Mechanism Research Of GSK-3β Regulating The Expression Of P21 To Promote The Progression Of Chordoma

Objective: The aim is to investigate the function of glycogen synthesis kinase(GSK-3β)in the proliferation of chordoma cells and tumorigenesis of chordoma,along with the role of GSK-3β-P21 pathway in the progression of chordoma.Methods: 1)41 pairs of chordoma and normal tissue specimens surgically resected from Guilin Medical school affiliated hospital were collected.The protein levels of GSK-3β in chordoma and adjacent normal tissues were detected by Western blot,and the expression of GSK-3β in chordoma and corresponding adjacent tissues were tested by IHC;2)The human chordoma cells U-CH1 were treated with different concentrations of Licl,GSK-3β inhibitor.The level of cell GSK-3β was detected by Western blot,and the cell proliferation ability by plate cloning experiment.When Licl is at 25mmol/L,the cell proliferation ability in different time periods after administration was measured by CCK-8.3)The effect of GSK-3β transfection for 24 hours was observed by fluorescence microscope,and the transfection efficiency of different fragments of GSK-3β in U-CH1 and JHC7,human chordoma cells,were detected by Western blot and qRT-PCR,selecting the fragment with best transfection efficiency for the following experiments.4)The experimental cells,U-CH1 and JHC7,were respectively divided into Si-NC and Si-GSK-3β groups,and the cell proliferation ability was detected by CCK-8 and plate cloning experiments;5)The experimental cells,U-CH1 and JHC7,were respectively divided into Si-NC and Si-GSK-3β groups,and the apoptosis of each group was analyzed by flow cytometry;6)The experimental cells,U-CH1 and JHC7,were respectively divided into Si-NC and Si-GSK-3βgroups,and the ability of the cells to regulate epithelial-mesenchymal transition(EMT)was detected by Western blot and Transwell chamber experiment;7)The experimental cells,U-CH1 and JHC7,were respectively divided into Si-NC and Si-P21 groups,and the cell proliferation ability was tested by CCK-8;8)The interaction between GSK-3β and P21 in U-CH1 and JHC7 was detected by Coimmunoprecipitation,qRT-PCR,Western blot and immunofluorescence.9)The experimental cells,U-CH1 and JHC7,were respectively divided into Si-GSK-3β and Si-GSK-3β+ Si-P21 groups,and the proliferation ability and apoptosis of each group were analyzed by CCK-8 and flow cytometry;10)Nude mouse model transplanted with Chordoma was established,and the interaction between GSK-3β and P21 was explored by measuring and recording tumor volume and Western blot.Results: 1)Compared with adjacent normal tissues adjacent to cancer,the level of GSK-3β in chordoma tissue was significantly higher(P<0.01);2)Western blot results showed that the GSK-3β level of cells was significantly decreased after the treatment of Licl(P<0.01).The results of plate cloning and CCK-8 confirmed that the cell proliferation ability and the number of colonies were significantly reduced after the treatment of Licl(P<0.05);3)Fluorescence results showed the cell transfection efficiency.The qRT-PCR and Western blot results revealed that the most efficient fragments in U-CH1 and JHC7 are respectively Si-GSK-3β-1 and Si-GSK-3β-2;4)The results of CCK-8 and plate cloning showed that after the transfection with GSK-3β,the proliferation ability of the two cells both decreased in various degrees(P<0.01);5)The results of flow cytometry showed that after the transfection of GSK-3β,the two kinds of cells underwent apoptosis in various degrees(P<0.01);6)The results of Western blot and Transwell chamber experiments showed that after the transfection of GSK-3β,the migration and invasion ability of the two kinds of cells decreased(P<0.01),and the epithelial-mesenchymal transition(EMT)process was inhibited(P<0.001);7)CCK-8 results showed that the down-regulation of p21 promoted the proliferation of chordoma cells;8)The results confirmed that GSK-3β and P21 could interact with each other;9)The results of CCK-8 and flow cytometry showed that the cell proliferation activity after co-transfection with GSK-3β and P21 was higher than that in the GSK-3β group alone.The down-regulation of P21 reversed the partial functions with the presence of Si-GSK-3β in chordoma;10)The results of nude mouse tumor transplantation model confirmed that GSK-3β interacts with P21 and the down-regulation of GSK-3β inhibited the proliferation of chordoma cells in vivo.Conclusions: This study confirmed that GSK-3β was defined as an oncogene in chordoma,changes of whose level affected cell proliferation,invasion and migration.P21 was recognized as its downstream target,and the GSK-3β-P21 pathway was involved in regulating the occurrence and development of chordoma.This study explored the role of GSK-3β-P21 signaling pathway in chordoma,further studying the pathogenesis of chordoma,providing a new direction and prognostic indicators for its clinical treatment.