In recent years, changes in microRNA (miRNA) expression have been detected
In recent years, changes in microRNA (miRNA) expression have been detected in almost all human cancer types, including glioblastoma (GBM). grade in GBM patients. miR-744 upregulation inhibited Dexamethasone kinase activity assay the proliferation, colony formation, migration, and invasion, in addition to inducing apoptosis of GBM cells was confirmed to be upregulated in GBM tissues, and this was inversely correlated with upregulation of miR-744 expression. Moreover, KCTD18 antibody knockdown exhibited similar inhibitory effects as miR-744 overexpression in GBM cells. Notably, recovered expression counteracted the tumor-suppressing roles of miR-744 in the malignant phenotypes of GBM cells. Taken together, these results demonstrate that miR-744 directly targets NOB1 to inhibit the aggressive behaviors of GBM cells. Hence, the miR-744/axis may be useful in the identification of novel therapies for GBM patients. siRNA and negative control siRNA (NC siRNA) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The overexpression plasmid pcDNA3.1-NOB1 (pc-NOB1) and empty pcDNA3.1 plasmid were constructed by the Chinese Academy of Sciences (Changchun, China). Dexamethasone kinase activity assay Cells were plated into 6-well plates at an initial density of 5 105 cells per well. Cell transfection was performed using Lipofectamine 2000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific) in accordance with the manufacturers protocol. The transfected cells were incubated at 37C with 5% CO2 for 6 h, and the transfection mixture was replaced with fresh DMEM containing 10% FBS. RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA of cell lines or tissue specimens was isolated using TRIzol? reagent (Invitrogen; Thermo Fisher Scientific), according to the manufacturers instructions. The concentration and quality of total RNA was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). For miR-744 detection, total RNA was converted into cDNA using a TaqManTM MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific). The synthesized cDNA was then subjected to quantitative PCR (qPCR) with a TaqMan microRNA assay kit (Applied Biosystems; Thermo Fisher Scientific). To quantify mRNA expression, reverse transcription was conducted using a Prime-Script RT Reagent Kit (Takara Bio, Dalian, China), followed by qPCR using Dexamethasone kinase activity assay the SYBR Premix Ex was predicted as the potential target of miR-744, and this association was then evaluated using a luciferase reporter assay. The 3-UTR of containing wild type (wt) and mutant (mut) miR-744 binding site was chemically constructed by Genepharma, cloned into the pmirGLO luciferase reporter vector (Promega Corporation, Madison, WI, USA) to generate pmirGLO-NOB1-3-UTR wt and pmirGLO-NOB1-3-UTR mut, respectively. For the reporter assay, cells were plated into 24-well plates at 1.0 105 cells per well. Lipofectamine 2000 was employed to co-transfect cells with miR-744 mimics/inhibitor or miR-NC/NC inhibitor and pmirGLO-NOB1-3-UTR wt or pmirGLO-NOB1-3-UTR mut, according Dexamethasone kinase activity assay to the manufacturers protocol. A total of 48 h after transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation). The luciferase activity was normalized to that of the firefly luciferase activity. Protein extraction and western blot analysis A total protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to isolate total protein from tissue specimens or cells according to the manufacturers instructions. The concentration of total protein was determined with a Bicinchoninic Acid Assay Kit (Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of protein were loaded onto 10% SDS-PAGE gels for electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). After blocking for 2 h with 5% fat-free milk, the membranes were incubated overnight at 4C with primary antibodies against NOB1 (cat. no. Dexamethasone kinase activity assay ab224619; 1:1,000 dilution) or GAPDH (cat. no. ab201822; 1:1,000 dilution; both from Abcam, Cambridge, UK). After that, the membranes were washed thrice with Tris-buffered saline and 0.05% Tween-20 (TBST) followed by incubation with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; 1:5,000 dilution; Abcam) at room temperature for 2 h. Finally, the protein signals were developed using an.