Knockdown of miR 92b decreased glioma cell prolifirelation, reduced apoptosis and up regulated the expression from the target, DKK3, whereas ectopic expression of miR 92b exhibited the opposite effects. Furthermore, miR 92b could regulate the expression of downstream genes in the Wnt beta catenin signaling pathway, such as Bcl2, c myc and p c Jun. These findings indicate that DKK3 is really a crucial target of miR 92b and that the microRNA may be critical therapeutic targets and survival predictors in glioma. Materials and strategies The human glioma tissue samples and their corresponding nontumorous tissues were 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 data have been collected from January 2008 to June 2013, which includes 9 grade I II tumors, 8 grade III tumors and 3 grade IV tumors. The glioma samples had been deep frozen making use of liquid nitrogen, stored at ?80 C and were quantified by Genuine time PCR. This study was authorized by the Institutional selelck kinase inhibitor Evaluation Board of Xinhua hospital. Individuals were followed by clinical and laboratory monitoring on a regular basis beginning at definitive diagnosis. Illness specific survival time was defined because the time from definitive diagnosis to illness certain death. Reagents The antibodies aganist c jun, phospho c jun, JNK, phospho JNK, DKK3, beta catenin, Bcl 2, B actin, caspase 3, Bax, c myc had been purchased from Santa Cruz Biotechnology. The dual luciferase reporter assay method, the PGL3 Promoter, the PGL3 Basic and PRL TK vectors had been purchased from Promega.
The miRNA mimics and siRNA have been bought from Biomics Biotechnologies. All other chemical compounds have 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 making use of Lipofectamine 2000 in accordance with the suppliers guidelines. MicroRNA microarrays Total RNA was extracted from eight glioma tissues using the miRVana miRNA Isolation Kit in accordance with the manufacturers guidelines. The samples have been subsequently submitted to Shanghai Biotechnology Corporation for array hybridization on an Agilent Human miRNA array.
Each and every microarray chip was hybridized using a single sample labeled with either Cy3 or Cy5. Background subtraction and normalization were performed. The raw data were de posited at Shanghai Biotechnology Corporation and haven’t been reported publicly up till the present moment. We chosen the miRNAs that exhibited a distinction in expression levels of at the very least two fold in between the glioma tissue samples and their correspond ing nontumorous tissues.