Th Title Loaded From File miR-326 has not been examined to date. In this study, we show for the first time that miR-326 potently and directly regulates NOB1. Furthermore, we demonstrate that miR-326 inhibits the activation of the MAPK pathway, which is one of the core pathways in glioma, and miR-326 overexpression impaired cell viability and the invasiveness of glioma cells. Taken together, these results establish miR-326 as a regulator of NOB1 expression and MAPK pathway activity in human glioma, with potential therapeutic implications.gave written informed consent according to a study protocol that was approved by Tissue Committee and Research Ethics Board of Second Military Medical University. Normal brain tissues were obtained from 8 patients who underwent surgical resections for reasons other than malignancy, such as cerebral trauma, for whom a partial resection of normal brain tissue was required as decompression treatment for their severe head injuries to reduce increased intracranial pressure under the permission of each of the patient’s family. The pathological diagnoses of all enrolled patients were confirmed by two different pathologists, according to the WHO grading system.Cell CultureHEK293T cells and human glioma cells A172, U373 and U87 obtained from the American Type Culture Collection (ATCC) were cultured in DMEM supplemented with 10 fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL of streptomycin. Cells were cultured at 37uC in a humidified atmosphere of 5 CO2.Materials 1315463 and Methods Tissue Preparation thics StatementThe specimens of the glioma patients used in this study were provided by the Shanghai Institute of Neurosurgery. All patientsFigure 1. Identification of miR-326 target sites within the NOB1 39-UTR. (A) Ideograph of NOB1 mRNA. One miR-326 binding site was detected in the NOB1 39-UTR. The sequence of wild-type (WT) and mutant (MT) miR-326 target sites in the NOB1 39UTR are shown. A point mutation (underlined) was made in the seed region to block the binding between miR-326 and mRNA. (B) A luciferase reporter assay was used to confirm the contribution of the four miR-326 target sites. U87 cells were co-transfected with luciferase reporter plasmids containing either WT or MT miR-326 target sites and miR-326 or miR-NC precursors. miR-326 and full-length wild-type NOB1 39UTR Title Loaded From File decreased luciferase activity. All results were derived from independent experiments performed in triplicate. miR-NC, non-effective control miRNA; *indicates a significant difference from the miR-NC precursor and co-transfected control plasmids (P,0.01). doi:10.1371/journal.pone.0068469.gMicroRNA-326 as a Tumor Suppressor in GliomaFigure 2. Overexpression of miR-326 down-regulated NOB1 mRNA and protein expression. (A) HEK293T cells were transfected with previously generated miR-326, miR-NC, NOB1 shRNA or untreated control vectors. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was probed with anti-NOB1 and anti-GAPDH antibodies. miR-326 significantly repressed NOB1 protein expression (B, C) The downregulation of NOB1 mRNA following miR-326 transfection was determined by RT-PCR (*p,0.01). doi:10.1371/journal.pone.0068469.gPlasmids Constructs and Luciferase Reporter AssayThe 39-untranslated region (39-UTR) of NOB1 and a mutation sequence were amplified by PCR using the primers that included a Bgl II restriction site on the 59 and 39 strands. The PCR products were inserted into the Bgl II sites of the pGL3-control vector (Pr.Th miR-326 has not been examined to date. In this study, we show for the first time that miR-326 potently and directly regulates NOB1. Furthermore, we demonstrate that miR-326 inhibits the activation of the MAPK pathway, which is one of the core pathways in glioma, and miR-326 overexpression impaired cell viability and the invasiveness of glioma cells. Taken together, these results establish miR-326 as a regulator of NOB1 expression and MAPK pathway activity in human glioma, with potential therapeutic implications.gave written informed consent according to a study protocol that was approved by Tissue Committee and Research Ethics Board of Second Military Medical University. Normal brain tissues were obtained from 8 patients who underwent surgical resections for reasons other than malignancy, such as cerebral trauma, for whom a partial resection of normal brain tissue was required as decompression treatment for their severe head injuries to reduce increased intracranial pressure under the permission of each of the patient’s family. The pathological diagnoses of all enrolled patients were confirmed by two different pathologists, according to the WHO grading system.Cell CultureHEK293T cells and human glioma cells A172, U373 and U87 obtained from the American Type Culture Collection (ATCC) were cultured in DMEM supplemented with 10 fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL of streptomycin. Cells were cultured at 37uC in a humidified atmosphere of 5 CO2.Materials 1315463 and Methods Tissue Preparation thics StatementThe specimens of the glioma patients used in this study were provided by the Shanghai Institute of Neurosurgery. All patientsFigure 1. Identification of miR-326 target sites within the NOB1 39-UTR. (A) Ideograph of NOB1 mRNA. One miR-326 binding site was detected in the NOB1 39-UTR. The sequence of wild-type (WT) and mutant (MT) miR-326 target sites in the NOB1 39UTR are shown. A point mutation (underlined) was made in the seed region to block the binding between miR-326 and mRNA. (B) A luciferase reporter assay was used to confirm the contribution of the four miR-326 target sites. U87 cells were co-transfected with luciferase reporter plasmids containing either WT or MT miR-326 target sites and miR-326 or miR-NC precursors. miR-326 and full-length wild-type NOB1 39UTR decreased luciferase activity. All results were derived from independent experiments performed in triplicate. miR-NC, non-effective control miRNA; *indicates a significant difference from the miR-NC precursor and co-transfected control plasmids (P,0.01). doi:10.1371/journal.pone.0068469.gMicroRNA-326 as a Tumor Suppressor in GliomaFigure 2. Overexpression of miR-326 down-regulated NOB1 mRNA and protein expression. (A) HEK293T cells were transfected with previously generated miR-326, miR-NC, NOB1 shRNA or untreated control vectors. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane, which was probed with anti-NOB1 and anti-GAPDH antibodies. miR-326 significantly repressed NOB1 protein expression (B, C) The downregulation of NOB1 mRNA following miR-326 transfection was determined by RT-PCR (*p,0.01). doi:10.1371/journal.pone.0068469.gPlasmids Constructs and Luciferase Reporter AssayThe 39-untranslated region (39-UTR) of NOB1 and a mutation sequence were amplified by PCR using the primers that included a Bgl II restriction site on the 59 and 39 strands. The PCR products were inserted into the Bgl II sites of the pGL3-control vector (Pr.
