhibition efficiency was screened out. DHFR gene expression declined obviously after the constructed plasmid was transfected into SKOV3cell. THE SECOND QUARTER:THE CONSTRUCTION OF siRNA DHFR LENTIVIRUS INTERFERENCE CARRIER AND ITS BIOLOGICAL FUNNCTION AND INFLUENCE IN OVARIAN CANCERObjective To construct dyhidrofolate reductase (DHFR) gene RNAi interference lentivirus carrier, to explore the relationship between the inhibition of DHFR gene and platinum drug resistance in ovarian cancer,which made the foundation for Resistance mechanisms in the therapy of ovarian cancer. Methods To design targeting hairpin siRNA of DHFR gene, select the best siRNA silent sequence, join to pGSCIL/GFP vector after anneal, adopt PCR to identify recombination sequence, transfect into SKOV3cell lines through liposomes, finally use fluorescence camera. Western blotting identify the interferential DHFR gene of ovarian cancer SKOV3cell was constructed successfully,named DHFR-pGSCIL-SKOV3cell. Adopting the flow cytometric to detect the cell apoptosis of DHFR-pGSCIL-SKOV3cells, pGSCIL-SKOV3cells and SKOV3cells in different cisplatin concentrations (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h,48h and72h),and the of cell periodic changes of theres cells under IC50cisplatin concentration (4.4ug/ml), we also used highly effective liquid phase method (HPLC) to test intracellular cisplatin concentration in different induction of cisplatin concentration (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h.48h and72h),what’s more.we further adopted transmission electron microscope to observe ultrastructural changes of these kinds of cells under induction of IC50cisplatin concentration (4.4ug/ml).The aim of this study is investigate how the downregulated DHFR gene expression affects biological actions of ovarian cancer cell in vitro.Results (1)Let double-strand nucleotide after annealing connect to pGSCIL/GFP vector, sequence result is correct. Transfect Virus particles into SKOV3cell, using western blotting identifed interference effect.(2)Through the flow cytometric to detect apoptosis changes of cells knocked DHFR gene,we found that DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3and SKOV3cell apoptosis rate increased with the raised cisplatin concentration (2.5ug/ml.5.0ug/ml,10.0ug/ml,20.0ug/ml) at24h、48h and72h,and the apoptosis rate of DHFR-pGCSIL-SKOV3was obvious higher than pGCSIL-SKOV3and SKOV3cells at24h and48h.(3)We adopted the flow cytometric to test different time period under IC50cisplatin concentration (4.4ug/ml) for three kinds of ovarian cancer cells cycle, and the results indicated that G1stage rate of DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3and SKOV3cells at24h、48h and72h were obviously higher than those of G2and S stage rates; However, at24h and48h, DHFR-pGCSIL-SKOV3cells G2/M and S phase rates were significantly increased while G1phase rates decreased,at72h, G2/M and S phase rates were significantly increased while G1phase rates decreased for all of three kinds of cells.(4)This experiment used HPLC method to detect intracellular cisplatin concentration, and the results showed that the cells in the cisplatin concentration (2.5ug/ml、5.0ug/ml) at24h and48h,the intracellular cisplatin content of DHFR-pGCSIL-SKOV3cell were significantly higher than pGCSIL-SKOV3and SKOV3cells; but cisplatin concentration (7.5ug/ml) at24h, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3cell were obviously lower than pGCSIL-SKOV3and SKOV3cells.while higher than pGCSIL-SKOV3and SKOV3cells at48h.(5)The experiment obeserved ultrastructural changes of three different cells induced by IC50cisplatin concentration(4.4ug/ml) in different time period through the electron microscope,which found that for DHFR-pGCSIL-SKOV3at24h, visible when mitochondria swelling, its ridge disappear, the empty bubble and atrophy, obvious endoplasmic reticulum expansion.ribosomes gathered together, rare golgiapparatus.At48h,some mitochondria were dissolve and disappeared, and a large number of white vesicles were appeared; at72h, the micro filament obvious increased and gathered together, mitochondrial structure also disappear.But the microfilament of the other two cells arranged disorderly, mitochondrial form diversification.However,for pGCSIL-SKOV3and SKOV3cells at24h and48h rare endoplasmic reticulum expansion, mitochondrial structure change is not obvious, and even existed2-3golgi apparatus; the microwire gathered together at24h and48h, and the number and structure of mitochondria had obvious change, for pWPI-SKOV3cells,which had rare microfilament, the number of mitochondria decreased but structure change is not apparent, we also saw seldom microfilament and obvious mitochondrial structure changes in SKOV3cells, expand and pyknotic mitochondrial structure existed at the same time; all of hese cells appeared expansion of endoplasmic reticulum and had rare normal organelles; at72h,there are inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in pWPI-SKOV3and SKOV3cells, and there were rare microfilament in DHFR-pWPI-SKOV3cells,nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells is on the verge of death.Conclusions We are succeed in constructing DHFR gene siRNA carrier, screen the effective target sequence, and then transfect into SKOV3cell.DHFR gene expression were significantly weakened. The research indicated thatknock out DHFR gene is related to cisplatin drug resistance in ovarian cancer,The results made the foundation to investigate the molecular mechanism of multidrug-resistance in tumor.