title: Neurotensin signaling stimulates glioblastoma cell proliferation by upregulating c-Myc and inhibiting miR-29b-1 and miR-129-3p.
abstract: BACKGROUND
  Neurotensin (NTS) and its primary receptor NTSR1 are implicated in cancer progression. Aberrant expression of NTS/NTSR1 contributes to the proliferation of glioblastoma cells; however, the mechanism is not fully understood.
  
  
  METHODS
  Microarray and real-time PCR were performed to identify the NTS-regulated micro (mi)RNAs. The targets of the miRNAs were identified by luciferase assays and immunoblot analysis. The c-Myc binding sites in the miR-29b-1 and cyclin-dependent kinase (CDK)4 promoters were identified through chromatin immunoprecipitation assay. Cell proliferation was evaluated by Cell Counting Kit-8 assay and flow cytometry analysis. An orthotopic xenograft model demonstrated the role of NTS/NTSR1 and miRNAs in glioblastoma growth in vivo.
  
  
  RESULTS
  Pharmacological inhibition or small interfering NTSR1 treatment blocked glioblastoma cell cycle progression in the G1 phase with a concomitantly decreased expression of CDK6, CDK4, and c-Myc. Knockdown of NTSR1 increased the expression of miR-29b-1 and miR-129-3p, which were responsible for the decreased CDK6 expression. NTS/NTSR1 signaling activated the transcription factor c-Myc in U87 cells, leading to increased CDK4 expression and repressed miR-29b-1 expression. Knockdown of NTSR1 decreased the glioblastoma growth in vivo and significantly prolonged the survival time of the tumor-bearing mice, an effect that can be largely reversed by antagomir.
  
  
  CONCLUSIONS
  Our study showed a novel regulatory mechanism of NTS/NTSR1, an upstream signaling of miRNAs and c-Myc, in glioblastoma progression. The inhibition of the NTSR1 function or the upregulation of miR-29b-1 and miR-129-3p expression impaired glioma cell proliferation. These results suggested that the NTS/NTSR1/c-Myc/miRNA axis may be a potential therapeutic target for glioblastoma therapy.
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Qing
authors.initials: Q
authors.lastname: Ouyang
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Ji
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Zhen
authors.initials: Z
authors.lastname: Dong
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Rui
authors.initials: R
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Lunshan
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Hongjuan
authors.initials: H
authors.lastname: Cui
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Minhui
authors.initials: M
authors.lastname: Xu
authors.affiliation: Department of Neurosurgery, Daping Hospital, Third Military Medical University, Chongqing, China (O.Q., Z.J., X.L., X.M., Y.L.); Biomedical Analysis Center, Third Military Medical University, Chongqing, China (C.G.); State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, Southwest University, Chongqing, China (D.Z., Y.R., C.H.); State Key Laboratory of Trauma, Burns and Combined Injury, Department one, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China (L.L.).
authors.forename: Liang
authors.initials: L
authors.lastname: Yi
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D035683:MicroRNAs / Q000235:genetics*; D009496:Neurotensin / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018028:Receptors, Neurotensin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: C532045:MIRN29a microRNA, human; D035683:MicroRNAs; C541915:Mirn129 microRNA, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018028:Receptors, Neurotensin; C114036:neurotensin type 1 receptor; D009496:Neurotensin
keywords: cell cycle; glioblastoma; miRNAs; neurotensin; signal transduction