The RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA). Total RNA was reverse transcribed into cDNA applying an oligo d(T) (Bio-Rad Laboratories, Inc., Hercules, CA, USA). cRNA was hybridized onto the Affymetrix HumanTable I. Distribution of chosen characteristics amongst sufferers with colon cancer and controls. Characteristic Number of people Age (years) 60 60 Sex Male Female Smoking status No Yes Drinking status No Yes Colon cancer, n ( ) 102 57 (55.88) 45 (44.12) 78 (76.47) 24 (23.53) 41 (40.20) 61 (59.80) 37 (36.27) 65 (63.73) Handle, n ( ) 57 31 (54.39) 26 (45.61) 46 (80.70) 11 (19.30) 31 (54.39) 26 (45.61) 33 (57.89) 24 (42.11) P-value0.009 0.Genome U133 Plus two.0 Array, staining was performed using a Fluidic Station450 and GeneChips have been scanned with the Affymetrix GeneChip Scanner 7G. Data had been quantified and featureextracted using Agilent Function Extraction software (version A.ten.7.three.1; Agilent Technologies, Inc., Santa Clara, CA, USA). Cell culture and cell transfection experiments. Human Caco2 cells were bought from Form Culture Collection with the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s minimal essential medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA), 10 fetal bovine serum (FBS; GE Healthcare Life Sciences) at 37 in five CO2 and 95 relative humidity. miRNA (miR)-766 mimics and negative manage mimics had been purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Subsequently, 20 ng/ml miR-766, anti-miR-766 mimics and damaging handle mimics were transfected into Caco2 cells making use of Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer’s protocol. Cell viability assay and evaluation of apoptosis. The cells (1×106 cell/ml) were incubated with medium containing MTT (five mg/ml) for 4 h and dissolved with 150 DMSO. The medium was removed and dissolved with 150 DMSO for 15 min at space temperature. The absorbance was measured at 490 nm utilizing a microplate reader. To analyze apoptosis, the cells were washed twice with ice-cold PBS and resuspended in 500 binding buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Subsequently, five annexin Vfluorescein isothiocyanate and five propidium iodide (each Nanjing KeyGen Biotech Co., Ltd.) were added and the cells were incubated for 15 min at room temperature in the dark. Apoptosis was analyzed applying a FACSort flow cytometer and quantified working with BD CellQuestTM Pro application (BD Biosciences, Franklin Lakes, NJ, USA).EXPERIMENTAL AND THERAPEUTIC MEDICINE 17: 4100-4108,Figure 1. Expression of miR766 in individuals with colon cancer. (A) Heat map and (B) reverse transcriptionquantitative polymerase chain reaction analyses of the expression of miR-766 in sufferers with colon cancer. (C) Overall and (D) disease-free survival rates of patients with colon cancer with differing expression levels of miR-766. ##P0.01 vs. Control. Manage, 57 normal volunteers; Colon cancer, 102 sufferers with colon cancer; miR/miRNA, microRNA.Cell migration assay. The Caco2 cells (1×105 cell/ml) had been seeded on 24-well plates and were added to the upper chamber of every migration Iodixanol Technical Information nicely (Corning Corporation, Corning, NY, USA). DMEM (500 ) with 20 FBS was added to the reduce chamber and incubated for 48 h at 37 . The reduced side were fixed with 75 icealcohol for 30 min and stained with 1 crystal violet answer for 1 h at area temperature. The cells have been counted under a fluorescence microscope (Axio version II, Carl Zeiss AG, Oberkochen, Germany). We.