| Ovarian carcinoma is the most lethal gynaecological malignancy in the USA. In2013, an estimated 22 240 new cases were diagnosed and 14 030 women died of thedisease. Ovarian cancer is frequently referred to as the ‘silent killer’, as the majorityof cases are diagnosed at an advanced stage. Improved understanding of the molecularpathogenesis of this disease and effective targeted therapies are needed to improvepatient outcomes. Based on the previous investigations in our laboratory, in this study,using immunohistochemical technique, gene cloning technology, lentivirus packagingtechnology, Western blot analysis, si RNA technology and MTT assay, weinvestigated the effect of protein kinase B(AKT2) regulating the proliferation ofovarian carcinoma cells through PKM2 with the purpose to lay the foundation forfinding the possible target for effectively therapy drug for ovarian cancer.In the first part, we preliminary verified the relationship between the expressionof AKT2 and PKM2 in ovarian cancer by immunohistochemical testing theexpression of AKT2 and PKM2 in 116 cases of ovarian cancer tissues and 50 cases ofnon-cancerous ovarian tissues, and then examining their relationship by statisticalanalysis. The results exhibited that Akt2 was over-expressed in 100 of 116 cases ofovarian tissues(over-expressed rate was 86.2%), whereas, the over-expression ofAkt2 in non-cancerous ovarian tissues was only 4 of 50(8%). The data from Fisher’sexact test exhibited that the statistically significant difference(P<0.001)had beendetected in the expression of Akt2 between ovarian cancer tissues and non-cancerousovarian tissues((P<0.001). The expression of AKT2 and PKM2 exhibited goodcorrelation in ovarian cancer tissues(R=0.505)(P<0.001).Based on the results of the first part, the role of AKT2 in regulation of thebiological behavior of ovarian cancer cells through PKM2 was further investigated inthe second and the third parts of this article.In the second part of this study, AKT2 c DNA was amplied by PCR, which wasthen cloned into lentiviral vector(p LVX-Neo-IRES-Zs Green) by Xba I and Xho Irestriction sites. AKT2 c DNA in this vector was then confirmed by DNA sequencing,and was then transfected into 293 T cells with the mixture of lentiviral packagingplasmid. After successfully obtained AKT2 recombinant lentivirus, this AKT2 recombinant lentivirus was then to be used to infect SKOV3 ovarian cancer cells.Western-blot was performed to identify and screen the AKT2 over-expressed SKOV3 cells. The results showed that the both AKT2 recombinant lentivirus expression vectorand AKT2 over-expressed SKOV3 ovarian cancer cells which were able to highlyefficient expression AKT2 had been successfully obtained, providing foundations forthe third part’s study.In the third part of this study, we focused on investigating the effect of AKT2over-expression on the proliferation of ovarian cancer cells as well as the effect ofknocked-down PKM2 on affection of AKT2 overexpressed SKOV3 cell ′sproliferation. The change of ovarian cancer cell’s proliferation was detected beforeand after knocked-down PKM2 expression in AKT2 over-expressed SKOV3 cells,and the results were then obtained. The result shows that the proliferation of ovariancancer cells in AKT2 over-expressed group was significantly higher than those inempty lentivirus infected group or uninfected ovarian cancer cells(P <0.001), whereas,the latter two groups exhibited no statistically significant difference(P>0.05). Theseresults suggest that AKT2 over-expression was able to increase the proliferation ofSKOV3 ovarian cancer cells, and knocked-down the expression of PKM2 in AKT2over-expressed SKOV3 cells was able to lead to decrease the proliferation of SKOV3 cells. |
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