Effect Of Akt Gene Expression On Proliferation Of Epithelial Ovarian Cancer Cells

Ovarian cancer is the most common malignant tumor in the female reproductive system,and it can develop at any age.Ovarian tissue composition is complex,about 90% of primary malignant tumors are epithelial ovarian cancer.In recent years,the incidence of ovarian cancer has increased year by year,and there has been a trend of younger age.Because its occurrence and development are occult and rapid,70% of the patients have entered the advanced stage and their 5-year survival rate is less than 40%.Advances in surgical techniques and the development of new chemotherapeutic agents and regimens have failed to significantly improve the prognosis of patients with ovarian cancer.Therefore,in-depth study of the molecular mechanisms involved in the development of ovarian cancer and the search for new clinically relevant prognostic markers are beneficial.Early detection of early warning signals and search for more effective treatment strategies to improve the survival and prognosis of patients with ovarian cancer.AKT is a serine/threonine protein kinase,also known as protein kinase B(PKB),which plays a key role in cellular signal transduction through the phosphorylation of multiple transcription factors and thus participates in various life activities of the organism.The PI3K/AKT/mTOR signaling pathway is widely present in various cells and is an important signal transduction pathway involved in cell proliferation,differentiation,and growth.AKT is a downstream effector of PI3 K.After being activated by PI3 K,AKT further activates its downstream factors such as FOXO1,mTOR NF-kB,GSK-3?,etc.,and regulates cell proliferation and apoptosis.It has been reported in related literature that the expression of AKT is closely related to the biological characteristics of gastric cancer,colorectal cancer and breastcancer cell proliferation,invasion,and metastasis.However,the correlation between AKT expression and epithelial ovarian cancer has rarely been reported.In this study,immunohistochemistry,in vitro cell culture,plasmid transfection,and Western blot were applied to analyze the effects of AKT on the proliferation of epithelial ovarian cancer cells in vitro,from cell culture,animal experiments,and clinical studies.In order to provide new data for elucidating the pathogenesis of epithelial ovarian cancer,it provides a new target for effective prevention and treatment of epithelial ovarian cancer.Part 1 Effect of AKT on Proliferation of Ovarian Cancer Cells andDownstream Signal Pathway ProteinsObjective: To investigate the effect of AKT on the proliferation of SKOV3 cells and its mechanism of action at the cellular level.Method: First,different ovarian cancer cell lines A2780,SKOV3,Ovcar3,HO8910,OVCA420,and CaoV3 were cultured.Western blot analysis was used to detect three different subtypes of AKT(AKT1,AKT2,and AKT3)in different ovarian cancer cell lines.Next,SKOV3 ovarian cancer cells were transiently and stably transfected with RNA interference technology to silence the highly expressed AKT subtypes in cancer cells,and further studied AKT by single solution cell proliferation assay and soft agar colony formation assays.Effect on proliferation of epithelial ovarian cancer cells.Finally,the expression of FOXO1,mTOR,NF-?B and GSK-3? protein in downstream signal transduction pathways after AKT3 gene silencing was detected by Western blotting.Results:1.Western Blot analysis was used to determine the expression of three subtypes of Akt in different ovarian cancer cell lines(A2780,SKOV3,Ovcar3,HO8910,OVCA420,CaoV3).The results showed that in different epithelial ovarian cancer cell lines,AKT1 and AKT3.The expression level was higher,while the expression level of AKT2 was lower,and AKT2 was hardly expressed in A2780 and CaoV3 cells.2.After down-regulating Akt1 and Akt3 genes in SKOV3 cells by plasmid transient transfection technology,the corresponding AKT1 in the transfected(siAkt1/si Akt3)cells was compared to the control and siVector groups.And AKT3 protein expression levels were significantly reduced(P<0.05).Plasmid transient transfection effectively silenced the expression of Akt1 and Akt3 genes in SKOV3 cells.3.Stably transfected SKOV3 cells with shRNA lentiviral vectors and silenced the Akt1 and Akt3 genes relative to control and empty vector plasmids(sh Akt1/shAkt3)in the transfection group(shAkt1/shAkt3)Protein levels were significantly lower(P<0.05).The stable transfection of Lentivirus successfully silences the expression of Akt1 and Akt3 genes in SKOV3 cells.4.Akt1 and Akt3 genes were successfully silenced in SKOV3 cells by plasmid transient transfection and lentiviral stably transfected techniques.SKOV3 cells were assayed for proliferative activity by single solution cell proliferation assay.The results showed silencing of Akt3 gene.The cell prolife-ration was significantly inhibited(P<0.05),but there was no significant change in the silent Akt1 gene(P>0.05).5.Using SKOV3 stably transfected technology to effectively silence the Akt1 and Akt3 genes in SKOV3 cells,the proliferation of SKOV3 cells was assayed by single solution cell proliferation assay.The results showed that silencing Akt3 gene can significantly inhibit cell proliferation.(P<0.01),but there was no significant change in silent Akt1 gene(P>0.05).6.Using the lentiviral stably transfected technique to effectively silence the Akt1 and Akt3 genes in SKOV3 cells,colony formation assay was used to measure cell proliferation.The results showed that the silencing Akt3 gene group was strongly inhibited in soft agar colony formation.The cell proliferation of SKOV3 cells was significantly inhibited(P<0.01),while there was no significant change in silent Akt1 gene(P>0.05).7.Effect of Akt3 silencing on FOXO1 expression and phosphorylated mTOR signaling.After the Akt3 gene was stably transfected into the SKOV3 cells by stably transfected cells,the proteins were extracted and analyzed byimmunoblot.The phosphorylation levels of downstream proteins FOXO1,mTOR,NF-?B and GSK-3? in the AKT signaling pathway were significantly reduced(P<0.05),but there was no significant change in the level of dephosphorylation.It was proved that AKT3 may affect the proliferation and growth of epithelial ovarian cancer by regulating the phosphorylation levels of downstream FOXO1,mTOR,NF-?B and GSK-3? proteins.Conclusion:1.AKT1 and AKT3 are highly expressed in different ovarian cancer cell lines,while low or no expression of AKT2.2.Successful transfection of Akt3 in SKOV3 cells with plasmid transient transfection and stably transfected with lentiviral virus can significantly inhibit SKOV3 cell proliferation.3.Akt3 gene may affect the proliferation and growth of epithelial ovarian cancer by regulating the phosphorylation of downstream FOXO1,mTOR,NF-?B and GSK-3? proteins.Part 2 Effect of Silencing Akt Gene on the Growth of SKOV3 Cells inNude MiceObjective: To further investigate the effects of silencing of Akt1 and Akt3 genes on the growth of transplanted tumors of SKOV3 cells in nude mice by animal experiments.Method: Ninety BALB/c-nude female nude mice with the same size and health status were randomly divided into groups(15 in each group).The group that silenced Akt1 gene was divided into blank control group(Vehicle group)and blank plasmid group(shVector group)and Akt1 gene silencing group(shAkt1 group);Akt3 gene silencing group: blank control group(Vehicle group),blank plasmid group(shVector group)and Akt3 gene silencing group(shAkt3 group).SKOV3 cells(Vehicle group),SKOV3 cells transfected with empty plasmids(shVector group),and SKOV3 cells(ShAkt1 group/shAkt3group)that silence the Akt1/Akt3 gene were injected subcutaneously.The volume of the transplanted tumor and the body weight change of the nudemice were monitored every 5 days.After 25 days,the mice were anesthetized by ether inhalation and the mice were killed by cervical dislocation.The tumors were completely detached and the tumor volume and weight were measured.Results:Nude mice weightThere was no statistical difference in body weight of nude mice measured at 5,10,15 and 20 days.The weights of nude mice in the 25-day Vehicle group,shVector group,and shAkt1 group were: 24.1±1.4 g,24.3±2.0 g,and22.3±1.5 g,respectively.The weight of nude mice in Vehicle group,shVector group and shAkt3 group were: 25.2±1.3g,25.7±2.0g and 24.7±1.2g,respectively.There was still no statistical difference between the two groups(P>0.05).2.Tumor volume in nude miceWith the advancement of the experimental process,the tumor volume of each group of nude mice gradually increased.On days 5,10,15,and 20,the volume of transplanted tumors in nude mice was not significantly different(P>0.05).After 25 days of feeding,the volume of tumors in Vehicle group,shVector group,and shAkt1 group were 593.15±53.46mm3,552.23±67.12mm3,and 463.18±43.3mm3,respectively.There was no significant change in tumor volume between the three groups(P> 0.05).The tumor volume in the25-day intact dissection group was 583.34±103.46mm3,531.45±117.1mm3,and 289.20±67.12mm3 in the Vehicle,shVector,and shAkt3 groups,respectively.Compared with the Vehicle group and the shVector group,the tumor volume in the shAkt3 group was significantly reduced(P<0.01).3.Tumor weight in nude miceAfter 25 days,nude mice were sacrificed by deodorization,and subcutaneous tumors were excised and weighed.The results showed that tumor weights in the Vehicle group,shVector group,and shAkt1 group were:1.8±0.4g,1.7±0.5g,and 1.6±0.2g,respectively.There was no statistical difference between the three groups(P>0.05).The tumor weights in Vehiclegroup,shVector group,and shAkt3 group were: 2.2±0.3g,1.8±0.4g,and0.4±0.3g,respectively.The tumor weight of shAkt3 group was significantly lower than that of the other two groups,with statistical significance(P<0.01).Conclusion:1.After silencing Akt3 gene,the transplanted tumor volume of SKOV3 cells in nude mice was significantly reduced and the weight was significantly reduced.2.Silencing Akt1 gene had no significant effect on the growth of transplanted tumor in nude mice.Part 3 Akt3 expression in human ovarian epithelial cancer tissueexpression and clinical significanceObjective: To detect the expression of AKT3 protein in epithelial ovarian cancer tissues,analyze the relationship between the expression of Akt3 protein and clinicopathological features of patients with ovarian epithelial cancer,and the influence on the prognosis of patients.Method: A total of 75 cases of ovarian epithelial carcinoma from May2003 to May 2011 in Shijiazhuang Maternal and Child Health Hospital and People's Hospital of Shijiazhuang were selected for immunohistochemical staining to detect AKT3 protein in epithelial ovarian cancer expression.Patients were collected for complete clinical information,including patient's age,stage,pathological type,cell grade,survival,etc.,and statistical analysis.Results:1.In 75 cases of epithelial ovarian cancer,46 cases showed positive expression of AKT3,accounting for 61.3% of the total sample.2.The positive rate of AKT3 in stage III-IV patients was significantly higher than that in stage I-II(P<0.01).The positive expression rate of AKT3 in high-grade cancer patients was significantly higher than that in low-grade cancer patients(P<0.01).There was no significant effect on the expression of AKT3 in different ages,pathological types,lymph node metastasis,and ascites(P>0.05).Using Kaplan-Meier(Log Rank test)analysis,the average survival time of Akt3 negative ovarian cancer patients was 42.4 months,significantly higher than the average survival time of AKT3 positive expression patients was 21.6months(P<0.05).Conclusions:1.The expression of AKT3 is closely related to clinical stage and tissue differentiation.The higher the clinical stage,the lower the degree of tissue differentiation and the higher the expression of Akt3,and vice versa.2.AKT3 expression was negatively correlated with the survival and prognosis of patients with ovarian cancer.The survival time of patients with Akt3 positive expression was shortened and the prognosis was poor.