| Background: NEK2 is a cell cycle-associated serine /threonine protein kinase involved in assembly of centrosomes,regulation of centrosome separation,and signal regulation of spindle assembly checkpoints to maintain genome stability.Abnormal expression and dysfunction of NEK2 will lead to tumorigenesis,and high expression of NEK2 is associated with malignant progression of tumors,drug resistance,and poor prognosis of tumors.Therefore,the abnormal expression of NEK2 can be used to indicate tumor progression and disease prognosis,and is a potential molecular therapeutic target with great development prospects.In the previous study,we designed and synthesized NEK2 inhibitors which have strong anti-tumor activity in vivo and in vitro for a variety of tumors including gastric cancer and breast cancer.The strength of the action is related to the high expression of NEK2 and KDM5 B.NEK2 inhibitor not only down-regulates the expression of NEK2,but also down-regulates KDM5 B,suggesting that NEK2 may directly or indirectly participate in the regulation of KDM5 B to play its biological function,and this finding has not been reported,and it is worthy of further discussion.Purpose: The aim of this study was to investigate the effects of NEK2 on the development of breast cancer and gastric cancer by constructing NEK2 silencing and NEK2 over-expression stable cell lines.To confirm the regulation of NEK2 on KDM5 B in the previous study,and further study the specific molecular regulation mechanism of NEK2 on KDM5 B.In order to clarify the important role of KDM5 B in targeting the inhibition of NEK2 against breast cancer and gastric cancer,and to discover a new signaling pathway that mediates the development of breast cancer and gastric cancer,it provides a new idea for the development of new anti-tumor drugs.Methods: Using bioinformatics methods to evaluate the correlation between NEK2 expression and pathological process,prognosis and patient survival of breast cancer;Detecting the relationship between NEK2 and KDM5 B by immunoprecipitation experiments;NEK2 silencing and NEK2 overexpressing breast cancer stable transfecting cell lines were constructed by CRISP/cas9 system and p-babe vector system respectively;NEK2 silencing and NEK2 overexpressing gastric cancer monoclonal stable transfecting cell lines were constructed by tet-on and tet-off lentiviral transfection systems respectively;Using Western blot to detect the changes of protein levels in cellular;CCK-8 and clony formation method were used to detect tumor cell proliferation;wound healing and Transwell test were used to detect tumor cell migration;PI staining and Annexin V-FITC method were used to detect cell cycle and apoptosis respectively;MTT assay was used to detect Sensitivity to chemotherapeutic drugs;tumor formation in nude mice was observed to the effect of interference with NEK2 on tumor growth.Results:(1)In breast cancer,high expression of NEK2 is associated with disease stage and poor prognosis.Silencing NEK2 inhibits proliferation and migration of breast cancer MDA-MB-468 cells,leading to G2/M arrest;while overexpression of NEK2 promotes proliferation and migration of breast cancer MCF-7 cells without affecting cell cycle.The knockdown of NEK2 resulted in a decrease in the level of NEK2 protein,and also significantly decreased the expression of KDM5 B and increased the expression of H3K4me3.However,the overexpression of NEK2 in MCF-7 cells increased the level of KDM5 B protein and decreased the expression of H3K4me3.Coimmunoprecipitation experiments showed that NEK2 did not directly interact with KDM5 B.Further studies found that NEK2 regulates KDM5B/H3K4me3 mainly by activating ?-catenin/Wnt signaling pathway.Silencing NEK2 inhibits the expression of bridge proteins ?-catenin and c-Myc,thereby reducing the transcription and expression of KDM5 B.Overexpression of NEK2 activates ?-catenin/Wnt signaling pathway,and the expression of ?-catenin and c-Myc is increased,the expression of KDM5 B is increased,and the demethylation of H3K4 is increased.After MCF-7 OE-NEK2 cells were treated with c-Myc inhibitor,the expression of c-Myc and its downstream KDM5B protein was significantly decreased,and the expression of H3K4me3 was increased,which did not affect the expression of ?-catenin in the upstream,but weakly inhibited the expression of NEK2.After MCD-7 OE-NEK2 cells were treated with KDM5 B inhibitor,the expression of KDM5 B was significantly decreased,and the expression of H3K4me3 was increased,which did not affect the expression of ?-catenin and c-Myc proteins.c-Myc and KDM5 B inhibitors can offset or alleviate the regulation of overexpression of NEK2 on KDM5 B to some extent.The results showed that NEK2 regulates the proliferation of breast cancer cells by activating ?-catenin/Wnt signaling pathway and regulating KDM5B/H3K4me3 expression.(2)The expression levels of NEK2 were higher in multiple gastric cancer cells.Silencing NEK2 inhibits proliferation and metastasis of MGC-803 gastric cancer cells,leading to G2/M arrest and apoptosis,increasing the sensitivity of gastric cancer cells to ABC substrate chemotherapeutic drugs,and slowing tumor growth in nude mice;overexpression of NEK2 The proliferation of SNU-1 gastric cancer cells increased the resistance to chemotherapeutic drugs,and tumor growth accelerated in nude mice.At the cellular level,silencing of NEK2 inhibited the expression of p-P38,p-AKT,and p-ERK proteins in the P38/MAPK classical signaling pathway,whereas overexpression of NEK2 significantly increased the expression of p-P38,p-AKT,and p-ERK proteins..NEK2 regulates gastric cancer growth by activating the P38/MAPK signaling pathway.Western blot analysis of gastric cancer in nude mice showed that silencing NEK2 inhibited the expression of ?-catenin and c-Myc protein,the expression of KDM5 B was significantly decreased,and the expression of H3K4me3 was increased.The results showed that in gastric cancer,NEK2 can also affect the proliferation of gastric cancer cells by activating ?-catenin/Wnt signaling pathway and regulating KDM5B/H3K4me3 expression.Conclusions: This topic for the first time systematically revealed the important role of NEK2 in the development of breast cancer and gastric cancer.Silencing NEK2 inhibits proliferation,migration and cell cycle arrest of breast cancer and gastric cancer cells,and increases the sensitivity of gastric cancer cells to chemotherapeutic drugs.Overexpression of NEK2 promotes the proliferation and migration of breast cancer cells and gastric cancer cells,and promotes the resistance of gastric cancer cells to chemotherapeutic drugs.NEK2 can regulate the protein expression of KDM5B/H3K4me3 by activating ?-catenin/Wnt signaling pathway,which affects the growth and migration of breast cancer and gastric cancer.This study systematically revealed the role of NEK2 in cell proliferation,metastasis,and drug resistance of breast cancer and gastric cancer for the first time,and first discovered the regulation of NEK2 on KDM5 B and its mechanism,and elucidated the development of NEK2 during tumorigenesis.The importance of the original innovative meaning provides new targets and ideas for the development of new anti-tumor drugs. |
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