Study On The Regulation Of Hypoxia And NKAP Gene On The Cytological Function Of Breast Cancer

Part I:NKAP affects the apoptosis of breast cancer cells through the PI3K/AKT signaling pathwayObjective:To NKAP plays an important role in transcriptional repression,T cell development,maturation and function acquisition,maintenance and survival of adult hematopoietic stem cells and RNA splicing.But people's understanding of it is still not enough,especially in the area of cancer.Breast cancer is a major public health problem in the world and is also the most common type of cancer in women.We want to explore whether NKAP affects the occurrence and development of breast cancer.Methods:The differences of NKAP expression between breast cancer patients and normal controls were analyzed by TCGA,GTEx and GEPIA databases,and their correlation with survival rate of breast cancer patients was analyzed.When NKAP was found to be differentially expressed in breast cancer and correlated with poor prognosis,we knocked down the expression of NKAP in MCF-7 breast cancer cells by RNAi technique.Followed by the use of CCK8 experiment and plate clone formation experiment to explore NKAP knockdown on cell proliferation,Transwell experiments revealed its role in cell migration,and the use of flow cytometry to detect apoptosis.In terms of mechanism,the signal pathways involved in finding a role for NKAP in breast cancer cell MCF-7 are sought by WB.Results:1.NKAP is highly expressed in breast cancer2.Knockdown of NKAP inhibits the proliferation and clonality of breast cancer cells MCF-73.Knockdown of NKAP inhibits MCF-7 cell migration4.Knockdown of NKAP can promote the apoptosis of MCF-7 cells by regulating the expression of caspase3-dependent apoptosis-related proteins5.Knockdown of NKAP affects the PI3K/Akt/mTOR pathway Conclusion:NKAP is differentially expressed in breast cancer and is closely related to the poor prognosis of patients with breast cancer.NKAP plays an oncogene role in glioma through the AKT/mTOR signaling pathway.Knockdown of NKAP can inhibit the proliferation and migration of breast cancer cell MCF-7 and promote its apoptosis.It may provide new ideas for the future treatment and prognosis of breast cancer.Part ?:The impact of NKAP in breast cancer under hypoxic conditionsObjective:In recent years,there is increasing evidence that the oxygen(02)content in tumor tissue is an important determinant of breast cancer metastasis.Ongoing tumor hypoxia leads to more malignant phenotypes.Understanding how to simulate an accurate hypoxic environment in vitro and establish a reliable,easy-to-manipulate hypoxic model is the first step toward studying the hypoxic tumor microenvironment.In this study,the optimal hypoxic model was found by culturing CoC12 and MCR-7 breast cancer cells at different concentrations in vitro.The subject of the previous experiments confirmed that NKAP is a potential prognosis of breast cancer patients poor indicators.In studying NKAP function,we found that hypoxia in breast cancer cells resulted in a significant increase in the expression of this gene,which was positively correlated with the expression of HIF-1?.Therefore,we hypoxia can up-regulate the expression of NKAP in breast cancer cells,and then regulate the growth and metastasis of tumor cells.The purpose of this study is to investigate the functional effects of hypoxia on breast cancer cells and clarify the role of new hypoxia target protein and NKAP in tumor hypoxia and its specific mechanisms to further explore new potential targets in the treatment of breast cancer.Methods:In this study,different concentrations of CoC12(50,100,150 and 200 ?M)and MCR-7 breast cancer cells were cultured together in vitro to find the optimal hypoxic model.We detected the expression of HIF-1?,CXCR4 and VEGF under different concentrations of CoC12 by qRT-PCR and WB to clarify the hypoxic microenvironment.Subsequently,the changes of biological behavior of breast cancer cells in hypoxic microenvironment were detected by micrograph and TUNEL assay.The effects of hypoxia on the proliferation of breast cancer cell MCF-7 were analyzed by CCK8 assay.The effect of hypoxia on NKAP expression in breast cancer cells MCF-7 was detected by WB.Knockdown of NKAP in hypoxic conditions to investigate the effects of hypoxia and NKAP knockdown on the proliferation and migration of breast cancer cells MCF-7.Results:1.CoCl2 induced the expression of HIF-la,CXCR4 and VEGF2.CoCl2 promotes MCF-7 cell apoptosis3.CoCl2 inhibited MCF-7 cell proliferation4.Hypoxia induces NKAP expressionConclusion:The hypoxia condition induced by CoC12 can significantly induce the expression of HIF-la,CXCR4 and VEGF in breast cancer cell line MCF-7,which is related to the length and concentration of CoCl2 treatment.In vitro hypoxia in tumor cells and its effect on cells is a dynamic process.CoC12 mimicked hypoxic conditions produce cytotoxicity in MCF-7 cells and induce apoptosis.Hypoxia can induce NKAP expression in breast cancer cells MCF-7.When hypoxia and NKAP knockdown were performed simultaneously,the inhibition of cell proliferation was exacerbated,but hypoxia prevented the promotion of NKAP knockdown on migration.These preliminary results indicate that crosstalk may exist in the signaling pathway of hypoxia signaling pathway and NKAP in breast cancer cell MCF-7.That is,there may be a synergistic effect in cell migration,and may have an antagonistic effect in cell proliferation and survival.NKAP is a very interesting target protein that is closely related to the development of breast cancer and hypoxia.The specific mechanism is still waiting for our exploration.