Effect Of Reduced Expression Of PARP-1 By RNA Interference On The Reversal Of EMT In Ovarian Cancer SKOV3 Cell Line And Sensitivity Of Cisplatin In Vitro And Vivo

Ovarian cancer is the common malignant tumor of female genital system, because of its high malignant degree, concealment, and progress rapidly, ovarian cancer is the most lethal gynecologic malignancy in the world. Due to a lack of specificity of screening, the majority of patients present with advanced disease (stage III or IV) at the time of diagnosis, and affect directly on the survival of patients. Current standard treatment of ovarian cancer in advanced stages, consists of complete cytoreductive surgery followed by chemotherapy, usually based on a platinum. The major impediment for the successful treatment is tumour relapse and chemoresistance. Although the improvement of surgery, the overall 5-year survival rate has no obvious improvement. Multidrug resistance has serious effect on the treatment, in order to achieve a good clinical therapeutic effect, we are urgent to solve the relapse and chemoresistance of ovarian cancer by boycotting the invision and enhancing the sensitivity of chemotherapy.Poly(ADP-ribose)polymcrase-1, as a monomer protease, is widely existed in most nucleus of eukaryotic cell, PARP-1 is involved in regulating cell differentiation, proliferation, tumor transformation and DNA damage repair. Studies have shown that PARP-1 expressed highly in a variety of malignance, including in ovarian cancer, thus we think that PARP-1 may be involved in tumor invasion and metastasis. Because EMT plays an important role in the invasion and metastasis of tumor cells, so we analyze PARP-1 may be associated with EMT in the occurrence of ovarian cancer.The enhancement of DNA damage repair ability is an important reason of chemotherapy resistance, when tumor cells are exposed to cytotoxic antitumor drug, resulting in DNA damage, which can reduce the sensitivity of chemotherapy. So we assume, inhibition of PARP-1 may enhance the sensitivity of tumour cells. Based on this assumption, we established PARP-1 stable transfected SKOV3 cell line, we intend to explore the biological behavior in vitro, observing the effect of invasion, EMT and the sensitivity of cisplatin, we also establish the ovarian cancer transplanted tumor animal model, further observing the sensitivity of cisplatin in vivo, providing theoretical and experimental basis for the targeted therapy of ovarian cancerPart I:Effect of reduced expression of PARP-1 by RNA interference on the reversal of EMT in ovarian cancer SKOV3 cell lineObjectives:1. To establish PARP-1 gene transfection and stable expression in ovarian cancer SKOV3 cell line.2. To explore the effect of invasion by inhibiting PARP-1 in ovarian cancer SKOV3 cell line.3. To explore the reverse effect of epithelial-mesenchymal transition by inhibiting PARP-1 in ovarian cancer SKOV3 cell line.Methods1.Gene transfection and stable expression in ovarian cancer SKOV3 cell line (RT-PCR and Western blot).2. The effect of inhibiting PARP-1 on the invasion detected by Transwell.3.The effect of inhibiting PARP-1 on the expressions of Vimentin and E-cadherin protein in ovarian cancer SKOV3 cell line by western blot.Results1. Transfection efficiency Lentiviral vector carries green fluorescent protein (GFP), which is used to screening successfully transfected cells. Observation of the cells using an phase contrast fluorescence microscope indicated that cells started to express GFP at 12h,24h,36h, after transfection and GFP expressed highest at 72h after transfection, transfection efficiency add up to more than 90%.2.PARP-1mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) after transfection 72h. Compared with untransfected group and NcSiRNA group, In the PARP-1SiRNA transfection group, the expression of PARP-1mRNA was significantly down-regulated(P<0.05), downregulation of PARP-1 mRNA (～38% with PARP-1 SiRNA1,81% with PARP-1 SiRNA2, P<0.05; P<0.01, respectively).3.Compared with untransfected group and NcSiRNA group, in the PARP-1 SiRNA transfection group, the expression of PARP-1 protein was significantly down-regulated(P<0.05) and protein (～12% with PARP-1 siRNA1,～44% with PARP-1 siRNA2, P<0.05, P<0.01, respectively) expression was observed. According to the results, PARP-1 siRNA2 was the most efficient and specific sequence to inhibit the expression of PARP-1 in SKOV3 cells and was used to finish the subsequent experiments.4. The invision potential of SKOV3 cells was examined by transwell chamber assay. In the PARP-1 SiRNA transfection group, the invasion of SKOV3cells were inhibited notablely as compared with the negative control group(NcSiRNA) and the untransfected group. The difference was significant(P<0.05), there was no difference between the NcSiRNA and the untransfected group. The results indicate that inhibiting the expression of PARP-1 could decrease the invision of ovarian cancer SKOV3 cells, also showed that PARP-1 maybe take part in the development of ovarian cancer.5.The effect of inhibiting PARP-1 on the expression of Vimentin and E-cadherin protein in ovarian cancer SKOV3 cell line by western blot Compared with the untransfected group and NcSiRNA group, in the PARP-1 SiRNA transfection group, the expression of Ecadherin protein was significantly up-regulated(P<0.05), there was no difference between the NcSiRNA and SKOV3 group(P>0.05). Compared with untransfected group and NcSiRNA group, in the PARP-1SiRNA transfection group, the expression of Vimentin protein was significantly down-regulated(P<0.05), there was no difference between the NcSiRNA and the untransfected group (P>0.05). The results indicate that inhibiting the expression of PARP-1 could make EMT reversal.Conclusions:1.Inhibition of PARP-1 may obviously downregulate the invision of ovarian cancer SKOV3 cell line.2.Inhibition of PARP-1 may reverse EMT.3.The expression of PARP-1 is probably associated with the invision and EMT in ovarian cancer SKOV3 cell line. PARP-1SiRNA transfection group, the expression of Vimentin protein was significantly down-regulated(P<0.05), there was no difference between the NcSiRNA and the untransfected group (P>0.05). The results indicate that inhibiting the expression of PARP-1 could make EMT reversal.Conclusions:1.Inhibition of PARP-1 may obviously downregulate the invision of ovarian cancer SKOV3 cell line.2.Inhibition of PARP-1 may reverse EMT.3.The expression of PARP-1 is probably associated with the invision and EMT in ovarian cancer SKOV3 cell line.Part Ⅱ:Effect of reduced expression of PARP-1 by RNA interference on the sensitivity of cisplatin in vitroObjectives:1. To explore the effect of proliferation by inhibiting PARP-1 via RNA interference in ovarian cancer SKOV3 cell line.2. To explore the effect of proliferation by inhibiting PARP-1 via RNA interference on the sensitivity of cisplatin in ovarian cancer SKOV3 cell line.3. To explore the effect of apoptosis, cell cycle, and ERK signaling pathway by inhibiting PARP-1 via RNA interference after treated with cisplatin.Methods1.The changes of cell clone formation ability of inhibiting PARP-1 via RNA interference in ovarian cancer SKOV3 cell line.2.The cell viability was detected by CCK8 after treated with different concentration of cisplatin.3.AnnexinV-FITC/PI test on flow cytometry and Hoechst 33258 staining were performed to detect apoptosis; Flow cytometry (FCM) to detect cell cycle; The ERK signaling pathway protein expressions were detected by western blot via inhibiting PARP-1 after treated with cisplatin.Results:1. The effect of proliferation by inhibiting PARP-1 expressionCell colony experiment showed that the clone formation ability of stable transfection SKOV3 cell was inhibited, cell grew slowly, PARP-1SiRNA group compared to NcSiRNA group, the difference was statistically significant(P<0.05), showing that inhibiting the expression of PARP-1 could affect the proliferation.2.The effect of proliferation by inhibiting PARP-1 expression after treated with cisplatinCell viability was determined by CCK-8, the cell survival rate was downregulated based on the higher concentration of cisplatin, also it appeared a dose-dependent manner in response to cisplatin treatment, downregulation of PARP-1 in SKOV3 cell via cisplatin treament, the cell viability was significantly inhibited, the difference was statistically significant(P<0.05), showing that inhibiting the expression of PARP-1 could enhance the sensitivity of cisplatin, also the proliferation ability was inhibited significantly.3.The morphological changes were visualized by Hoechst 33258 staining and AnnexinV-FITC/PI test on flow cytometry was performed to detect apoptosis.We found that the morphological changes of apoptosis would take place after treated with cisplatin, nuclei turned pale and uniform distribution, in the PARP-1 SiRNA group, the number of apoptosis was higher than NcSiRNA group, we found the nuclei condensed with blue light under fluorescence microscope, showing the characteristics of cell pycnosis, and the number of apoptotic cells increased significantly, the difference was statistically significant(P<0.05). AnnexinV-FITC/PI test on flow cytometry showed that apoptosis rate was increased slightly as compared with NcSiRNA group, the difference was statistically significant(P<0.05), after treated with cisplatin, the rate of apoptosis increased significantly compared with NcSiRNA group, It showed that inhibiting PARP-1 could enhance the chemical toxicity effect on cisplatin treatment, increased the apoptosis of SKOV3 cells and strengthened the effect on promoting apoptosis of SKOV3 cells.4. Flow cytometry (FCM) to detect cell cycleEvery group in the cell cycle distribution was different, after inhibiting the expression of PARP-1, G0/G1 phase cells increased slightly, S phase cells reduced slightly, when NcSiRNA group was treated with cisplatin, the distribution of cell cycle in G0/G1 phase cells increased higher than the NcSiRNA group or the PARP-1 SiRNA group, it showed that only PARP-1 SiRNA was not obvious to the cell cycle distribution. Suppression of PARP-1 after cisplatin treatment with 48 hours, the cell cycle arrested significantly in G0/G1 phase and showed a significant reduction of S phase and G2/M phase cells.5.ERK signaling pathway was detected by western blotThe NcSiRNA and PARP-1 SiRNA group cells were exposed to 10uM cisplatin for 48h, we found that cisplatin inhibited phosphorylation of ERK in SKOV3 cells, however, inhibition of PARP-1 expression by RNAi induced even lower pERK1/2 expression, while the total ERK 1/2 was unaffected. It showed that inhibiting PARP-1 expression could enhance the inhibition of ERK signaling pathway.6. U0126 determine the influence of ERK 1/2 activity on cell viabilityU0126 is a specific ERK inhibitor, first, lOuM U0126 was used to for 2h prior to cisplatin treament, then the SKOV3 cells were exposed to lOuM cisplatin, then we found the cell viability was downregulated significantly. It showed that U0126 could effectively enhanced PARP-1 SiRNA mediated cisplatin sensitivity.Conclusions:1.Inhibition of PARP-1 may downregulate the proliferation of SKOV3 cell.2.Inhibition of PARP-1 could enhance the cytotoxicity induced by cisplatin, decrease the cell viability, and decrease the resistance of cisplatin.3.Inhibition of PARP-1 by RNA interference could make SKOV3 cell morphological changes, a higher apoptosis rate, the number of apoptosis increased higher, inhibition of PARP-1 could enhance the effect on promoting apoptosis of SKOV3 cell induced by cisplatin.Every group in the cell cycle distribution was different, after inhibiting the expression of PARP-1, G0/G1 phase cells increased slightly, S phase cells reduced slightly, when NcSiRNA group was treated with cisplatin, the distribution of cell cycle in G0/G1 phase cells increased higher than the NcSiRNA group or the PARP-1 SiRNA group, it showed that only PARP-1 SiRNA was not obvious to the cell cycle distribution. Suppression of PARP-1 after cisplatin treatment with 48 hours, the cell cycle arrested significantly in G0/G1 phase and showed a significant reduction of S phase and G2/M phase cells.5.ERK signaling pathway was detected by western blotThe NcSiRNA and PARP-1 SiRNA group cells were exposed to 10uM cisplatin for 48h, we found that cisplatin inhibited phosphorylation of ERK in SKOV3 cells, however, inhibition of PARP-1 expression by RNAi induced even lower pERK1/2 expression, while the total ERK 1/2 was unaffected. It showed that inhibiting PARP-1 expression could enhance the inhibition of ERK signaling pathway.6. U0126 determine the influence of ERK 1/2 activity on cell viabilityU0126 is a specific ERK inhibitor, first, lOuM U0126 was used to for 2h prior to cisplatin treament, then the SKOV3 cells were exposed to lOuM cisplatin, then we found the cell viability was downregulated significantly. It showed that U0126 could effectively enhanced PARP-1 SiRNA mediated cisplatin sensitivity.Conclusions:1.Inhibition of PARP-1 may downregulate the proliferation of SKOV3 cell.2.Inhibition of PARP-1 could enhance the cytotoxicity induced by cisplatin, decrease the cell viability, and decrease the resistance of cisplatin.3.Inhibition of PARP-1 by RNA interference could make SKOV3 cell morphological changes, a higher apoptosis rate, the number of apoptosis increased higher, inhibition of PARP-1 could enhance the effect on promoting apoptosis of SKOV3 cell induced by cisplatin.Part Ⅲ:Effect of reduced expression of PARP-1 by RNA interference on the sensitivity of cisplatin in vivoObjectives:1. To inhibit the PARP-lgene expression in nude mice by PARP-1SiRNA carried by lentiviral vector, transplanting tumor model of ovarian cancer was established.2. To explore the effect of sensitivity induced by cisplatin via inhibiting PARP-1 gene expression in nude mice.3.To explore the effect of proliferation induced by cisplatin via inhibiting PARP-1 in nude mice.Methods:100ul SKOV3 ovarian cancer cells with a concentration of 1×107/ml each was inoculated in BALB/c(nu/nu) in nude mice subcutaneous side through cell transplantation and successful establishment of ovarian cancer nude mice model was made.20 mice were selected when transplanted tumor was eaqual or more than 5mm in diameter, and were randomly divided into control group(Physiological saline 0.1ml), cisplatin group(2mg/kg), cisplatin(2mg/kg)+lentiviral vector control group(virus content of 1 x 109pfu) and cisplatin (2mg/kg)+PARP-1 lentiviral vector(virus content of 1 x 109pfu),5 rats in each group. Cisplatin or physiological saline by abdominal cavity administration and lentiviral vector by intratumoral injection, once every 72h with total 3 times. The longest path and the shortest path of the tumor were measured every three days after injection, mean tumor volume and weight of each group were calculated, we observed the changes that the mice bearing tumor were treated with cisplatin in combination with suppression of PARP-1. Ki67 protein expressions in tumor tissues from each group were detected by immunohistochemistry, and the proliferation was also observed after the treatment of cisplatin.Results:1.Transplanting tumor model of ovarian cancer was established after cell inoculation with the lentency of 2 weeks, the rude rice was in a good condition. We continuously observed the transplanted tumour shape, tumor growth was relatively uniform early, later tumour shape turned irregular, hard, and boundary fuzzy. After 4 weeks of treatment, the nude rices were killed, we immediately removed the tumour, and made pathological section, we observed the characteristics of morphology by HE staining, the size and the shape were irregular, the nucleus was bigger, interstitial edema and cellular atypia, we confirmed ovarian cancer nude rice tumor model was established.2.1n order to expore the effect of cisplatin and PARP-1SiRNA on the growth of transplanted nude rice, cisplatin or physiological saline was injected by abdominal cavity administration, while the lentiviral vector was injected by intratumoral injection. We found that the growth turned more slowly in PARP-1SiRNA than in NcSiRNA group, so did tumour volume.28days later, tumor volume on average of blank control group, cisplatin chemotherapy group, ciaplatin chemotherapy+ lentiviral vector control group were 576mm3,339mm3,341 mm3; and that of cisplatin chemotherapy+PARP-1 lentiviral vector group was 213mm3, far less than the other groups, the difference was statistically significant(P<0.05). Tumor weight on average of blank control group, cisplatin chemotherapy group, cisplatin chemotherapy +lentiviral vector control group were 1.0g,0.56g,0.57g; and that of cisplatin chemotherapy+PARP-1 lentiviral vector group was 0.21g, far less than the other groups, the difference was statistically significant(P<0.05). The inhibition rate of tumour on average of cisplatin chemotherapy group, cisplatin chemotherapy+lentiviral vector control group were 33.6%,32.3%; and that of cispatin chemotherapy +PARP-1 lentiviral vector group was 59.4%, far more than the other groups, the difference was statistically significant(P<0.05).3.1mmunohistochemistry showed that the mice bearing tumor were treated with cisplatin in combination with vector of PARP-1SiRNA, which significantly decreased the expression of Ki67 protein.Conclusions:Sensitivity of ovarian cancer cell to cisplatin could be significantly improved by RNA interference suppressing the expression of PARP-1 in vivo.