| ?Backgroud and aim?Ovarian cancer is one of the three major malignant tumors of the female reproductive system.However,the incidence rate accounts for 20% to 30% of gynecologic malignancies,the mortality rate is highest among gynecological tumors.There are many signaling pathways involved in the development of ovarian cancer,and the most important one is the phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)signaling pathway.AKT is at the center of this pathway.It can be activated by multiple pathways,and can also regulate downstream related molecules.It plays an important role in multiple pathways associated with malignancy.AKT molecules are divided into 3 subtypes,AKT1/2/3.Among them,AKT2 is most closely related to invasion,metastasis and survival of cancer cells.Pyruvate kinase M2(PKM2)is often overexpressed in cancer cells,leading to increase glucose uptake,accumulate the glycolytic metabolites,and reprogram the cellular metabolism,promoting cancer cell growth and survival.In addition to its function of regulating tumor metabolism,PKM2 can also interact with intracellular molecules as a protein kinase,transcriptional coactivator,etc.,regulate the transcription of genes,and is closely related to the role of cancer cells in resisting apoptosis and others that promote the development of cancer.In our previous study,immunohistochemistry showed that ovarian cancer often overexpresses AKT2 and PKM2 compared with adjacent normal tissues,and both of them have statistically positive correlation(P< 0.05).Then,by using immunoprecipitation and MALDI-TOF/TOF MS and MS,we identified that PKM2 is one of new substrates of AKT2.In this study,whether PKM2 participates in regulation of the proliferation,invasion and survival of ovarian cancer cells will be investigated through elevated or decreased the expression of PKM2.Next,we will investigate the new mechanisms of AKT signaling pathways through PKM2 on the regulation of ovarian cancer,which will lead to provide the important evidence for development of new anti-cancer drugs.?Methods?SKOV3 and HEY ovarian cancer cells were selected as the research object.Firstly,LMO transduction and siRNA knockdown techniques were used to up-regulate or down-regulate PKM2 and observe its effect on the biological function of ovarian cancer cells.Then AKT2 and PKM2 lentiviral expression vectors were used to transduce ovarian cancer cells to overexpress AKT2 and PKM2,respectively,and then transfected with shPKM2 and shAKT2 to establish AKT2 overexpression stable knockdown PKM2 and PKM2 overexpression stable knockdown AKT2 The effects of different factors on the biological function of ovarian cancer cells were observed in ovarian cancer cell lines.Then the cells were treated with the PI3 K signaling pathway activator EGF,the inhibitor LY294002,and the mTOR inhibitor Rapamycin,and their effects on the expression of PKM2 were observed.The expression of downstream molecules(NF-?B,Bcl-XL,H3 histone,STAT3)after different treatments was detected by Western-Blot.Finally,the cells in each group of established ovarian cancer SKOV3 cells were injected into the female Babl/c nude mice through the tail vein to observe the number of lung metastatic nodules and the expression of related molecules in the nodules of each group by Western-Blot.?Result?1.CCK-8 and clone formation experiments showed that compared with the control groups,overexpression of PKM2 could increase the proliferation ability of ovarian cancer cells,the difference was statistically significant(P<0.01),and siPKM2 knockdown could inhibit the proliferation ability of ovarian cancer cells(P< 0.05);2.Flow cytometry results showed that compared with the control groups,PKM2 could promote G1 phase to S phase transition of ovarian cancer cell cycle(P< 0.05),and siRNA interference with PKM2 could block ovarian cancer cells in G1/S phase(P< 0.05);3.Flow cytometry results showed that over-expression of PKM2 could increase the ability of ovarian cancer cells to induce apoptosis induced by serum starvation.Knockdown of PKM2 reduced the ability of ovarian cancer cells to induce apoptosis induced by serum starvation.It is suggested that PKM2 also inhibits apoptosis in ovarian cancer cells;4.Western-Blot results showed that over-expression of PKM2 in ovarian cancer cells can increase the expression of CCND1 and decrease the expression of CDKN1 A.Knockdown of PKM2 can decrease the expression of CCND1 and increase the expression of CDKN1 A.5.Compared with the human immortalized epithelial ovarian cancer cell line HOSEpiC,the human ovarian cancer cell lines A2780,CAOV-3,COC1,HEY,HO8910,OVCAR3 and SKOV3 all showed high expression of AKT2(P< 0.05);6.siRNA interference with AKT2 will down-regulate the expression of PKM2.Changes in the expression of AKT1 and AKT3 did not affect the expression of PKM2 protein;7.We successfully constructed AKT2 overexpression and stable knockdown of PKM2,PKM2 overexpression and stable knockdown of AKT2 ovarian cancer cell lines;8.Over-expression of AKT2 can up-regulate the expression of PKM2 in ovarian cancer cells;shRNA interference with PKM2 did not inhibit the expression of AKT2 in ovarian cancer cells with over-expressing AKT2;Silencing AKT2 can inhibit the expression of PKM2 in ovarian cancer cells over-expressing PKM2;9.shRNA interference with AKT2 inhibits the migration and invasion of ovarian cancer cells mediated by PKM2;10.shRNA interference with AKT2 can inhibit PKM2-mediated anti-apoptosis in ovarian cancer cells with serum starvation;11.The expression of PKM2 is regulated by PI3 K,an upstream molecule of AKT2,and the expression of PKM2 is also regulated by mTOR,a downstream molecule of AKT2;12.shRNA interference with AKT2 can inhibit the number of lung metastatic nodules and metastatic foci caused by PKM2-overexpression in nude mice;13.Western-Blot found that mTOR,STAT3 and NF-?B may be downstream molecular targets of the AKT2/PKM2 pathway that promote the progression of ovarian cancer.In particular,STAT3 and NF-?B may serve as downstream molecular targets of PKM2.?Conclusion?1.PKM2 can increase the proliferation ability of ovarian cancer cells;PKM2 can promote G1 phase to S phase transition of ovarian cancer cells;PKM2 can increase the anti-apoptosis induced by serum starvation in ovarian cancer cells.Overexpression of PKM2 could increase the expression of CCND1 and decrease the expression of CDKN1 A.2.Human ovarian cancer cell lines often have high expression of AKT2;AKT2 is an upstream molecule of PKM2.In vitro experiments have shown that AKT2 regulates the capacity of anti-apoptosis induced by serum starvation,invasion and metastasis of ovarian cancer cells through PKM2.Both PI3 K and mTOR regulate the expression of PKM2.In vivo experiments showed that AKT2 regulates the number of lung metastatic nodules and the area of metastases of ovarian cancer cells in nude mice through PKM2.mTOR,STAT3 and NF-?B may be downstream molecular targets of the AKT2/PKM2 pathway that promote the progression of ovarian cancer.In particular,STAT3 and NF-?B may serve as downstream molecular targets of PKM2. |
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