Construction Of Gene-modified Ovarian Cancer Cells And Effect Of PTK2 On Cell Biological Behavior Of Ovarian Cancer

IntroductionOvarian cancer（OC）is one of the most common malignancies of the female reproductive system,with a morbidity of 9.1/100,000 in developed countries and5.0/100,000 in developing countries worldwide.The mortality rate of ovarian cancer ranks the first place among gynaecological malignancies.According to the American cancer society,there were about 290,000 new cases and 180,000 deaths of ovarian cancer worldwide in 2018.Ovarian epithelial cancer（EOC）accounts for the vast majority of OC,and OC patients have a long five-year survival rate when diagnosed early.Although ovarian cancer can be treated with surgery combined with chemotherapy drugs,the five-year survival rate hovers around 30 percent due to frequent recurrence and metastasis.Tumor initiation and progression are closely related to tumor suppressor/oncogene genes.In recent years,molecular targeted therapy has developed rapidly.The biological therapy model represented by molecular targeting treatment can regulate tumor progression at the molecular level,bringing hope for improving the prognosis of ovarian cancer patients.Therefore,it is urgent to explore the molecular mechanism related to the occurrence and metastasis of ovarian cancer,explore the potential targets of molecular targeted therapy for ovarian cancer,and improve the early diagnosis rate of ovarian cancer patients.The tumor suppressor gene p53 is located at 17p13.1,which is a gene with the highest mutation rate in human cancer cells.It has been reported that p53 is mutated in more than 50%of poorly differentiated epithelial ovarian cancer.Overexpression of oncogene c-Myc can lead to cell proliferation,differentiation and malignant transformation.It has been reported that c-Myc amplification exists in about 30%of ovarian tumors.Protein tyrosine kinase（PTK2）,also known as focal adhesion kinase（FAK）,is located at chromosome 8q24.3.PTK2 is up-regulated in a variety of solid tumors in humans and plays a role in promoting cancer.It has been found that PTK2overexpression is highly correlated with p53 mutation in human breast cancer.PTK2N-terminal domain and p53 N-terminal domain can be interacted directly.PTK2 and c-Myc regulate tumor cell invasion,and inhibition of integrin/PTK2 signaling axis and c-Myc synergistically regulate malignant biological behaviors of ovarian cancer.Interestingly,the data in the TCGA showed that in a group of ovarian serous carcinoma patients,three genes including PTK2,c-Myc and p53 change at the same time.Therefore,we hypothesized that the simultaneous overexpression of oncogene c-Myc and PTK2 in mouse ovarian epithelial cells with defective p53 function(p53^-/-)(PTK2+c-Myc+p53^-/-)could induce the transformation of ovarian epithelial cells to form ovarian cancer cells.Firstly,modified genes were transfected into mouse ovarian epithelial cells using lentivirus vector system,in a single or combination way,the cells with potential tumorigenic ability was planted in the ovarian tissue in mice,ovarian epithelial tumor in mice model was established,and the effect in tumor formation and metastasis were further observed.Molecular targeted therapy for ovarian cancer has attracted extensive attention and research in recent years.Based on the above studies in this subject,PTK2 plays an important role on cell biological behavior of ovarian cancer.Therefore,the study of PTK2 as the treatment target will guide us to better understand the process of the occurrence and development of ovarian cancer,and bring new ideas for molecular targeted therapy of ovarian cancer.GSK2256098 is a small molecule inhibitor that targets PTK2 kinase activity and Y397 phosphorylation.We knocked out PTK2 using CRISPR/Cas9 system,and chose GSK2256098 to block PTK2 Y397 phosphorylation to evaluate for the first time the effect and possible mechanism of PTK2 on cell biological behaviors of ovarian cancer cells.We are expected to provide research basis and theoretical support for molecular targeting and molecular mechanism research of ovarian cancer.This topic is divided into three parts.Part Ⅰ:Construction of gene modified mouse ovarian cancer cell lineObjectiveThis part aims to construct p53^-/-+c-Myc+PTK2 cell line,and establish epithelial ovarian cancer mouse model.Materials and Methods1.The material1.1 Cell line:C57BL/6 mouse epithelial ovarian cells.1.2 Plasmid:p53 gene knockout plasmid,c-Myc gene overexpression plasmid and PTK2 gene overexpression plasmid.1.3 Animals:4-week NSG immunodeficient female mice.2.Methods2.1 The plasmids were transformed and extracted.2.2 The lentiviral vector system was used to package and transfer p53 gene knockout vector,PTK2 and c-Myc gene overexpression vector into normal mouse ovarian epithelial cells individually or in combination.2.3 Stable transfected cell lines and groups were establishedAccording to plasmids transfection,this project was divided into 8 groups:p53^-/- transfection group（Control group）.Western blot was used to detect p53,c-Myc and PTK2 protein levels in 8 groups of cells.2.4 MTT method,monolayer cell clonogenesis test and soft agar cell clonogenesis test were used to detect the proliferation and colony formation ability of ovarian epithelial cells in 8 groups of genetically modified mice.Transwell migration and invasion assays were used to detect the invasion and migration ability of 8 groups of genetically modified cells.2.5 The gene-modified cell lines with potential tumor-generating capacity were selected,and the cells were implanted into ovarian tissue of immunodeficient NSG mice,and the tumor-forming effect and metastasis were observed.2.6 SPSS 22.0 was used for statistical analysis,the results were expressed as（x±s）,and the normality test was conducted,and 0.05 was taken as the test level.Results1.Expression of PTK2,c-Myc and p53 proteins in mouse ovarian epithelial cellsCompared with the control group（0.06±0.02）,PTK2 protein expression increased in PTK2 group（1.22±0.11）,PTK2+c-Myc group（1.11±0.11）,p53^-/-+PTK2group（0.86±0.09）,and p53^-/-+c-Myc+PTK2 group（1.34±0.16）,the differences were statistically significant（P<0.001）.Compared with the control group（0.08±0.02）,c-Myc protein expression levels were significantly increased in c-Myc group（0.56±0.04）,PTK2+c-Myc group（1.01±0.12）,p53^-/-+c-Myc group（0.76±0.09）,and p53^-/-+c-Myc+PTK2 group（1.23±0.15）（P<0.001）.Compared with the control group group（0.04±0.01）decreased,the differences were statistically significant（P<0.001）.2.Proliferation of gene modified mouse epithelial ovarian cellsThe results of MTT experiment showed that,compared with the control group and other 6 groups of genetically modified cells,the proliferation capacity of cells in the p53^-/-+c-Myc+PTK2 group was significantly increased（P<0.01）.Compared with group and PTK2+c-Myc group increased（P<0.01）.Compared with the control group,the proliferation ability of p53^-/-,c-Myc and PTK2 groups showed no significant difference during the whole test period（P>0.05）.3.Colony formation of gene modified mouse epithelial ovarian cellsThe results of monolayer colony formation experiment showed that the number of colony in p53^-/-+c-Myc+PTK2 group（93.67±6.11）was higher than control group（4.33±1.53）and other 6 groups（P<0.001）.The results of soft agar cell colony group（82.67±6.81）was higher than that in control group（6.67±1.53）and the other 6groups（P<0.001）.4.Results of cell migration and invasion of gene modified mouse epithelial ovarian cellsThe results of cell migration experiments showed that the number of cell migration in the p53^-/-+c-Myc+PTK2 group（85.33±5.03）was higher than that in the control group（16.00±1.00）and the other 6 groups（P<0.001）.The results of cell invasion experiment showed that the number of cell invasion in p53^-/-+c-Myc+PTK2group（60.30±6.11）was greatly higher than control group（11.00±1.00）and the other c-Myc group（21.00±2.65）were different from the control group（11.00±1.00）（P<0.05）.There were no significant differences of cell invasion among PTK2 group（12.33±0.58）,c-Myc group（14.00±1.00）and control group（11.00±1.00）（P>0.05）.5.Objective cell line selectedBased on the above results,cell proliferation,colony formation,migration and invasion ability in the p53^-/-+c-Myc+PTK2 group was significantly higher than the other groups,although monolayer cell colony formation,invasion and migration group,but there were no statistical difference of soft agar colony formation ability we speculated that the p53^-/-+c-Myc+PTK2 group cells have potential tumorigenic mouse model construction.6.Luciferase gene marked mouse ovarian epithelial cellsThe luciferase gene was separately transfected into normal mice epithelial cells were greater than 10⁶,indicating fluorescent gene had been integrated into the cellular DNA in mice,and can be used for subsequent transplantation tumor model.7.Tumor formation of p53^-/-+c-Myc+PTK2 cells in NSG mice p53^-/-+c-Myc+PTK2 cells were injected into the ovarian tissue of NSG mice.After 12 weeks,tumor formation in ovarian tissue of mice in the p53^-/-+c-Myc+PTK2 metastasis,including liver,spleen and intestine.ConclusionsIn this part,the p53^-/-+c-Myc+PTK2 cell line was successfully constructed,and the p53^-/-+c-Myc+PTK2 cell line was used to successfully establish a mouse ovarian cancer model.This cell line is expected to be used to study the molecular mechanism of the occurrence and development of ovarian cancer.Part Ⅱ: Role of PTK2 in the proliferation,migration and invasion of ovarian cancer cellsObjective To investigate the role of PTK2 in cell proliferation,migration and invasion of ovarian cancer cells.The materials and Methods 1.The material 1.1 Cell lines: SKOV3,OVCAR8 cells were used in this study.1.2 Plasmid: PTK2 gene knockout plasmid.2.Methods 2.1 Statistical analysis of PTK2 expression in ovarian cancer tissues and its relationship with prognosis.The expression of PTK2 in ovarian cancer and adjacent tissues was detected by immunofluorescence.2.2 lentiviral vector was used to package PTK2 gene knockout plasmid.The PTK2 gene knockout plasmid transfected cell line was established.At the same time,small molecule inhibitor GSK2256098 was used to inhibit PTK2 key activation site（p-FAK）Y397 for targeted PTK2 activation,and the control group was DMSO control.Western blot was used to detect the expressions of PTK2 and p-FAK in human ovarian cancer cell lines.2.3 MTT and cell colony experiments were used to detect changes in cell proliferation and colony formation,transwell migration/invasion experiment and cell wound healing experiment were used to detect the changes of cell invasion and migration ability after PTK2 gene was knocked out and PTK2 activation was inhibited by GSK2256098.2.4 The statistical method is the same as the part I.Results 1.High level of PTK2 expression in ovarian cancer tissues is associated with the prognosis of ovarian cancer patients PTK2 was strongly stained in the cytoplasm of tumor cells,and was highly expressed in ovarian cancer tissues compared with adjacent tissues.2.PTK2 expression was decreased in SKOV3 and OVCAR8 cells after PTK2 knockout or inhibited by GSK2256098 After PTK2 was knocked out,PTK2 expression could not be detected in SKOV3 and OVCAR8 cell lines.The inhibitory effect of GSK2256098 on（p-FAK）Y397 in ovarian cancer cells was significant when the concentration of GSK2256098 was 40μmol/L（P<0.001）.3.PTK2 knockout or inhibited by GSK2256098 reduced proliferation of ovarian cancer cells MTT results showed that the proliferation ability of SKOV3 and OVCAR8 cells after PTK2 knockout was lower than control group（P<0.05）,and the proliferation ability of cells after PTK2 activity was inhibited by GSK2256098 was lower than DMSO group（P<0.05）.4.PTK2 knockout or inhibited by GSK2256098 reduced colony formation of ovarian cancer cells The results of monolayer cell clone formation experiment showed that the number of cell colony in the PTK2 knockout group was greatly lower than control group（P<0.05）,and the number of cell clones in GSK2256098 group was lower than DMSO group（P<0.05）.5.Knockout of PTK2 or inhibition of PTK2 activity with GSK2256098 reduced the migration and invasion ability of ovarian cancer cells The migration and invasion cell numbers of SKOV3 and OVCAR8 cells after PTK2 knockout was greatly lower than control group（P<0.05）,and the migration and invasion number of cells after PTK2 activity was inhibited by GSK2256098 was lower than DMSO group（P<0.05）.Conclusions PTK2 is an oncogene in ovarian cancer.PTK2 knockout or targeted inhibition reduces the ability of ovarian cancer cells to proliferate,colone,invade and migrate.Part Ⅲ: The role of PTK2 in tumor formation and metastasis of ovarian cancer cells in miceObjective To investigate the role of PTK2 in tumor formation and metastasis of ovarian cancer cells in mice.The material and Methods 1.The material Animals: 4-week-old immunodeficient female NSG mice were selected.2.Methods 2.1 PTK2 gene knockout and control SKOV3 cells were implanted in NSG mice to build human ovarian cancer mice model.2.2 The human OVCAR8 ovarian cancer cells were implanted in 10 mice ovarian tissues.One group was treated with GSK2256098（Intragastric administration,75 mg/kg/d,treatment 5 days a week,a total of 4 weeks）,using DMSO in control group.2.3 The statistical method is the same as the part I.Results 1.PTK2 knockout reduced ovarian tumor growth and metastasis in mice The in vivo imaging system was used weekly to monitor tumor growth and metastasis.The results showed that the maximum fluorescence intensity of average tumor cells after 4 weeks was significantly lower in the knockout PTK2 group than in the knockout control group（P<0.001）.Tumor tissue metastasis was found in liver,spleen,intestine of the control group,while no tumor tissue was found in the PTK2 knockout group.2.Inhibition of PTK2 activity reduced ovarian tumor growth and metastasis in mice The mean maximum fluorescence intensity of tumor tissues in the GSK2256098 treatment group was significantly lower than control group（P<0.001）.The weight of ovarian cancer tissue in mice in the GSK2256098 treatment group was lower than control group（P < 0.001）.After targeted inhibition of PTK2 activity with GSK2256098,no metastasis was found in the mice,while metastatic tumors in the liver,spleen and intestine were found in the control group.Conclusions PTK2 promoted the formation and metastasis of ovarian cancer in mice,and PTK2 knockout or targeted inhibition reduced the formation and metastasis of ovarian cancer in mice.