The Study On The Effect Of Polycomb-like 2 On The Proliferation Of Glioma Cell

Objective To study the effect of polycomb-like 2(PCL2)on the biological characteristics of glioma cell proliferation,and to explore the role and possible mechanism of PCL2 in the development of glioma.Methods 1.We use The Cancer Genome Atlas(TCGA)database to detect the expression of PCL2 in various tumors.117 cases of clinical glioma(WHO ?-?)were collected,and PCL2 expression and localization were detected by immunohistochemical staining.2.To construct adenoviral vectors that overexpress the PCL2 gene and interfere with the PCL2 gene,and transfect into human glioma U87 cell line and U251 cell line respectively.Western blot test was used to detect PCL2 protein expression level;3.Morphological changes were observed after intracellular transfection of PCL2 with an inverted microscope;The cell growth curve was drawn by CCK8 experiment;Cell proliferation ability was determined by EdU proliferation detection method;Plate colony formation experiment was used to detect formation ability of the tumor cell colony;Cell cycle and apoptosis were detected by the flow cytometry;Western blot method was used to detect the expression of the core protein EZH2,SUZ12 and EED of the PRC2 complex and the methylation levels of H3K27me3,H3K9me2,H3K4me2 under the conditions of over-expressing PCL2 and interfering with PCL2,4.Using EZH2 inhibitors 3-deazaneplanocin A(DZNep)to interfere with U87 cells overexpressing the PCL2 gene,to detect tumor cell proliferation,colony forming ability,and expression levels of PRC2 core proteins EZH2,SUZ12 And EED.Results 1.The PCL2 gene is expressed differently in 31 tumors in the TCGA database: diffuse large B-cell lymphoma(BLCA),esophageal cancer(ESCA),and glioblastoma(GBM)and other high expressions;the expressions in acute myeloid leukemia(LAML),ovarian serous cystadenoma(OV),and lung cancer(LUAD and LUSC)were lower than corresponding normal tissues.Immunohistochemical examination of 117 human glioma tissues revealed that PCL2 was positively expressed in the nucleus,and its expression increased with the glioma grade(P <0.05).2.The adenoviral vectors of over-expressed PCL2 group(hPCL2 group)and interference PCL2 group(shRNA PCL2 group)were successfully constructed.GFP fluorescence showed the efficiency of virus infection was high.Detection of intracellular protein levels of PCL2 gene showed that when the MOI value in two groups was 50,the expression tendency of protein in both groups were stable,and the infection time of two groups was 48 h in the subsequent experiments..3.Inverted microscope observation,CCK8 experiment,EdU cell proliferation detection,and plate colony formation experiments showed that compared with the blank control group(NC)and the empty vector group(Vector),cells in the overexpressing PCL2 group(hPCL2 group)tended to aggregate,and cell viability,proliferation ability,and colony forming ability were enhanced(P <0.05,P <0.01),and cell activity,proliferation ability,and colony forming ability of the PCL2 group(shRNAPCL2 group)were reduced(P <0.05,P <0.01);cell cycle Detection showed that the percentage of cells in the S phase of the hPCL2 group increased(P <0.05),while that of the cells in the shRNA PCL2 group decreased relatively,and that of the G0/G1 phase increased(P <0.05).Apoptotic fluorescence staining showed that the cell morphology of the hPCL2 group was regular and full.The cell membranes of the shRNAPCL2 group were shrunk and the nuclei inside were red stained,showing the appearance of apoptotic status.The cell flow cytometry analysis of the apoptotic cell ratio showed that compared with the NC group and the Vector group,The proportion of apoptotic cells in the hPCL2 group decreased(P <0.05),and the proportion of apoptotic cells in the shRNAPCL2 group increased significantly(P <0.01).4.Western Blot detection of PRC2 member protein levels and histone methylation expression: Compared with the NC group and the Vector group,the expression of EZH2 and EED in the hPCL2 group increased(P <0.05),the expression of SUZ12 decreased(P <0.05),and H3K4me2 The levels of H3K27me3 and H3K27me3 increased,and the levels of H3K9me2 decreased(P <0.05).In contrast,the expressions of EZH2,SUZ12,and EED in the shRNAPCL2 group decreased(P <0.05),the levels of H3K4me2 and H3K27me3 decreased,and the levels of H3K9me2 increased.5.DZNeP interfered with U87 cells in hPCL2 group.CCK8 experiment showed that cell viability and colony forming ability decreased(P <0.05)after the intervention of DZNeP in hPCL2 group(DZ-hPCL2 group).Compared with the control group(NC)and the negative control group(DMSO),the expressions of EZH2,SUZ12 and EED in the DZ-hPCL2 group showed a downward trend(P <0.05).Conclusion 1.PCL2 gene is highly expressed in gliomas.2.Overexpression of PCL2 gene can promote proliferation of glioma cell,up-regulate the expression of PRC2 core proteins EZH2 and EED,promote histone H3K27me3 methylation,and inhibit H3K9me2 and H3K4me2 methylation.3.Interfering with the PCL2 gene promotes glioma cell apoptosis,down-regulates the expression of EZH2 and EED,inhibits the histone H3K27me3 methylation,and promotes the methylation of H3K9me2 and H3K4me2.4.DZNeP can reduce the protein expression of SUZ12 and EED by inhibiting EZH2,and reduce the proliferation of glioma cells caused by PCL2 gene overexpression.