Mechanism Of ARHGAP30 Regulating Epithelial Ovarian Cancer Through PI3K/AKT/mTOR Signaling Pathway

Background and Objective:Ovarian cancer is a gynecological malignancy that is difficult to detect early and has a very low 5-year survival rate.The 5-year survival rate of patients is about 47%because there is no effective screening method in the early stage,mainly due to incomplete initial surgery and resistance to platinum-based chemotherapy in the later stage.ARHGAP30 is a member of the Rho GTPase activating protein family,which controls the switches of many signal transduction pathways in eukaryotic cells and produces a variety of biological effects.Recent studies have found that Rho A and PI3 K in Rho GTPase are jointly involved in the angioproliferative response of endothelial cells and form pathological vascular remodeling.Recent studies have shown that ARHGAP30 is involved in tumor progression.It is known that in about 70% of EOC,the PI3K/AKT/m TOR pathway is upregulated,leading to overactivation of signaling pathways related to angiogenesis,cell survival and metabolism.GEPIA(Gene Expression Profiling Interactive Analysis)reminds that ARHGAP30 is abnormally expressed in ovarian cancer and expresses high in ovarian cancer.However,is there any difference in the expression of ARHGAP30 in EOC,and is there a close relationship between the expression of ARHGAP30 and EOC,EOC and PI3K/AKT/m TOR pathway,and ARHGAP30,EOC,PI3K/AKT/m TOR pathway? Whether these affect tumor invasion,proliferation,and invasion is unclear and requires further study.This experiment aims to study the mechanism of ARHGAP30 on human epithelial ovarian cancer,explore the specific mechanism of ARHGAP30 regulating PI3K/AKT/m TOR signaling pathway,and clarify the mutual regulation of ARHGAP30 and PI3K/AKT/m TOR signaling pathway in EOC,Provide new treatment ideas and directions for EOC treatment.Method:1.Detection of ARHGAP30 m RNA and protein expression level in 81 EOC tissues and their normal ovarian tissues by q RT-PCR and immunohistochemistry;2.q RT-PCR was used to detect the expression of ARHGAP30 and m RNA in normal ovarian cells(IOSE80)and epithelial ovarian cancer cells(SKOV3,OVCAR3,and A2780);ARHGAP30-specific si RNA was designed,divided into NC group,and transfected into SKOV3,OVCAR3 cells groups,RT-PCR detection of its transfection efficiency;3.After knocking down ARHGAP30 in SKOV3 and OVCAR3 cells,the proliferation ability of si ARHGAP30 cells was detected by Ed U method;the apoptosis of si ARHGAP30 cells was detected by flow cytometry;the invasion and migration ability of si ARHGAP30 cells was observed by Transwell;4.After knocking down the expression of ARHGAP30 in SKOV3 and OVCAR3 cells,and after treating SKOV3 and OVCAR3 cells with Buparlisib,Changes in the expression levels of key proteins in the PI3K/AKT/m TOR signaling pathway(p-PI3 K,p-AKT,p-m TOR);5.Western blot was used to detect the expressions of cell metastasis,proliferation and apoptosis markers after knockdown of ARHGAP30 and pathway inhibitor Buparlisib in SKOV3 and OVCAR3 cells;6.After knocking down the expression of ARHGAP30 in EOC cells(SKOV3and OVCAR3)in vivo validation(animal experiments),the expression of tumor appearance,tumor volume and tumor weight in nude mice.Result:1.The abundance of ARHGAP30 m RNA and protein in EOC tissue was significantly higher than that in normal ovarian tissue(P <0.01);2.The m RNA level of ARHGAP30 in EOC cells(SKOV3,OVCAR3,A2780)was significantly higher than that in human normal ovarian epithelial cells(IOSE80);After SKOV3 and OVCAR3 cells were transfected with si ARHGAP30 to interfere with ARHGAP30 expression,ARHGAP30 m RNA levels were significantly knocked down;3.After knocking down the expression of ARHGAP30,in SKOV3 and OVCAR3 cells,the cell invasion,proliferation,and migration abilities were significantly decreased,and cell apoptosis was significantly increased;4.Knockdown of ARHGAP30 expression and pathway inhibitor Buparlisib can reduce the expression of key proteins in the PI3K/AKT/m TOR signaling pathway after treatment of SKOV3 and OVCAR3 cells,and Buparlisib shows the same effect as ARHGAP30 knockdown;5.Knockdown of ARHGAP30 expression and pathway inhibitor Buparlisib can reduce the expression of cell proliferation(ki67),metastasis(MMP9)and apoptosis(Bax)markers after treatment of SKOV3 and OVCAR3 cells,Buparlisib showed the same effects as ARHGAP30 knockdown Effects;6.After knocking down the expression of ARHGAP30 in epithelial ovarian cancer cells(SKOV3 and OVCAR3),the expression of tumor appearance,tumor volume,and tumor weight were significantly decreased(in vivo verification).Conclusion:1.The expression of ARHGAP30 was significantly increased in EOC tissues and cells;2.After knocking down the ARHGAP30,in SKOV3 and OVCAR3 cells,the cells abilities of invasion,migration,proliferation were significantly reduced,and cells apoptosis were significantly increased;and this conclusion has been further confirmed by nude mouse animal experiments(in vivo);3.After knocking down ARHGAP30 in EOC cells,it can significantly inhibit cells invasion,migration and proliferation and induce cells apoptosis by regulating PI3K/AKT/m TOR signaling pathway.This result provide new insight into the mechanism of proliferation,invasion and potential malignant potential in EOC,and provide new idea and direction for the treatment of EOC.