Background MicroRNAs (miRNAs) are small noncoding RNAs that potentially play a

Background MicroRNAs (miRNAs) are small noncoding RNAs that potentially play a critical role in tumorigenesis. miR-320b provides new insights into the pathophysiology of CRC proliferation, and identifies miR-320b as a novel therapeutic target for the treatment of CRC. and was directly targeted by miR-320b, and buy Raltitrexed (Tomudex) that overexpression of miR-320b in CRC cells decreased both mRNA production, and protein expression of gene function, and that increasing miR-320b expression levels may provide a novel approach for CRC treatments. Methods Tissue samples and cell lines A total of 48 CRC tissue samples and their adjacent non-tumor tissues were obtained from Department of Colorectal Surgery, Changhai Hospital (Shanghai, China) for qRT-PCR analysis. All tissue samples were obtained surgically and immediately snap frozen and stored in liquid nitrogen until use. The study protocol was approved by Shanghai Changhai Hospital Ethical Committee, and written informed consent was obtained from all subjects before the study was conducted. Additionally, five normal colorectal tissues were obtained from non-cancer patients by colonoscopy. For the experiments, cell lines including HCT-116, SW-480, SW-620, LoVo and HEK293 were used and were purchased from American Type Culture Collection (ATCC). SW-480 and SW-620 were cultured in Leibovitzs L-15 medium containing 10?% FBS. HCT-116 and LoVo cells were cultured in Hams F12K medium containing 10?% FBS, and HEK293 cells were cultured in DMEM medium containing 10?% FBS. All cells were maintained at 37?C in a humidified atmosphere with 5?% CO2. RNA quantification Total RNA was isolated using a Trizol extraction kit (Life Technologies, USA) according to the manufacturers instructions. Purified mRNA and miRNAs were detected by qRT-PCR assay using All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, USA). U6 small RNA was used as an internal control for normalization and quantification of miR-320b expression. As an internal control -actin was measured for normalization and quantification of c-Myc expression. Luciferase reporter assay The luciferase reporter was constructed by cloning human cDNA sequence into pMIR-Report (Ambion, Austin, USA). Wild type or mutant mRNA fragments buy Raltitrexed (Tomudex) were amplified and cloned into the luciferase reporter via Spe and Hind sites. Luciferase reporter assays were performed as following, HEK293 and SW-480 cells were plated in a 96-well plate and co-transfected with 50 nM single-stranded miRNA mimics, or negative control oligonucleotides, with 10?ng of firefly luciferase reporter and 3?ng of pRL-TK (Promega, USA) using the JetPRIME reagent (Polyplus-transfection). Cells were harvested 48?h after the transfection and analyzed using Dual-Luciferase Reporter Assay System (Promega, Japan). Oligonucleotide and plasmid transfection RNA oligos were chemically synthesized and purified by (Genepharma, China). Sense sequence of human miR-320b mimics was 5- AAA GCU GGG UUG AGA GGG CAA -3 and antisense sequence was 5- UUG CCC UCU CAA CCC AGC UUU U-3. Negative control oligonucleotides were 5-AAU UCU CCG AAC GUG UCA CTT-3 and 5-GUG ACA CGU UCG GAG AAU UTT-3. buy Raltitrexed (Tomudex) The transfections were performed with INTERFERin reagent (Polyplus-transfection). The final concentration of miRNA was found to be 50 nM. To generate pGL3-c-MYC constructs, the coding DNA sequence fragment of was amplified and inserted into the growth of CRC cells, the MTT assay was used. A total of 5??103 cells were seeded into each well of 96-well plates and transfected with miR-320b mimics or negative control oligonucleotides at a final concentration of 50 nM respectively. On the buy Raltitrexed (Tomudex) day of measurement, 100?l of spent medium was replaced with an equal volume of fresh medium CXCR4 containing buy Raltitrexed (Tomudex) 0.5?mg/ml MTT. Plates were incubated at 37?C for 4?h, then the medium was replaced with 100?l of DMSO (Sigma, USA), and were then shaken at room temperature for 10?min. Absorbance was then measured at a wavelength of 570?nm. Tumorigenicity assay in Non Obese Diabetic (NOD) mice All mice were cared and maintained according to.