To Determine The Expression Of AKT3in Breast Cancer Tissue And To Investigate The Effect Of AKT3on The Biological Behaviors Of Breast Cancer Cells

Breast cancer is one of the most common malignancies in women worldwide，and is also one of main dead causes of women. Based on a large number of literatureand the previous work of our laboratory, in this study, immunohistochemistry,adenovirus AKT3expression vector transduction, Western blot and DNA ladderassays were applied to determine the expression of AKT3in breast cancer tissue andto investigate the effect of AKT3on the biological behaviors of breast cancer cellswith the purpose to provide a foundation for understanding the potential mechanismof AKT3’s action and discovering the possible therapeutic target of drug.In the first part of this study, the expression of AKT3in80cases of invasivebreast ductal cancer tissue (IBDC) was determined by immunohistochemistry, and therelationship between AKT3expression and the clinical pathological characteristic ofpatients with invasive breast ductal cancer was then analyzed. The results showed thatafter de-paraffin tissues incubated with anti-AKT3antibody, positive samples stainedin cytoplasm of the IBDC cells were detected. AKT3was over-expression in57of80cases (71.20%) IBDC tissue compared to levels in non-neoplastic tissues P<0.01(Wilcoxon Signed Ranks Test). Over-expression of AKT3was not correlated withpatients’ age, nor with tumor size neither, but closely related with TNM stage andlymph node metastasis. Therefore, the expression of AKT3was closely related withpoorer prognosis in IBDC.Based on the first part results of this study, the second and the third parts of thisstudy focused on investigation the roles of AKT3on the biological behaviors ofIBDC cells.In the second part of this study, RT-PCR was used to amply AKT3cDNAobtained by first reverse transcript total RNA from aborted fetal brain tissue thenPCR. AKT3cDNA was inserted into adenovirus shuttle vector,pShuttle-IRES-hrGFP-1, by XhoI and the Bgl II restriction sites. The recombinantshuttle vectors were confirmed by DNA sequencing, then transformed intoEscherichia coli BJ5183carrying backbone plasmid PAdeasy-1to obtain adenovirus plasmid through homologous recombination. The adenovirus plasmid wastransfected into293A cells to form adenovirus particle. Followed, the adenovirusparticles were transduced into IBDC BT474cells. Western blot was carried out toanalyze the expressed effect of AKT3adenovirus expression vectors in BT474cellsand to obtain the best cells that had the highest AKT3expression. The resultsshowed that the AKT3adenovirus expression vector were successfully constructedand western blot confirmed that AKT3had a good expression in BT474cells,providing foundations for the third part’s study.In the third part of this study, the activity of cell proliferation before and aftertransfected with AKT3gene was detected by MTT assay. The apoptostic change ofBT474post-AKT3gene transfection was detected by DNA ladder, and thetumorigenicity of the BT474cells before and after transfected with AKT3gene wastested by the colony formation in soft agar assay. The results showed that theproliferation activity, and the tumorigenicity of BT474cells were increased and cellapoptosis was decreased after the cells had been transfected with AKT3gene. Theanalystical results of statistics indicated the discrepancy was significant betweenBT474cells transduced with pAd-AKT3vector and pAd-GFP empty vector (P＜0.05).The preliminary results indicate AKT3might cause the changes of cell proliferation,apoptosis and tumorigenicity, and the molecular mechanism of its action is stillunclear, which needs to be further investigated.