The Study Of Inactivating Mutations Of IK Gene And Its Loss Of Protein Expression In Endometrial Cancer

Endometrial cancer is one of three common gynecological oncologies.Its morbidity and mortality have been increasing in the recent 10 years.Now it is the most common gynecological oncology.Endometrial cancer can be divided into two types according to the Pathology types.Type I,which is estrogen dependent,accounts for most of the diseases.Its ratio is about 70-80%.Most of the type I endometrial cancer have a good prognosis.Endometrioid endometrial cancer is its primary pathology type.Although the total prognosis of type I endometrial cancer is correlated to International Federation of Gynecology and Obstetrics(FIGO)stages and tumor grades,individual patients with this type show different responses to therapy and have statistically significantly different clinical courses.Therefore,single pathological grouping is insufficient to predict clinical course for type I endometrial cancer.New grouping method need to be confirmed.Our previous study showed that cluster IV,which consisted of advanced and high grades diseases,had better survival rate that cluster II,which consisted of early stages and low grades diseases.In addition,IK gene was strikingly mutated in cluster IV.So IK mutations may contribute to this kind of better clinical survival.There is no study about IK cytokine in endometrial cancer.Further study about type I EC in The Cancer Genome Atlas(TCGA)database showed that most of the IK gene mutational types were frameshift.This kind of mutation could cause deficiency of IK protein.In addition,IK gene mutation was negatively associated with IK m RNA levels and positively associated with the survival of EC patients.So in this study,human endometrial cancer cell line Ishikawa and KLE cells were selected to investigate how IK knockdown play roles in endometrial cancer and its possible functional mechanisms in vitro.Section I The roles of IK knockdown in cell growth of endometrial cancer cellsObjective:To investigate how IK knockdown influences the growth of endometrial cancer cellsMethods: Si RNA transfection method was used to knock down the protein expression of IK and western blot method was chose to detect IK expression level after 24 h,48h and 72 h.Cell viability assay(CCK-8),colony formation assay were used to detect how IK knockdown regulate the cell viability and cell proliferation.Anexin V-FITC/PI staining and trypan blue assay were used to detect how IK knockdown regulate cell apoptosis and cell death.PI staining was used to detect how IK knockdown regulate cell cycle.Western blot method was used to detect cell autophagy related proteins.Acridine orange dye staining was used to evaluate the autophagy.Cell apoptosis inhibitor Q-VD-OPH and cell autophagy inhibitor 3MA were used to investigate whether they could influence cell death after knock down of IK protein level.Results: IK protein expression level was decreased by IK si RNAs transfection,especially after 48 h and 72 h transfection.IK knockdown could inhibit the cell viability and proliferation and caused G2/M phase arrest in Ishikawa and KLE endometrial cancer cell lines.The protein expression of cell cycle related protein cyclin D1 was decreased.IK knockdown could activate caspase 3,7,8 and 9 and increase the protein level of anti-apoptotic protein BCL-2.Similarly,the protein level of cleaved PARP was decreased.Then IK knockdown caused cell apoptosis and cell death.IK knockdown could lead to the transformation from LC3 BI to LC3 BII and decrease the expression level of autophagy related protein P62.There were more acridine orange dye staining positive cells.Cell apoptosis inhibitor Q-VD-OPH could decrease the cell death ratio while cell autophagy inhibitor 3MA could increase the cell death ratio.Conclusion: IK knockdown could inhibit the normal growth of endometrial cancer cells,inhibit mitosis and induce apoptotic cell death through the intrinsic mitochondria dependent signaling pathway and the extrinsic death receptor dependent signalingpathway.Section II The roles of IK knockdown in DNA damage repair pathway of endometrial cancer cells and its functional mechanisms studyObjective: To investigate how IK knockdown influences DNA damage repair pathway and its possible functional mechanisms.Methods: Seventy-two hours after si RNAs transfection,epi-Quick in situ DNA damage assay kit,?-H2 AX immunostaining method and western blot method were used to detect whether IK knockdown caused DNA damage in Ishikawa and KLE endometrial cancer cells.Homologous recombination assay and non-homologous end joining assay were used to detect whether IK knockdown influenced their activities.When DNA damage happened,protein mass spectrum analysis and immunoprecipitation method were used to investigate which DNA repair related proteins could bind with IK and further immunoprecipitation assays were used to investigate how IK knockdown influence the protein binding activity.Cell viability assay,cell apoptosis assay and cell death assay were used to detect whether IK knockdown could sensitize the endometrial cancer cells to cisplatin treatment.Results: Seventy-two hours after si RNAs transfection,IK knockdown could cause DNA damage in Ishikawa and KLE endometrial cancer cell lines.More ?-H2 AX foci were found after IK was knocked down.Western blot results also showed increased protein expression of ?-H2 AX.IK knockdown inhibited homologous recombination and non-homologous end joining pathways.When DNA damage happened,protein mass spectrum analysis data showed IK could bind with Ku80.Further immunoprecipitation result confirmed this result.In addition,IK knockdown inhibited the heterodimerazition of Ku80 and Ku70.IK knockdown could sensitize endometrial cancer cells to cisplatin treatment.IK si RNAs transfection and cisplatin could decrease cell viability and induce cell apoptosis and cell death significantly.Conclusion:IK knockdown could cause DNA damage and inhibit DNA repair pathway through regulating the heterodimerazition of Ku80 and Ku70.IK knockdown could sensitize endometrial cancer cells to cisplatin treatment.Taken together,the main mutational type of IK gene is frameshift,which is a kind of inactivating mutations.IK mutation was significantly associated with its m RNA expression.In addition,IK mutation had positive correlation with clinical survival of endometrial cancer patients.In vitro study showed that IK knockdown could inhibit DNA repair pathways and induce DNA damage and then inhibit the normal growth of endometrial cancer cells.The functional mechanism was that IK knockdown could inhibit the heterodimerazition of Ku80 and Ku70.And also IK knockdown could sensitize endometrial cancer cells to cisplatin treatment.IK can be considered as a new therapeutic target for endometrial cancer treatment in future.