Clinical specimens
Tumor tissues and adjacent tissues from CRC patients (n = 20) were collected from China-Japan Union Hospital of Jilin University and all the participants signed the consent forms. Among the 20 patients, 11 patients were male and 9 patients were female. The average age of included patients was 59.75±9.00 years old. The enrolled patients were pathologically diagnosed as CRC and did not undergo preoperative radiotherapy and/or chemotherapy prior to resection. The project was authorized by the Ethical and Scientific Committee of China-Japan Union Hospital of Jilin University. All samples were stored at -80 °C until subsequent analysis.
Cell culture
Human CRC cell lines including SW460, HCT116, HT-29, LoVo, Colo205, SW480 and normal colon epithelial cell line NCM460 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium supplmented with 10% fetal bovine serum (Gibco, Carlsbad, CA), 100U/ml penicillin and 100mg/ml streptomycin. Cells were normally maintained at 37°C in an incubator with 5% CO2 until use.
Cells transfection
The sequence of BCL2 was inserted into vector pcDNA3.1 (Santa Cruz, Dallas, TX) for its ectopic expression in HCT116 and LoVo cell lines. Constructions, miR-34b-5p mimic, miR-34b-5p inhibitor and miR-NC mimic were delivered into CRC cells by Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to manufactures instructions.
Establishment of stable cell lines
Three shRNAs sequences targeting LINC02418 and negative control sequence were inserted into HuSH shRNA GFP Lenti Cloning Vector (Origene, Rockville, MD) following commercial guidance. The lentivirus was packaged with usage of 293T cells following common protocol.
Cell counting kit-8 analysis and colony formation assay
For CCK-8 experiment, the cells were seeded at density of 5 ×104 cells per well on 48-well plate. Cells were harvested at 12h, 24h, 48h and 72h, and cell proliferation was assessed using cell counting kit-8 (Beyotime, Shanghai, CHA) according to the manufacturer’s instructions. The optical density (OD) at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA).
For colony formation assay. Total number of 3000 cells were seeded in 6-well plates and maintained in RPMI 1640 medium containing 10% FBS. After culture of 14 days, cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO) for another 5 min till manually counting of visible colonies.
Xenograft assay
Male BALB/c nude mice (5~6 weeks old) were maintained under specific pathogen-free facility and were manipulated according to protocols approved by the China-Japan Union Hospital of Jilin University. All the mice were randomly divided into four groups, and each group contained 5 mice.
HCT116-sh-LINC02418, HCT116-sh-NC, LoVo-sh-LINC02418 and LoVo-sh-NC cells (2×106 cells) resuspended in 200 μl of medium were subcutaneously inoculated into nude mice. After 7 days post-injection, tumor size was measured every 3 days, and the tumor volumes were recorded. After 21 days post-injection, mice were sacrificed by cervical dislocation. Tumors were separated from mice and the weight of tumor was measured. The survival curve analysis was not involved in this experiment.
Transwell migration and invasion assay
Cells migration assays were performed in a 24-well transwell chamber (CoStar, Badhoevedorp, Netherlands). Cells were plated and allowed to migrate through 8 μm-pore sized polycarbonate membrane. The chamber for invasion assay was pre-coated with 1 mg/ml Matrigel (Sigma-Aldrich, St. Louis, MO). A number of 5×104 cells were added to the upper chamber of the transwells with FBS-free medium and the lower chamber was filled with 500 μl RPMI 1640 medium containing 10% FBS. After 24h incubation, the cells were fixed by 4% formaldehyde for 10 min, stained by 0.1% crystal violet for 20 min. Images were captured under microscope.
Quantitative reverse transcription PCR (qRT-PCR) assay
Total RNA was extracted from clinical tissue and CRC cells by TRIzol reagent (Invitrogen, CA, USA). RNA reverse transcription for mRNA and miRNA was performed using Prime ScriptTM RT Master Mix (Takara, Otsu, Japan) and TaqMan MicroRNA Reverse Transcription system (Thermo Fisher Scientific, MA, USA), respectively. The quantitative PCR was carried out by SYBR Premix Ex Taq II (TaKaRa Biotechnology, Dalian, China). Commercial miRNA qRT-PCR primers for miR-34b-5p and U6 were purchased by RiboBio (Guangzhou, China). The available primers sequences were as follows: LINC02418-F: 5’-ATTTCCATGGCGTTTCTCAC, LINC02418-R: 5’-AGGCAGGAGAATTGCTTGAA; BCL2-F: 5’-GGCATCTTCTCCTTC CAG-3’, BCL2-R:5’-CATCCCAGCCTCCGTTAT-3’; GAPDH-F:5’-ACAACTTTGGTATCGTGGAAGG-3’, GAPDH-R:5’GCCATCACGCCACAGTTTC-3’. Applied Biosystems 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA) was used for RNA quantification assay. U6 and GAPDH were used as internal control of miRNAs and mRNAs, respectively. Fold change was calculated by the 2−△△Ct method.
Luciferase assay
The fragments containing the binding sites or the mutated sites were synthesized and inserted into a pGL3-basic vector for dual luciferase assay. HCT116 and LoVo Cells were seeded in a 12-well plate and co-transfected with reporter plasmids and miR-NC/miR-34b-5p mimic. After 48 hours, cells were harvested, dual-luciferase reporter assays were performed according to the protocol using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI) on a GloMax 20/20 luminometer (Promega, Madison, WI).
Fluorescence in situ hybridization (FISH) assay for miRNA
HCT116 and LoVo cells reaching 70% confluency were fixed in 4% paraformaldehyde for 20 min at room temperature, followed by permeabilized treatment in 70% ethanol at 4°C overnight. For cellular miR-34b-5p detection, FISH assay was conducted following previous procedures [26]. Specific Digoxigenin (DIG)–labeled locked nucleic acid (LNA) probe against miR-34b-5p was purchased from QIAGEN (Hilden, Germany). The 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA, USA) was adopted to stain the cell nucleic.
Immunohistochemistry (IHC) assay
The tumor samples from CRC patients were fixed in 10% formaldehyde, embedded in paraffin and then sectioned into slices. For IHC assay, tumor slices were firstly deparaffinized and rehydrated. After washing, slices were treated with H2O2 to reduce the endogenous peroxidase activities. Slices were then incubated with primary antibody against BCL2 (Abcam, Cambridge, UK) overnight at 4 °C. With three times washing in PBS, the slides were incubated with secondary streptavidin-horseradish peroxidase-conjugated antibody (Jackson Immuno Research, PA, USA) for 1h, and reacted with 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (Yeasen Biotech, Shanghai, China) for 5 min. Finally, slides were counterstained with hematoxylin for 1 minutes, dehydrated and mounted with neutral gum.
Western Blot
Protein samples were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA). The membranes were blocked in 5% nonfat milk. Primary antibodies including anti-GAPDH (1:2000, Abmart, Shanghai, CHN), anti-BCL2 (1:500, Abcam, Cambridge, UK), anti-Caspase 9 (1:1000, Cell Signaling Techonolgy, MA, USA), anti-cleaved-Caspase 9 (1:500, Cell Signaling Techonolgy, MA, USA), anti-Caspase 3 (1:1000, Abcam, Cambridge, UK), and anti-cleaved-Caspase 3 (1:1000, Abcam, Cambridge, UK) were used for incubation overnight at 4°C. The membranes were incubated with HRP-conjugated secondary antibody (1:3000, Jackson Immuno Research, PA, USA) at room temperature for 1h and the protein bands were detected by Pierce Fast Western Blot Kit (Thermo Fisher Scientific, MA, USA).
Flow cytometric analysis
Apoptosis assays of HCT116 and LoVo cells were performed using Annexin V-FITC/propidium iodide (AV/PI) Apoptosis Detection Kit (Abcam, Cambridge, UK) according to the commercial instruction. Cells were incubated with ice-cold 75% ethanol at 4℃ overnight, followed by resuspending in 500 µl of 1× Annexin V Binding Buffer. Then, cells were stained with 5 μl Annexin V/FITC and 5 μl Propidium Iodide (PI) in the dark for 15 min at the room temperature. Apoptosis rates were examined and analyzed by flow cytometry using FACS Calibur (BD Biosciences, CA, USA).
Statistical analysis
Statistical data analysis was conducted using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA). Experiments were carried out in triplicate and the data was displayed as mean ± SD. Statistical analysis was conducted using Student’s t-test or one-way analysis of variance. Statistical significance was considered when p < 0.05.