And maintained under specific pathogen-free conditions in Second Military Medical Title Loaded From File University. When the female BALB/cnu mice were 7? weeks of age, each mouse was inoculated with 1.56107 U373 cells transfected with miR-326 or miR-control or NOB1 shRNA in 0.2 mL of medium subcutaneously in the forelimb, the mouse injected mock-infected cells as control. Tumor sizes were measured every three days in two dimensions using a caliper, and the volume (mm3) was calculated using the formula V = 0.5* larger diameter *(smaller diameter)2. The tumors were excised and weighed from the sacrificed mice after 21 days. All procedures involving animals were approved by the Animal Care and Use Committee in Second Military Medical University.Statistical AnalysisThe Student’s IF formamidase from Helicobacter pylori (PDB accession code 2E2L), and t-test was used for statistical analysis in assays performed on glioma cell lines. For experiments of glioma tissue samples, relative expression levels of NOB1 mRNA for each group normal brain, low-grade glioma (LGG) and high-grade glioma (HGG) were expressed as mean 6 SE, the Mann-Whitney U test was used to compare the differences between groups. When studying the relationship between NOB1 expression and patients’ prognosis, we first grouped glioma patients of all grades to those live longer than 24 months and those live less than 24 months, Mann-Whitney U test was then applied to compare the expression of NOB1 between these two groups. Then the prognosis in lowgrade glioma and high-grade glioma patients were 18204824 also analyzed 1315463 separately. Fisher’s exact test was used to compare the immunolabelling results of NOB1 between high-grade and low-grade gliomas. SPSS 15.0 (SPSS Inc, Chicago, USA) was used for the statistical analysis and a significance level of P,0.05 was used to evaluate the difference between groups.Measurement of Phosphorylation of Signaling ProteinsThe changes in phosphorylation of selected proteins in certain of signaling pathways were analyzed with Proteome Profiler Array kit (ARY003; R D Systems, Minneapolis, MN) according to the manufacturer’s instructions. In brief, human A172 and U373 glioma cells were grown, and then infected with miR-326 precursor, control precursor or NOB1-shRNA. At the designated times, each dish was washed twice with phosphate-buffered saline and processed according to the kit protocol. Incubations with the array contained 300 ug of lysate protein. Net integrated pixel density for each spot (an average of duplicate spots after subtraction of average background density) was determined by densitometry and analyzed using Quantity One (ISBE, Sheffield,Figure 8. Expression of NOB1 protein in glioma and normal brain tissue samples. Immunohistochemical staining of normal brain tissue (A, B), grade I (C, D), grade II (E, F), grade III (G, H) and grade IV (I, J) glioma tissue specimens expressing NOB1. NOB1 staining was stronger in high-grade gliomas than that in low-grade gliomas. No significant staining was observed in normal brain tissues. doi:10.1371/journal.pone.0068469.gMicroRNA-326 as a Tumor Suppressor in GliomaFigure 9. Schematic diagram illustrating the interplay among miR-326, NOB1 and the MAPK pathway in glioma. miR-326, as a tumor suppressor by targeting NOB1, decreased the tumorigenesis of glioma cells in vivo and in vitro through the modulation of the MAPK pathway. Overexpression of miR-326, which suppresses the expression of NOB1, activates the MAPK patheay by increasing the phosphorylation of ERK1/2, JNK and p38 MAPK, which inhibits the cel.And maintained under specific pathogen-free conditions in Second Military Medical University. When the female BALB/cnu mice were 7? weeks of age, each mouse was inoculated with 1.56107 U373 cells transfected with miR-326 or miR-control or NOB1 shRNA in 0.2 mL of medium subcutaneously in the forelimb, the mouse injected mock-infected cells as control. Tumor sizes were measured every three days in two dimensions using a caliper, and the volume (mm3) was calculated using the formula V = 0.5* larger diameter *(smaller diameter)2. The tumors were excised and weighed from the sacrificed mice after 21 days. All procedures involving animals were approved by the Animal Care and Use Committee in Second Military Medical University.Statistical AnalysisThe Student’s t-test was used for statistical analysis in assays performed on glioma cell lines. For experiments of glioma tissue samples, relative expression levels of NOB1 mRNA for each group normal brain, low-grade glioma (LGG) and high-grade glioma (HGG) were expressed as mean 6 SE, the Mann-Whitney U test was used to compare the differences between groups. When studying the relationship between NOB1 expression and patients’ prognosis, we first grouped glioma patients of all grades to those live longer than 24 months and those live less than 24 months, Mann-Whitney U test was then applied to compare the expression of NOB1 between these two groups. Then the prognosis in lowgrade glioma and high-grade glioma patients were 18204824 also analyzed 1315463 separately. Fisher’s exact test was used to compare the immunolabelling results of NOB1 between high-grade and low-grade gliomas. SPSS 15.0 (SPSS Inc, Chicago, USA) was used for the statistical analysis and a significance level of P,0.05 was used to evaluate the difference between groups.Measurement of Phosphorylation of Signaling ProteinsThe changes in phosphorylation of selected proteins in certain of signaling pathways were analyzed with Proteome Profiler Array kit (ARY003; R D Systems, Minneapolis, MN) according to the manufacturer’s instructions. In brief, human A172 and U373 glioma cells were grown, and then infected with miR-326 precursor, control precursor or NOB1-shRNA. At the designated times, each dish was washed twice with phosphate-buffered saline and processed according to the kit protocol. Incubations with the array contained 300 ug of lysate protein. Net integrated pixel density for each spot (an average of duplicate spots after subtraction of average background density) was determined by densitometry and analyzed using Quantity One (ISBE, Sheffield,Figure 8. Expression of NOB1 protein in glioma and normal brain tissue samples. Immunohistochemical staining of normal brain tissue (A, B), grade I (C, D), grade II (E, F), grade III (G, H) and grade IV (I, J) glioma tissue specimens expressing NOB1. NOB1 staining was stronger in high-grade gliomas than that in low-grade gliomas. No significant staining was observed in normal brain tissues. doi:10.1371/journal.pone.0068469.gMicroRNA-326 as a Tumor Suppressor in GliomaFigure 9. Schematic diagram illustrating the interplay among miR-326, NOB1 and the MAPK pathway in glioma. miR-326, as a tumor suppressor by targeting NOB1, decreased the tumorigenesis of glioma cells in vivo and in vitro through the modulation of the MAPK pathway. Overexpression of miR-326, which suppresses the expression of NOB1, activates the MAPK patheay by increasing the phosphorylation of ERK1/2, JNK and p38 MAPK, which inhibits the cel.