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 in vitro 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 Leibovitz’s L-15 medium containing 10 % FBS. HCT-116 and LoVo cells were cultured in Ham’s 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.
Total RNA was isolated using a Trizol extraction kit (Life Technologies, USA) according to the manufacturer’s 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 β-actin was measured for normalization and quantification of c-Myc expression.
Luciferase reporter assay
The luciferase reporter was constructed by cloning human c-MYC cDNA sequence into pMIR-Report (Ambion, Austin, USA). Wild type or mutant c-MYC mRNA fragments were amplified and cloned into the luciferase reporter via Spe I and Hind III 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’. 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 c-MYC was amplified and inserted into the KpnI and XhoI sites of the pGL3 construct. All constructs were verified by direct sequencing. Finally, the transfections were conducted using INTERFERin reagent (Polyplus-transfection, France). The final concentration of plasmids was diluted to 100 ng.
To measure in vitro 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 day of measurement, 100 μl of spent medium was replaced with an equal volume of fresh medium containing 0.5 mg/ml MTT. Plates were incubated at 37 °C for 4 h, then the medium was replaced with 100 μl 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 the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Second Military Medical University, Shanghai. Cholesterol-conjugated miR-320b mimics and negative control oligonucleotides transfected SW-480 cells (1 × 106) were suspended in 150 μl PBS and then injected subcutaneously into either side of the posterior flank of the same Non obese diabetic (NOD) 6 week old mice. Mice were examined every three days over a course of 4 weeks. Tumor volume (V) was monitored by measuring the length (L) and width (W) of the tumor with calipers and was calculated with the formula V = (L × W2) × 0.5.
Protein from tissue and cells was separated in a 12 % SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked with 5 % non-fat milk and incubated with anti-c-Myc antibody, anti-Cyclin D1 (Santa Cruz, CA) or anti-β-actin antibody (Sigma, CA, USA). After being washed extensively, a goat anti-mouse secondary antibody (Pierce, IL, USA) was added to the system. The proteins were detected using ECL reagents (Pierce).
Immunohistochemical (IHC) staining
Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in graded series of ethanol followed by heat induced epitope retrieval in citrate buffer (PH 6.0). Sections were incubated at 4 °C overnight with monoclonal antibodies against c-Myc (Santa Cruz, CA) and Ki-67 (Cell Signaling Technology, Danvers, MA). Immunostaining was performed using ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (code K 5007, DakoCytomation, Glostrup, Denmark) according to the manufacturer’s instructions. Subsequently, sections were counterstained with hematoxylin (Dako) and mounted in dimethyl benzene.
All statistical analyses were carried out using the SPSS 16.0 statistical software package. Continuous variables were expressed as mean ± SEM. Differences between groups were calculated with Student’s t test. A two-tailed P value test was used with a P value of < 0.05 considered statistically significant.