| BackgroundAbout 80%of intracranial primary malignant tumor are gliomas.Glioblastoma multiforme(GBM)is one of the most lethal types of human cancer.Despite numerous advances in chemotherapeutic protocols,GBM therapy still relies on the palliative efficacy of a combination of surgery,radiation and temozolomide.Due to GBM’s extremely malignant characteristics,such as robust invasiveness,rapid proliferation and increased angiogenesis,patients with GBM have a median survival time of only 14 months.G protein-coupled receptor(GPCR)is the most diverse type of receptor superfamily,which is characterized by 6 transmembrane helical structures and mediates multiple ligands,and is closely related to the occurrence and development of multiple tumors.G protein-coupled receptor kinases(GRKs)belong to the serine/threonine kinase family that bind and phosphorylate agonist-activated G protein-coupled receptors(GPCRs),leading to their desensitization.Seven GRK subtypes(GRK1-7)have been characterized and divided into three groups according to their sequence homology.Rhodopsin kinases or visual GRK subfamily(GRK1 and GRK7),the β-adrenergic receptor kinases subfamily(GRK2/GRK3)and the GRK4 subfamily(GRK4,GRK5 and GRK6).The distribution of GRK4 is limited and mainly exists in the testis,which can phosphorylate sperm on the surface of GPCR.There is also a small amount of GRK4 expression in parts of brain tissue and kidney.Studies demonstrate that GRK expression and activity are impaired in many pathological conditions.Among the GRKs,GRK5 is the most studied due to its involvement in several pathologic conditions,including cancer.Indeed,GRK5 is able to both inhibit cancer progression,through the desensitization of GPCR and non GPCR-receptors(PDGFR,PGE2R,TSH),and induce tumor growth,acting on non-receptor substrates(NPM1,AUKA,p53),depending on its subcellular localization and on the type of cancer.Chromosome translocation in the region of 10q24 has been observed in several tumors,including glioma,suggesting that an alteration of genes in this region can affect tumor progression.Since GRK5 maps on chromosome 10 at the region of q24,it is likely that there is a correlation between an alteration in GRK5 expression and certain glial tumors.The NF-κB family of widely expressed transcription factors,regulate cell cycle progression,cell proliferation and apoptosis in tumors.A previous study reported that NF-κB regulates GBM secretion.Inhibition of the NF-κB pathway resulted in the downregulation of IL8,IL6,CCL2,IL-1β and CXCL14 and in the inhibition of their related pathways.GRK5 is an important regulator of NF-κB signaling,and it can enhance its activity.Conversely,p50 and p65 can also directly bind to GRK5 DNA in the nucleus,thus upregulating the GRK5 level.However,the role of GRK5 in the GBM is so far poorly understood,which prompted us to examine the correlation between GRK5 expression and NF-κB signaling in gliomas.We examined the expression level and subcellular localization of GRK5 in glioma tissues at different levels,and analyzed its distribution characteristics.The effect of downregulation of GRK5 on the biological activity of glioma cells was studied by in vitro and in vivo experiments.Using the NF-κB stimulator:phorbol myristate acetate(PMA),the correlation between GRK5 and NF-κB signaling pathway in glioma,and the effect of GRK5 knockdown on the downstream products of NF-κB(CCL2,IL6 and IL8)was further studied.ObjectivesPart I Study on the expression and localization of GRK5 in gliomas1.To investigate the expression of GRK5 in glioma tissues at different pathological levels;2.To investigate the cell localization and distribution characteristics of GRK5 in glioma tissues;3.To investigate the relationship between GRK5 and glioma stem cells Part Ⅱ Study on the functions of GRK5 and the internal relationship of GRK5-NF-κB signaling pathway in glioma cell1.To investigate the expression of GRK5 in glioma cell lines and establish GRK5 konckdown(GRK5-KD)stable cell lines.2.To investigate the effect of GRK5 on the biological activity(including proliferation,migration,invasion and apoptosis)of glioma cells by in vitro and in vivo experiments.3.To investigate the correlation between GRK5 and NF-κB signaling pathway in glioma,as well as the influence of GRK5 down-regulation on the downstream products of NF-κB signaling pathway.MethodsPart Ⅰ Study on the expression and localization of GRK5 in gliomas1.GRK5 expression levels in 10 normal brain specimens and 110 glioma specimens of different pathological levels were detected by western blotting,real-time PCR and immunohistochemistry.2.The subcellular localization and distribution characteristics of GRK5 in glioma tissues,as well as the relationship between GRK5 and tumor blood vessels were analyzed by immunohistochemistry and immunofluorescence;3.The co-expression of GRK5 and tumor stem cell marker CD133 was observed by double immunofluorescence.Part Ⅱ Study on the functions of GRK5 and the internal relationship of GRK5-NF-κB signaling pathway in glioma cell1.The expression levels of GRK5 in the three glioma cell lines of U87,U251 and T98G were detected by western blotting and real-time PCR.And the most suitable cell line for subsequent functional experiments were selected.2.GRK5-KD and NC stable cell lines was generated with lentivirus transfection method.Cell proliferation was detected by using CCK 8 assay.Cell migration was detected by using the scratch assay.Cell invasion was detected by using matrigel transwell assay.Cell apoptosis was detected by flow cytometry.Through the establishment of nude mouse transplantation tumor model,research GRK5 expression change in environment impact on the proliferation of glioma.A tumor xenograft model was established in female BALB/c athymic mice,to study the effect of GRK5 on glioma proliferation in vivo.3.Using the NF-κB stimulator PMA,the correlation between GRK5 and NF-κB signaling pathway in glioma was detected by western blotting.And the influence of GRK5 down-regulation on the downstream products of NF-κB signaling pathway were quantitatively analyzed by ELISA kit.ResultsPart Ⅰ Study on the expression and localization of GRK5 in gliomas1.qRT-PCR data showed that GRK5 was abnormally increased in glioma tissues compared to normal brain tissues.And the expression level of GRK5 in patients with high-grade gliomas(WHO Ⅲ/Ⅳ)was significantly higher than that in low-grade gliomas(WHO Ⅱ).Western blotting also showed that the GRK5 protein level was upregulated in gliomas.There was an association between higher GRK5 expression and high malignancy in glioma specimens.2.Immunohistochemical analysis showed that GRK5 was mainly confined in the cytoplasm,and its degree of enrichment in the cytoplasm of LGGs was low,whereas in the cytoplasm of HGGs,the degree of enrichment was high.Our data indicated that GRK5 expression was positively correlated with pathological grade.We also found a positive correlation between GRK5 and Ki-67 by immunohistochemical analysis.3.By immunohistochemistry,we found a positive correlation between GRK5 and the vessel marker CD34.Furthermore,GRK5 expression was detected close to the lumen of several tumor blood vessels in human gliomas,especially in the high-grade glioma tissue.4.By dual immunofluorescence analysis(8 WHO Ⅲ samples and 15 WHO IV samples),GRK5/CD133 co-expressing cells were detected in several High grade glioma specimens.It was found that most CD 133+ cells co-expressed GRK5 in HGGs.Part Ⅱ Study on the functions of GRK5 and the internal relationship of GRK5-NF-κB signaling pathway in glioma cell1.The relative expression of GRK5 in different cell lines(U87,U251 and T98G)was detected.We found that the GRK5 mRNA level was relatively higher in U251 cells than that in the other glioma cell lines.Using lentivirus transfection,a GRK5-knockdown stable cell line was generated.2.The transwell assay showed that the down-regulation of GRK5 significantly inhibited cell invasion.On the other hand,the wound healing assay showed that GRK5 knockdown slowed the wound closure speed of tumor cells.3.The effect of GRK5 on glioblastoma cell proliferation was examined by the CCK-8 assay.The viability of GRK5-KD cells significantly decreased compared to NC cells.The role of GRK5 in vivo was confirmed by the tumor xenograft model.After 30 days of subcutaneous inoculation,the cell suspension inoculation developed into a solid tumor xenograft in each mouse.The growth curve of tumor xenografts showed that the down-regulation of GRK5 slowed tumor growth in vivo,and no statistical difference was observed between NC and U251 groups.The average tumor weight in mice of the NC group was higher than that in mice of the GRK5-KD group.These findings suggested that GRK5 may promote the growth of gliomas in vitro and vivo.4.To further confirm the role of GRK5 in glioblastoma cells,the number of apoptotic cells was determined by flow cytometry.Using Annexin V-PE/7-amino-actinomycin D double-stained U251 cells,the externalization of phosphatidylserine was detected by flow cytometry.The percentage of apoptotic cells significantly increased in GRK5-KD cells(42.35±3.56)%compared with those of the NC(4.45±0.51)%and U251 group(2.68±0.31)%.The Bcl-2 family is an important regulatory factor in various apoptotic pathways.The loss of GRK5 down-regulated the Bcl-2 protein level.These data suggested that GRK5 may have the ability to induce apoptosis in glioblastoma cells.5.The effect of GRK5 on the protein level of NF-κB pathway was detected by using NF-κB stimulant PMA.Using the known NF-κB stimulator PMA,the relationship between GRK5 and the NF-κB pathway in gliomas was investigated by western blotting analysis.After 24h of PMA treatment,the expression of phosphorylated IκBα(pIκBα),p50,p65 and GRK5 was increased,suggesting that PMA can increase the levels of GRK5-NF-κB signaling proteins in glioblastoma cells(independent-samples t test).Next,the depletion of GRK5 decreased the levels of plKBa,p65 and p50 in glioblastoma cells,and this effect was partially reversed after treatment with PMA.This study showed an interaction between GRK5 and NF-κB signaling in glioblastoma cells.6.In addition,ELISA was used to quantify the levels of CCL2,IL-6 and IL-8 in the CM of glioblastoma cells,which are the downstream cytokines and chemokines regulated by NF-κB signaling.Data analysis showed that all of the three secretory products decreased in the CM of glioblastoma cells after GRK5 knockdown.However,treatment of cells with PMA reversed this effect by increasing their levels.ConclusionPart Ⅰ Study on the expression and localization of GRK5 in gliomas1.We confirmed that the abnormal high GRX5 expression in gliomas is positively correlated with the pathological grade of glioma.2.GRK5 may be important for angiogenesis in gliomas as well as for maintaining of the tumorigenic potential of GSCs.Part Ⅱ Study on the functions of GRK5 and the internal relationship of GRK5-NF-κB signaling pathway in glioma cell1.biological activities(proliferation,migration,invasion and apoptosis)were altered in gliomas due to the loss of GRK5,indicating an oncogene-like role for GRK5 in the pathogenesis and development of glioma.2.GRK5 interacts with classic tumor signaling pathway NF-κB in glioma.3.Down-regulation of GRK5 inhibits downstream cytokines and chemokines of the NF-κB pathway. |
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