title: Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness.
abstract: BACKGROUND
  Few studies have associated microRNAs (miRNAs) with the hedgehog (Hh) pathway. Here, we investigated whether targeting smoothened (SMO) with miR-326 would affect glioma biological behavior and stemness.
  
  
  METHODS
  To investigate the expression of SMO and miR-326 in glioma specimens and cell lines, we utilized quantitative real-time (qRT)-PCR, Western blot, immunohistochemistry, and fluorescence in situ hybridization. The luciferase reporter assay was used to verify the relationship between SMO and miR-326. We performed cell counting kit-8, transwell, and flow cytometric assays using annexin-V labeling to detect changes after transfection with siRNA against SMO or miR-326. qRT-PCR assays, neurosphere formation, and immunofluorescence were utilized to detect the modification of self-renewal and stemness in U251 tumor stem cells. A U251-implanted intracranial model was used to study the effect of miR-326 on tumor volume and SMO suppression efficacy.
  
  
  RESULTS
  SMO was upregulated in gliomas and was associated with tumor grade and survival period. SMO inhibition suppressed the biological behaviors of glioma cells. SMO expression was inversely correlated with miR-326 and was identified as a novel direct target of miR-326. miR-326 overexpression not only repressed SMO and downstream genes but also decreased the activity of the Hh pathway. Moreover, miR-326 overexpression decreased self-renewal and stemness and partially prompted differentiation in U251 tumor stem cells. In turn, the inhibition of Hh partially elevated miR-326 expression. Intracranial tumorigenicity induced by the transfection of miR-326 was reduced and was partially mediated by the decreased SMO expression.
  
  
  CONCLUSIONS
  This work suggests a possible molecular mechanism of the miR- 326/SMO axis, which can be a potential alternative therapeutic pathway for gliomas.
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Wenzhong
authors.initials: W
authors.lastname: Du
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Lingchao
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Zhijin
authors.initials: Z
authors.lastname: Dou
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Xuhui
authors.initials: X
authors.lastname: Lei
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Liang
authors.initials: L
authors.lastname: Chang
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Jinquan
authors.initials: J
authors.lastname: Cai
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Yuqiong
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Dongbo
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Ying
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Yongli
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China (W.D., X.L., Z.D., X.L., L.C., J.C., Y.C., D.Y., Y.S., Y.L., C.J.); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (L.C.).
authors.forename: Chuanlu
authors.initials: C
authors.lastname: Jiang
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D005909:Glioblastoma / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D053208:Kaplan-Meier Estimate; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D035683:MicroRNAs / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D000072086:Smoothened Receptor; D015996:Survival Rate; D015854:Up-Regulation
chemical_list: D053823:Hedgehog Proteins; C545322:MIRN326 microRNA, human; D035683:MicroRNAs; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor
keywords: SMO; glioma; hedgehog; miRNA; stemness