The Study On The Mechanism Of Neurotensin Receptor 1 Induces Cell Invasion Of Glioblastoma

Background:Human glioblastoma is the most common and lethal malignant tumor of central never system,which accounts for approximately 60% of adult primary malignant brain tumors.Despite glioblastoma multiforme(GBM)patients are treated with surgical resection,radiation therapy,chemotherapy and targeted therapy,the median overall survival of patients with GBM remain 8-15 months.High invasiveness is one of the hallmarks of human glioblastoma,which is closely related to the aggressiveness and the poor prognosis of patients with glioblastoma.Local invasive growth of glioblastoma complicates complete surgical resection,and the remnant glioblastoma mass inevitably leads to easy recurrence and high mortality after surgery,therefore,the radical resection of glioblastoma is not curative.In addition to migratory activity,proteolytic degradation of the extracellular matrix(ECM)is a critical element of cancer invasion.In general,cancer cells produce matrix metalloproteinases(MMPs)and urokinase-type plasminogen activator to degrade the extracellular matrix components.Moreover,tumor invasion is controlled by the local microenvironment and involves autocrine factors and paracrine factors.The neurotensin receptor 1(NTSR1)stimulation triggers the activation of various signaling pathways,such as Rho GTPases,focal adhesion kinases,and extracellular signal regulated kinase,leading to gene transcriptional activation and tumor growth.In our previous work,we found that NTSR1 is commonly overexpressed in human glioma,and the expression levels of NTSR1 are correlated positively with the pathological grade of glioma,while negatively with the prognosis of patients with glioma.Several reports implicate that once activated by ligands,NTSR1 signaling is involved in a variety of processes such as cell proliferation,cell apoptosis and the stemness maintenance of glioma,and these effects can be abolished by SR48692(a specific antagonist of NTSR1)or NTSR1 knockdown.In our previous report,we have reported that NTSR1 is critical for glioma invasion,and ERKpathway seem to be involved in this process,however,the molecular mechanisms underlying NTSR1 induced glioma invasion need further investigation.MicroRNAs(miRNAs)are a class of short and single stranded non-coding RNA,which bind to targets with near perfect complementarity to bring about the translational repression or transcript degradation of targets.Accumulating evidence has showed that miRNAs participate in the progression of cancers,and which have been categorized either as oncogenic miRNAs and tumour suppressor miRNAs depending upon their function.In particular,it has been shown that miRNAs are aberrantly expressed in glioma and involved in glioma proliferation.However,the relationships between miRNAs and glioblastoma invasion need further investigation.Objective:1.To test the effects of NTSR1 on the cell migration and invasion of glioblastoma.2.To investigate the molecular mechanism of NTSR1-induced glioblastoma cell migration and invasion.Materials and methods:1.siRNAs were used to knocked down the levels of NTSR1,Jun,and SOCS6 in U87 MG and U118 MG cells.Jun vector was used to overexpress the levels of Jun in U87 MG and U118 MG cells.Western blotting was used to detect the levels of NTSR1,Jun,and SOCS6.CCK-8 assay was performed to test the proliferation of U87 MG and U118 MG cells.The wound-healing assay was performed to detect the migratory activity of U87 MG and U118 MG cells.The transwell assay was used to detect the invasive activity of U87 MG and U118 MG cells.ChIP assay was used to test the interaction of Jun and miR-494 promoter.The shNTSR1-U87 MG and shNTSR1+miR-494-U87 MG cells were used to establish xenografts.MRI was used to observe the tumor volumes.H&E staining was used to observe the invasive activity of xenografts.Kaplan-Meier was performed to detect the overall survival time of tumor-bearing mice.Results:1.NTSR1 promotes the migration and invasion of glioblastoma cell lines.Silencing NTSR1 didn’t suppress cell proliferation within 24 hours,however,siNTSR1 suppressed the migratory activity of U87 MG and U118 MG cells.SiNTSR1 attenuated the invasive activity of U87 MG and U118 MG cells.The average volumes of xenografts were smaller in the shNTSR1-U87 MG group compared with the sc-shRNA-U87 MG group.The sc-shRNA-U87 MG group grew as extensively diffuse tumor cell clusters invading deep into the brain parenchyma.In contrast,NTSR1 knockdown produced tightly packed glioblastoma with relatively smooth borders.In addition,NTSR1 knockdown prolonged the overall survival time of tumor-bearing mice.2.NTSR1 regulates Jun/miR-494/SOCS6 signaling pathway.NTSR1 knockdown suppressed the expression levels of miR-494 and transcription factor Jun in U87 MG and U118 MG cells.ChIP analysis with a primer set covering the Jun binding site confirmed the physical association of Jun with miR-494 promoter by using an Jun antibody in U87 MG cells.Jun knockdown significantly suppressed the expression levels of miR-494,however,Jun overexpression promoted the expression levels of miR-494 in U87MG and U118 MG cells.Targetscan,pictar,and microrna databases showed that the3’ UTR of SOCS6 comprising three regions that matched the seed sequences of miR-494.The luciferase activity assay showed that miR-494 significantly suppressed luciferase activity in the WT reporter but not the MT reporter.SiNTSR1 elevated the expression of SOCS6 in U87 MG and U118 MG cells with sc-miRNA transfection,while miR-494 upregulation suppressed the expression of SOCS6 in both sc-siRNA and siNTSR1 backgrounds.Anta-miR-494 restored SOCS6 protein levels in U87 MG and U118 MG cells with sc-siRNA transfection rather than in U87 MG and U118 MG cells with siNTSR1 transfection.NTSR1 knockdown increased the levels of SOCS6 protein,however,miR-494 suppressed the expression levels of SOCS6.3.NTSR1 induces glioblastoma migration and invasion via miR-494/SOCS6 axis.MiR-494 upregulation enhanced glioblastoma cell migration and invasion in U87 MG and U118 MG cells,and restored the migratory and invasive activity of siNTSR1-transfected glioblastoma cells.Anta-miR-494 suppressed the cell migration and invasion of U87 MG and U118 MG cells with sc-siRNA transfection.SOCS6 knockdown promoted the migratory and invasive activity of U87 MG and U118 MG cells,and restored the migratory and invasive activity of U87 MG and U118 MG cells with siNTSR1 transfection.The average volumes of xenografts were larger in the shNTSR1+miR-494-U87 MG group compared with the shNTSR1-U87 MG group.The shNTSR1+miR-494-U87 MG group generated orthotopic glioblastoma was more aggressive than that of the shNTSR1-U87 MG group.miR-494 upregulation attenuated the survival advantage of the shNTSR1-U87 MG group.Conclusions:In summary,we reported that NTSR1 knockdown suppressed glioblastoma invasion in vitro and in vivo.NTSR1 regulated Jun/miR-494/SOCS6 axis in glioblastoma,and this axis was critical for NTSR1-induced glioblastoma migration and invasion.This is a novel mechanism that could be exploited to develop a new therapeutic strategy for human glioblastoma.