Cell Function Research Of Four Ovarian Cancer Candidate Pathogenic Genes

Background and purpose: Ovarian cancer(OC)is one of the five most common cancers in women and is the number one cancer of the reproductive system that causes women to die.According to statistics,about 20% to 25% of the occurrence and development of ovarian cancer are related to genetic factors.The 5-year survival rate of patients with advanced ovarian cancer is only 25% to 40%.According to the previous whole exome sequencing and sanger sequencing results of the research group,seven point mutations were screened in the TP53,PTEN,CREBBP and NOTCH2 genes in this experiment.The mutations were performed in cell functional experiments to investigate the effects of these 4 genes and 7 point mutations on protein expression,proliferation,migration and invasion of ovarian cancer cells.Methods: The ovarian cancer A2780 cell line without endogenous TP53,CREBBP and NOTCH2 protein expression was selected for cell experiment of the gene,and the ovarian cancer CAOV3 cell line without endogenous PTEN protein expression was selected for cell gene experiment.These genes were divided into wild-type group,mutant group and empty vector group.Construct wild-type plasmids of the TP53,PTEN,CREBBP and NOTCH2 genes,and use the point mutation kit to construct the selected TP53 G199 X,TP53 V157 fs,PTEN H122 D,PTEN Q22 X,CREBBP L1850 fs,CREBBP P1373 S and NOTCH2 P2113 S 7 point mutant plasmids.Liposome transfection was used to transfect into A2780 and CAOV3 cell lines,respectively.(1)Western blotting was used to detect the effect of mutant groups of each genome on protein expression of ovarian cancer cells;(2)CCK-8 experimental method was used.Detect the effects of wild-type and mutant groups of each genome on the proliferation of ovarian cancer cells;(3)Use scratch tests to detect the effects of wild-type and mutant groups of each genome on the migration of ovarian cancer cells;(4)Transwell cell invasion experiments were used to detect the effects of wild-type and mutant groups of each genome on the invasion of ovarian cancer cells;(5)Dual-luciferase assay was used to detect the activity of NOTCH2 wild-type group and P2113 S mutant group on Notch signaling pathway impact.Results:(1)Western blot experiments showed that: PTEN H122 D,CREBBP P1373 S,and NOTCH2 P2113 S mutations did not affect protein expression,while TP53 G199 X,TP53 V157 fs,PTEN Q22 X,and CREBBP L1850 fs mutations could not normally express the protein.(2)Over-expression of wild-type TP53,PTEN or CREBBP can inhibit the proliferation,migration and invasion of ovarian cancer cells,while over-expression of mutant TP53,PTEN or CREBBP can promote the proliferation,migration and invasion of ovarian cancer cells,however,Overexpression of wild-type or mutant NOTCH2 can promote the proliferation,migration and invasion of ovarian cancer cells.(3)The luciferase activity in ovarian cancer cells in the NOTCH2 wild-type group and the P2113 S mutant group was significantly increased compared with the empty vector group,and the promoter activity of CBF1 in ovarian cancer cells in the NOTCH2 P2113 S mutant group was significantly reduced compared with the wild-type group.Conclusion: Through cell experiments,it was shown that none of the missense mutations screened out affected protein expression,while nonsense mutations and frameshift mutations could cause protein deletion.Wild-type TP53,PTEN and CREBBP can inhibit the proliferation,migration,and invasion of ovarian cancer cells,and all mutant types of TP53,PTEN,CREBBP and NOTCH2 and NOTCH2 wild-type can enhance the malignancy of ovarian cancer cells used in this experiment.The NOTCH2 P2113 S mutant only partially activates the Notch signaling pathway in A2780 ovarian cancer cells.