To Investigate The PLK4 Expression And Its Effect On Proliferation And Drug-resistance In Glioma

Objectives:To explore the PLK4 expression through the data cohort and investigate its correlation with the pathological grade,survival of patients and chemo-sensitivity in glioma.RNA interfere and pheno-type assays were employed to ensure that the effect of PLK4 on proliferation and TMZ chemo-sensitivity of glioma cells.Further we confirmed the mechanism that PLK4 binds I?K? to trigger NF-?B/BCL2 anti-apoptosis signal pathway to affect the proliferation and TMZ chemo-sensitivity,as our research group predicted previously.Hence PLK4 may be a new therapeutic access to TMZ-resistant glioma.Methods:1.The RNA-seq data from CGGA was applied to analyse the PLK4 mRNA expression in glioma according to the WHO grades(II?III?IV).Meanwhile,the correlation with PLK4 expression and overall survival as well as the chemo-sensitivity of patients was explored.2.Human glioblastoma cell lines U87 MG,U251,LN229 and U87 EGFRvIII were taken to measure the PLK4 mRNA expression by real-time PCR.Further we have collected human glioma tissues of different pathological grades to mesure the PLK4 mRNA expression by real-time PCR,as well the PLK4 protein level by Western blot in each sample.3.Specific PLK4-targeted siRNA was constructed and transfected to the U87 MG and LN229 cell lines to measure the knockdown efficiency.Immunofluorescence assay was applied to observe the effect on centriole of the deleption of PLK4.Colony formation and MTT assay were implemented to evaluate the proliferation and MTT assay was used to determine the chemo-sensitivity to TMZ of glioma cells.4.Nude mice intracranial glioma model was established with U87 MG cells,then control group,PLK4-KD group TMZ-treat group and Co-treat group were set to verify that down-regulated PLK4 makes the increasd sensitivity to TMZ of glioma cells.5.NF-?B-targeted luciferase reporter gene plasmid pNF?B-luc was constructed to verify that NF-?B is the downstream effect target of PLK4 by Luciferase Assay.6.The cellular location of PLK4 and I?K? was observed with the immunofluorescence assay.And we used the Western blot and immunofluorescence assay to detect the level of I?B? protein and translocation of NF-?B-p65 respectively after PLK4 down-regulated.Consequently we confirmed that PLK4 can bind I?K?by forming complex to lead the degradation of I?B? and promote NF-?B-p65 entering into the nucleus from cytoplasm.7.The PCR primers of BCL2 family of anti-apoptosis gene were designed and real-time PCR was implemented to detect the changes of BCL-2 anti-apoptosis mRNA expression level after knockdown of PLK4,comfirming that down-regulated PLK4 can surpress the BCL-2 anti-apoptosis activity and enhance the chemo-sensitivity to TMZ.Results: 1 The analysis of mRNA profile in the CGGA dataset showed that the expression of PLK4 mRNA was positively correlated with the WHO pathological grade of glioma.Patients with high PLK4 status have significantly decreased survival,and the difference in PLK4 status in high-grade glioma has a significant effect on the patients' overall survival.Patients with low PLK4 status have significantly prolonged survival under chemo-therapy treatment but those with high PLK4 status don't.Therefore we proved that PLK4 expression varies in glioma and correlates with the pathological grade,prognosis and chemo-sensitivity.2.The PLK4 mRNA level in glioma cell lines was higher compared with normal cell,especially in U87 and LN229.PLK4 mRNA expression was detected in human glioma tissues,and the high grade glioma has a significant higher PLK4 status compared with the low grade glioma.However,we found that there was no significant revelance with the PLK4 protein level and pathological grade.3.PLK4 was down-regulated in target-siRNA transfected glioma cell lines.The number of centrioles was decreased after PLK4 down-regulated.MTT assays and colony formation assays revealed that the proliferation of glioma cells was reduced and the sensitivity to chemotherapy increased significantly.4.U87MG-induced nude mice intracranial glioma model revealed that treatment combined with PLK4-inhibited and TMZ made the tumor grow slower and prolonged the survival of animals significantly.The HE stainning showed that the size of tumor in Co-treated group was smaller than the control group.The IHC assays showed that the expression quantity of Ki-67 was decreased significantly.5.Luciferase reporter gene experiment showed that in the PLK4 down-regulated glioma cell lines,the fluorescence intensity of NF-?B was reduced than that of normal cells,demonstrating that NF-?B was the downstream effect target of PLK4.6.Through immunofluorescence stainning we found the co-location of PLK4 and I?K? and demonstrated that PLK4 can combine with I?K? forming a complex.The p65 subunit of NF-?B was translocated from nucleus to cytoplasm.7.The western blot showed that the expression level of I?B? was up-regulated after PLK4 down-regulated in the glioma cells,indicating that PLK4 can phosphorylate I?K? and degrade I?B?,thus leading to the activation of downstream signaling pathway.8.After PLK4 down-regulated in the glioma cell,the mRNA expression quantity of BCL2-A1 and BCL2-L1 in the BCL2 anti-apoptosis family were decreased,consequently we verified that PLK4 activated the downstream NF-?B/BCL2 anti-apoptosis pathway.Conclusion: 1.PLK4 is highly expressed in glioma,and the expression of PLK4 is negatively related to the patient's prognosis,especailly in the high grade glioma.2.PLK4 is highly expressed in glioma,and the expression level of PLK4 mRNA was positively correlated with pathologic grade.However,there is no significant correlation between protein expression level and pathologic grade.3.Low-PLK4 expression enhauces the sensitivity to chemo-therapy and prolongs the survival of patients.4.The reduction of PLK4 expression can inhibit the proliferation of glioma cells and enhance sensitivity to TMZ chemotherapy.5.PLK4 can be combined with I?K? to activate the NF-?B signaling pathway and promote the expression of BCL2 anti-apoptotic gene,so as to reduce the sensitivity of glioma cells to TMZ chemotherapy,which is consistent with the prediction of our research group.6.Combination of PLK4-inhibited and TMZ manifests a therapeutic effect on glioma.