Knockdown of miR 92b decreased glioma cell prolifirelation, lowered apoptosis and up regulated the expression in the target, DKK3, whereas ectopic expression of miR 92b exhibited the opposite effects. Additionally, miR 92b could regulate the expression of downstream genes on the Wnt beta catenin signaling pathway, for example Bcl2, c myc and p c Jun. These findings indicate that DKK3 is really a important target of miR 92b and that the microRNA might be critical therapeutic targets and survival predictors in glioma. Components and techniques The human glioma tissue samples and their corresponding nontumorous tissues had been collected at the time of surgical resection at the Division of Pediatric Neurosurgery, Xinhua Hospital, Shanghai Jiao Tong University.
Twenty frozen glioma specimens with clinical information had been collected from January 2008 to June 2013, which includes 9 grade I II tumors, eight grade III tumors and 3 grade IV tumors. The glioma samples have been deep frozen utilizing liquid nitrogen, stored at ?80 C and have been quantified by Real time PCR. This study was approved by the Institutional selleck inhibitor Review Board of Xinhua hospital. Patients have been followed by clinical and laboratory monitoring on a regular basis starting at definitive diagnosis. Illness particular survival time was defined because the time from definitive diagnosis to illness distinct death. Reagents The antibodies aganist c jun, phospho c jun, JNK, phospho JNK, DKK3, beta catenin, Bcl two, B actin, caspase 3, Bax, c myc were bought from Santa Cruz Biotechnology. The dual luciferase reporter assay program, the PGL3 Promoter, the PGL3 Standard and PRL TK vectors were bought from Promega.
The miRNA mimics and siRNA had been bought from Biomics Biotechnologies. All other chemical substances had been from Sigma Aldrich unless otherwise stated. Cell cultures and transfection The human glioma cell lines U251, U87, A172 and SHG44, and human astrocytes, have been maintained in RPMI 1640 medium with selleckchem 10% fetal bovine serum at 37 C in a humid atmosphere wih 5% CO2. Cell transfection was performed applying Lipofectamine 2000 in accordance with the producers directions. MicroRNA microarrays Total RNA was extracted from eight glioma tissues making use of the miRVana miRNA Isolation Kit in accordance with the manufacturers instructions. The samples were subsequently submitted to Shanghai Biotechnology Corporation for array hybridization on an Agilent Human miRNA array.
Each and every microarray chip was hybridized with a single sample labeled with either Cy3 or Cy5. Background subtraction and normalization have been performed. The raw data had been de posited at Shanghai Biotechnology Corporation and have not been reported publicly up till the present moment. We selected the miRNAs that exhibited a distinction in expression levels of a minimum of two fold among the glioma tissue samples and their correspond ing nontumorous tissues.