| Objectives and significance:Ovarian cancer has the highest mortality rate of all gynecological tumors.The etiology of ovarian cancer is complex,genetic factors play a major role in its development and progression,involving the inactivation of tumor suppressor genes and the activation of oncogenes.Due to the lack of appropriate ovarian tumor model,the study of the mechanism of ovarian cancer is limited.p53 gene mutation and functional loss are one of the most common genetic abnormalities in ovarian cancer.C-Myc gene has a high detection rate in ovarian cancer.Previous studies have found that ASAP1 gene plays an important role in the development of ovarian cancer.In this study,p53 gene of the primary ovarian epithelial cell was knocked out by CRISPR/Cas9 system.p53-/-、c-Myc and ASAP1were transduced into primary mouse ovarian epithelial cells individully and in combination by lentiviral vector system to determine their roles in tumorigenesis of ovarian cancer.Cells with tumorigenic potential were injected into NSG mouse subcutaneously to observe the tumor formation.To further study the role of ASAP1 gene in human ovarian cancer,ASAP1overexpression vector was transfected into SKOV3 and OVCAR3 cell lines to analyze its function on ovarian cancer.This research has developed a mouse ovarian epithelial cell model for recapitulating human ovarian cancer development and progression.The model was designed to transfer three genes to the mouse ovarian epithelial cell,individually and in combination.We have demonstrated that combinations of genetic lesions p53-/-、c-Myc and ASAP1 can induce ovarian tumor in mouse ovarian epithelial cells.These three genes are commonly present in human ovarian carcer.The model can simulate the occurrence and development of ovarian cancer,will be useful for studying the initiation and progression of ovarian cancer,identifying ovarian tumor markers,and establishing parameters for distinguishing a variety of biological behaviors of ovarian cancer to different therapeutic methods that target specific molecular pathways change in ovarian cancer.Our data indicate that ASAP1 plays as an oncogenic role in ovarian cancer cells and may contribute to tumor metastasis and chemoresistance,thus provide a novel target for ovarian cancer therapy.Material and methods:p53 knockout vector was constructed using CRISPR/Cas9system,lentiviralCRISPRv2-p53 vector.C-Myc overexpression vector pLenti-Myc and ASAP1 overexpression vector pLenti-ASAP1 and lentiviralCRISPRv2-p53 were packaged with lentiviral vector system.p53-/-、c-Myc and ASAP1 was transduced into primary mouse ovarian epithelial cells individully and in combination to study the biological characteristics of transgenic mouse epithelial cells:the protein expression were detected by western blot,MTT assay was used to detect the growth and proliferation ability of transgenic cells,the cell cycle was detected by flow cytometry,and the cell colony formation ability of transgenic cells was analyzed by single layer culture and soft agar.Mouse ovarian epithelial cells transduced with p53-/-+Myc+ASAP1 gene were injected into NSG mouse subcutaneously to detect the tumor formation.Lentiviral vector mediated ASAP1 over-expressed in ovarian cancer SKOV3and OVCAR3.Cell migration,invasion,proliferation,were examined using transwell,matrigel assays and MTT,respectively.The expression of marker proteins involved in epithelial to mesenchymal transition（EMT）were also detected.Colony formation was tested through cells in monolayer culture and anchorage-independent growth in soft agar.The cells apoptosis was also determined by chemotherapy drug paclitaxel inducing.Results:（1）The p53 gene of mouse ovarian epithelial cells was knocked out by CRISPR/Cas9system.ASAP1 and c-Myc genes can be transducted into mouse ovarian epithelial cells efficiently using the lentiviral vector system.Target genes DNA can be integrated into the genome of host cells.（2）The cell proliferation assay showed that p53-/-group was significantly higher than that of the control group（P<0.05）;p53-/-+Myc and ASAP1+p53-/-groups were significantly higher than that of the control group（P<0.05）;ASAP1 and c-Myc groups were not significantly different from that of control group（P>0.05）;ASAP1+Myc group was significantly higher than that of the control、ASAP1、c-Myc and control groups（P<0.05）;the proliferation rate of p53-/-+Myc+ASAP1 cells was significantly higher than that of other groups（P<0.05）,especially in the late time of detection.（3）The cell cycel detection showed that the G1 phase ratio of p53-/-、p53-/-+Myc、p53-/-+ASAP1、Myc and ASAP1 groups was significantly lower than the control group（P<0.05）,the S phase ratio of p53-/-+Myc、Myc and ASAP1 groups was significantly higher than that of control group（P<0.05）;the G1 phase proportion of p53-/-+Myc+ASAP1 group was significantly lower than other groups（P<0.05）,and the S phase proportion was significantly higher than the other groups（P<0.05）.（4）Soft agar showed that the cell colony formation ability of p53-/-was significantly higher than that of the control group（P<0.05）.There was no significant difference in the colony formation ability between p53-/-+Myc、p53-/-+ASAP1、ASAP1、c-Myc and ASAP1+Myc groups and the control group（P>0.05）.The colony formation ability of p53-/-+Myc+ASAP1 cell line was significantly higher than that of other groups（P<0.05）.（5）In monolayer culture,the number of cell colonies in p53-/-、myc and ASAP1groups were significantly more than that of the control group（P<0.05）.The number of cell colonies in ASAP1+Myc group was significantly higher than that of the control group（P<0.05）,while the number of cell colonies in p53-/-+myc and p53-/-+ASAP1 groups were significantly lower than that of the control group（P<0.05）.The colony formation ability of p53-/-+Myc+ASAP1 cell line was significantly higher than that of other groups（P<0.05）.（6）Xenograft experiment showed that p53-/-+Myc+ASAP1 cell lines can form tumor in NSG mouse.The expression of myc and ASAP1 increased and the expression of p53decreased in tumor tissues and cells isolated from tumor tissue in NSG mice.（7）ASAP1 overexpression dramatically increased cell migration and invasion of SKOV3 and OVCAR3 ovarian cancer cells than the control group（P<0.0001）,the proliferation and colony formation ability were significantlyenhanced（P<0.05）.The expression of E-cadherin protein in ASAP1 overexpressing cells was significantly lower than that in the control group（P<0.05）,the expression of N-cadherin and vimentin protein was significantly higher than that of the control group（P<0.05）.In addition,apoptosis data showed that overexpression of ASAP1 decreased the sensitivity of ovarian cancer cells to paclitaxel-induced apoptosis（P<0.05）.Conclusion:（1）Tumor suppressor gene p53 knockout in mouse ovarian epithelial cells resulted in the disorder of cell cycle,cell proliferation,colony formation ability enhancement;under p53knockout background,over expression of c-Myc gene has no effect on cell function,while overexpression of ASAP1 promoted cell proliferation and cell cycle disorder,but did not change the cell contact inhibition effect.（2）Overexpressing c-Myc or ASAP1 gene in mouse ovarian epithelial cells induced cell cycle disorder,increased monolayer colony formation ability,but had no effect on cell proliferation and transformation;while c-Myc gene and ASAP1 gene overexpression together did not have cumulative effect on cell function.（3）Knockout p53 gene and overexpression c-Myc gene and ASAP1 gene,induced primary mouse ovarian epithelial cells transformation,with tumor cells characteristics:cell cycle disorder,cell proliferation out of control,contact inhibition effect lost,growed well in suspended state.p53-/-+Myc+ASAP1 transgenic mouse ovarian epithelial cells could form tumor in NSG mice.（4）ASAP1 gene acted as“driver genes”in ovarian tumor,lentiviral vector mediated ASAP1 expression promoted cell migration and invasionin in ovarian cancer cells.In addition,ASAP1 promoted cell proliferation,survival and inhibited chemotherapy drug paclitaxel induced cell apoptosis.Moreover,ASAP1 expression promoted epithelial to mesenchymal transition（EMT）by upregulating the mesenchymal cell markers N-cadherin and vimentin,while downregulating epithelial cell marker E-cadherin in both ovarian cancer cell lines. |
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