| Research background:Malignant ovarian cancer has the highest mortality rate in all gynecological cancers.The five-year survival rate of patients is less than 30%.Studies have found that many signaling molecules are associated with cancer,especially changes in the expression or activity of kinases.The disorders of these signaling molecules play an important role in the occurrence and development of cancer.The phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/rapamycin mammalian target protein(mTOR)signaling pathway involved in protein kinase B(AKT)is one of the most frequently altered signaling pathways in human cancers.It has become the most attractive target in anti-cancer treatment.As a kinase,AKT also affects multiple processes in the cell that promote tumorigenesis.AKT2 is one of three subtypes of AKT,and is a gene encoding a tyrosine kinase,which is highly expressed in 8 ovarian cancer cell lines and highly expressed in 40% of primary ovarian cancer.AKT2 is involved in the complex signal transduction pathway of cell proliferation,metastasis and survival,and its overexpression is also associated with lower and worse survival.Pyruvate kinase M2(PKM2)is highly expressed in proliferating cells,especially tumor cells,and its enzymatic activity undergoes complex regulation to adapt cells to different physiological states.In addition to its role as a pyruvate kinase,PKM2 also acts as a transcriptional coactivator for protein kinases and genes involved in cell proliferation,migration and apoptosis.In our previous research work,PKM2 was unreported downstream substrate of AKT2 by immunoprecipitation + mass spectrometry and reverse immunoprecipitation with anti-phospho-AKT substrate antibody.In view of the important role of AKT and PKM2 in malignant tumors,we try to explore the the roles and mechanisms of of AKT2 and PKM2 and their interaction in ovarian cancer cell migration and growth.Research purposes:This study is to explore the role and the mechanism of action of AKT2 in the migration and growth of ovarian cancer cells by PKM2 from three levels of cells,animal models and clinical specimens,so as to elucidate the new mechanism of AKT2 and PKM2 in ovarian cancer.This mechanism can provide experimental clues for determining biomarkers for ovarian cancer diagnosis and targeted therapeutic targets.Research methods:1.Collect clinical ovarian cancer specimens,detect the expression of AKT2,PKM2 and related proteins by immunohistochemistry,and analyze the correlation of each molecule expression,and analyze the relationship between each molecule and tumor development degree and prognosis;2.Selecting representative ovarian cancer cell lines SKOV3 and HEY,up-regulating or down-regulating the expression of AKT2 and PKM2 at the cellular level by lentiviral transduction,siRNA transfection or shRNA transfection.Through the scratch test,CCK-8 test,glucose and lactic acid content determination,the effects of AKT2 and PKM2 on the migration and growth of ovarian cancer cells were studied,the role of AKT2 and PKM2 in the development of ovarian cancer was explored,the relationship and the regulation mechanisms between the two molecule was explored;3.Western-Blot was used to explore the effect of AKT2 or PKM2 expression on the related upstream and downstream proteins in ovarian cancer cells,and the relationship between AKT2 and PKM2 in related signaling pathways and the possible molecular mechanisms involved;4.SKOV3 cells were injected subcutaneously into nude mice to observe the effect of AKT2 on the tumorigenesis of nude mice through PKM2,and to further clarify the expression and changes of related proteins and possible molecular mechanisms.Research result:1.The immunohistochemical results of clinical ovarian cancer specimens showed that the positive rates of AKT2,PKM2,STAT3,B-cell lymphoma-XL(BCL-XL),Transforming growth factor-beta-induced factor 2(TGIF2)and Jumonji domain containing 5(JMJD5)in ovarian cancer tissues were higher than those in normal tissues,and their expression differences were statistically significant(P < 0.001).By χ2 test and multi-factor logistic regression analysis,the expression of AKT2 and PKM2,AKT2 and JMJD5,PKM2 and TGIF2,PKM2 and STAT3 in ovarian cancer tissues were correlated;2.Analysis of clinicopathological features χ2 test results showed that AKT2 overexpression and PKM2 overexpression were associated with survival and metastasis of ovarian cancer patients(P<0.05).Both AKT2 and PKM2 were associated with tumor size in ovarian cancer patients(P<0.05),and TGIF2 expression was correlated with TNM stage of ovarian cancer(P<0.05);Cox model multi-factor survival analysis showed that there were correlations between the survival of ovarian cancer patients and metastasis(HR: 2.796,95%CI:1.350-5.791,P=0.006)(P<0.01);between the survival of ovarian cancer patients and AKT2 overexpression(HR:1.624,95%CI:1.058-2.493,P=0.027)(P<0.05);between the survival of ovarian cancer patients and PKM2 overexpression(HR: 1.853,95% CI: 1.128-3.043,P = 0.015)(P < 0.05);3.Kaplan-Meier survival analysis showed that the difference in survival function between patients with PKM2 overexpression(positive 2+ and above)and those with low positive and negative(positive 1+ and below)was statistically significant(Log Rank P=0.016,P< 0.05);AKT2 overexpression(positive 2+ and above)patients with low positive and negative(positive 1+ and below)survival function difference was statistically significant(Log Rank P = 0.032,P <0.05);indicating PKM2 and Overexpression of AKT2 is closely related to poor prognosis in patients;4.Immunofluorescence and Western-Blot results showed that the stable SKOV3 and HEY cell lines overexpressing AKT2 and overexpressing PKM2 was successfully constructed.And successfully constructed SKOV3 and HEY cell lines that overexpression AKT2 and down-regulate PKM2 expression,SKOV3 and HEY cell lines that overexpression PKM2 and down-regulate AKT2 expression;5.Western-Blot results showed that when the expression of AKT2 in ovarian cancer cells increased,the expression level of PKM2 also increased,and when the expression of AKT2 in ovarian cancer cells was silenced,the expression level of PKM2 also decreased.This indicates that AKT2 regulates the expression of PKM2;however,the expression of PKM2 does not affect the expression of AKT2 in ovarian cancer cells,indicating that PKM2 is a downstream molecule of AKT2.At the same time,other molecules were detected,and the expression levels of STAT3 and p-STAT3 were positively correlated with the expression of AKT2 and PKM2,indicating that STAT3 might be the downstream molecule of AKT2 and PKM2.The expression of JMJD5 and TGIF2 was not affected by the expression of AKT2 and PKM2.The effect of this indicates that JMJD5 and TGIF2 are not downstream molecules of AKT2 and PKM2;6.The results of scratch repair experiments showed that ovarian cancer cells overexpressing AKT2 or overexpressing PKM2 could increase their migration ability,and relatively,Silencing the expression of AKT2 or PKM2 or down-regulating the expression of AKT2 in overexpressing cells of PKM2 may decrease the migration ability of cells.Combined with previous results such as Western-Blot,it was shown that AKT2 can alter cell migration ability by regulating the expression of PKM2;7.The results of CCK-8 assay showed that ovarian cancer cells overexpressing AKT2 or overexpressing PKM2 could increase their proliferation ability,while silencing the expression of AKT2 or PKM2 or down-regulating the expression of AKT2 in overexpressing cells of PKM2 would inhibit their proliferative ability.Combined with previous results such as Western-Blot,it was shown that AKT2 can change the cell proliferation ability by regulating the expression of PKM2;8.The results of determination of glucose and lactic acid in the medium showed that overexpression of AKT2 or overexpression of PKM2 in ovarian cancer cells increased the glucose consumption of cells,while silencing the expression of AKT2 or PKM2 or down-regulating the expression of AKT2 in overexpressing cells of PKM2 reduced glucose consumption in ovarian cancer cells;Overexpression of AKT2 or overexpression of PKM2 in ovarian cancer cells can increase the production of lactic acid in cells,while silencing the expression of AKT2 or PKM2 or down-regulating the expression of AKT2 in overexpressing cells of PKM2 may reduce lactic acid production in ovarian cancer cells.Combined with previous studies,it was shown that AKT2 can change the ability of cellular aerobic glycolysis by regulating the expression of PKM2,and the aerobic glycolysis of cells is on the rise;9.Subcutaneous tumor formation in nude mice showed that overexpression of AKT2 or overexpression of PKM2 in ovarian cancer cells can make tumors grow faster and become larger;while silencing the expression of AKT2 or PKM2 or down-regulating the expression of AKT2 in overexpressing cells of PKM2,tumor growth will be slower and the volume will be smaller.Combined with previous studies,it was shown that AKT2 regulates tumor growth by regulating the expression of PKM2.Conclusion:1.The expression of AKT2,PKM2,STAT3,BCL-XL,TGIF2 and JMJD5 in ovarian cancer tissues is higher than that in normal tissues,which can be used as the clinical diagnostic targets.There were correlations between AKT2 and PKM2,AKT2 and JMJD5,PKM2 and TGIF2 or STAT3 expression in ovarian cancer tissues;2.The expression of AKT2 and PKM2 is associated with survival and metastasis of ovarian cancer patients;The expression of AKT2 and PKM2 is correlated with the tumor size of patients with ovarian cancer;The expression of TGIF2 was correlated with the TNM stage of ovarian cancer.3.Overexpression of PKM2 or AKT2 is associated with poor survival and prognosis in patients;4.Overexpression of AKT2 or PKM2 genes can improve the migration ability of ovarian cancer cells,and improve the cell growth ability by increasing the ability of cellular aerobic glycolysis.PKM2 may be a downstream molecule of AKT2 and is an upstream molecule of STAT3.PKM2 is regulated by AKT2 and phosphorylates STAT3,which regulates the development of ovarian cancer by regulating the expression of STAT3. |
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