Hypoxic Glioma-Derived Exosomes Deliver MicroRNA-151a-3p To Induce Invasion And Proliferation By Targeting PDE4D

Objective:The purpose of this study is to explore how H-GDEs mediate the EMT of GBM cells,and to reveal the molecular mechanism by which H-GDEs significantly increases the ability to invasion,migration,proliferation and EMT in GBM,in order to produce new targets and strategies for precision chemotherapy.Methods:1.The culture media of normoxic and hypoxic glioma U87-MG cell lines were used for exosomes isolation with exosomes isolation kit;High throughput sequencing combined with transcriptomics were performed to detect the differentially expressed microRNAs and downstream target genes in exosomes derived from hypoxic and normoxic glioma cells.Then use lentivirus vector to knock down miR-151a-3p and over-express PDE4D.2.The expressions of CD9,CD63,TSG101 between normoxic and hypoxic condition were detected by Western blot,and the expression of HIF-1α was measured by Western blot to verify the hypoxia condition was successfully established.Then we knockdown HIF-1α by HIF-1α-siRNA and qPCR was performed to demonstrated the expression of miR-151a-3p in cell and exosomes.To identify the role of PDE4D in gliomas,we analyzed PDE4D expression in gliomas and normal sample data derived from TCGA using the GEPIA platform.The pathological samples of patients with different grades of glioma were collected,the results revealed that the expression of PDE4D was significantly increased in glioma samples,especially in GBM.Moreover,Kaplan-Meier survival analysis demonstrated that patients with low PDE4D expression had a significantly longer overall survival(OS)than those with high PDE4D expression.3.High-content screening cell counting,Edu and colony formation assay was used to detect cell proliferation in experimental group and control group.The scratch healing experiment,transwell chamber experiment and the adhesion rate of cells was used to evaluate the effect of miR-151a-3p and PDE4D on GBM cell invasion and migration.Stained with rhodamine phalloidin and F-actin was labeled and observe the change of its morphology under high-content screening.The changes of EMT-related proteins(Ecadherin,N-cadherin and Vimentin)were detected by Western blot,qPCR and immunofluorescence.4.miR-151a-3p and PDE4D interaction detection:The direct binding site of miR151a-3p to PDE4D was detected by luciferase reporter gene and RIP kit.The change of PDE4D expression level after miR-151a-3p knock down was detected by Western blot,and correlation analysis between the miR-151a-3p and PDE4D was conducted based on clinical data.Protein interaction detection:Co-localization of PDE4D and FAK was detected by cellular immunofluorescence.The interaction between PDE4D and FAK proteins was verified by protein immunoprecipitation.5.Animal experiments:glioma xenograft transplanted tumor model was established by injecting U251-Luc cells into armpit of nude mice.Tumor development was assessed every three days after inoculation.The tumor volume is calculated by V=0.5 × ab2,where "a" and "b" represent the longest and shortest diameters respectively.Finally,the expression of E-cadherin,N-cadherin,Vimentin and Ki-67 in each group was detected by immunohistochemistry and immunofluorescence.Results:1.The concentration and diameter of exosomes isolated under hypoxia and normoxia conditions meet the needs of subsequent tests,and the surface markers of exosomes are specific.It is proved that the method of exosomes isolation is effective and reliable.2.Based on the TCGA database,the expression level of PDE4D mRNA in glioma tissue was significantly higher than that in normal samples.At the same time,in the cohort of glioma patients in this study,miR-151a-3p and PDE4D can be used as an independent predictor of the prognosis of patients,the expression level of PDE4D and miR-151a-3p with an obviously negative correlation of the worse prognosis of patients(p<0.0001).3.H-GDEs significantly promote the ability of proliferation,invasion and migration.The epidermal marker E-cadherin protein expression was significantly downregulated,mesenchymal marker N-cadherin and Vimentin protein was significantly upregulated treated with H-GDEs,H-GDEs promoted the occurrence of EMT in GBM cells.Effect of H-GDEs can be reversed by the exosome inhibitor GW86494.Exo-miR-151a-3p is specifically expressed and secreted by hypoxic GBM cells and acts as an oncogene miRNA in GBM,miR-151a-3p-knockdown U251 cells were mainly negatively correlated with invasion,migration,proliferation and EMT.Knockdown of miR-151a-3p result in the significantly regulation of epidermal marker E-cadherin expression,and the mesenchymal marker N-cadherin and Vimentin significantly decreased,miR-151a-3p-knockdown inhibited the occurrence of EMT in GBM cells.5.A luciferase reporter assay,RIP detection kit and Western blot analysis was conducted to confirmed have the direct binding between miR-151 a-3P and PDE4D,and there exists interaction between them.6.PDE4D overexpression showed that the ability to proliferation,invasion was significantly enhanced in GBM cell.the epidermal marker E-cadherin protein expression was significantly downregulated,mesenchymal marker N-cadherin and Vimentin protein was significantly upregulated with PDE4D overexpression,which demonstrated PDE4D overexpression promoted the occurrence of EMT in GBM cells.7.U251 cells that were transfected with sh-miR-151a-3p(depletion)or ov-PDE4D(overexpression)either alone or with PDE4D(overexpression)lentivirus construct,we performed functional rescue assays to confirm further that the observed PDE4Dmediated phenotypes were facilitated by dysregulation of exo-miR-151a-3p expression.Our data demonstrate that PDE4D can be cyclised by miR-151a-3p.8.We performed GO and KEGG analyses found that differentially Expressed microRNAs and downstream Genes were enriched in Focal adhesion signal way,we performed confocal microscope and Co-IP experiments and confirmed that PDE4D colocalized with FAK in the cytoplasm,and PDE4D had direct interaction with FAK.The co expression of PDE4D and FAK was found in the immunofluorescence of glioma patients’ tissue and C57 mouse glioma,which was positively correlated with the invasion,migration and malignancy of glioma.9.In vivo,miR-151a-3p knockdown significantly inhibited the growth of subcutaneous xenograft model in vivo,PDE4D overexpression significantly promoted the growth of subcutaneous xenograft model in vivo.Immunohistochemical and IF results showed that the expression of Ki-67,N-cadherin,vimentin was lower in the miR-151a-3p knockdown group.the expression of E-cadherin was higher in the miR151a-3p knockdown group.PDE4D overexpression group promoted the expression of Ki-67,N-cadherin,vimentin,and inhibited the expression of E-cadherin.Conclusion:H-GDEs promotes normoxia glioma cells invasion,migration,proliferation and EMT in vitro.The significantly increased expression of miR-151a-3p and PDE4D in GBM is significantly correlated with the prognosis of patients and can be used as a prognostic marker for GBM patients.