A human normal colonic epithelial cell line (NCM460) and 5 human colon cancer cell lines (LoVo, SW620, HT29, HCT116 and SW480) were purchased from Nanjing Cobioer Biotechnology Co. Ltd. (Nanjing, Jiangsu, China). All of the CRC cell lines were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin. All cells were maintained in an incubator at 37°C with 5% CO2 in a humidified atmosphere 21.
Transfection and lentivirus transduction
The miR-654-5p inhibitor and its negative control (Ctrl) were purchased from RiboBio, and oligonucleotide transfection was carried out using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A short hairpin RNA (shRNA) targeting LINC01124 was designed by GenePharma (Shanghai, China) and cloned into the Prnat-u6.1/Neo plasmid (Biovector, Beijing, China). To establish a cell line with stable silencing of LINC01124, the plasmid carrying sh-LINC01124 or sh-Ctrl was cotransfected with packaging vectors to produce pseudotyped lentiviruses designated Lv-sh-LINC01124 and Lv-sh-Ctrl. The lentiviruses were concentrated by ultracentrifugation and then used to infect CC cells 21.
Quantitative real-time PCR
After transfection, total RNA was extracted from cells by TRIzol extraction (Invitrogen; Thermo Fisher Scientific, Inc.). Then, RNA samples were reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara). RT‑qPCR analyses were performed with SYBR Green (Takara). The results were normalized to the expression of glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH). Relative gene expression levels were calculated using the 2−ΔΔct method 16.
Cell proliferation analysis
For CCK-8 assays, 2000 cells were seeded into 96-well plates in 100 μl of complete medium and cultured for 1, 2, 3, 4, and 5 days. At each time point, 10 μL of the CCK‐8 solution (Dojindo, Kumamoto, Japan) was added into each well, and the absorbance was then measured at 450 nm using a microplate reader after 2 h at 37°C. The cell viability was measured by the absorbance. For colony formation assays, cells were seeded into 12-well plates at a density of 500/well and incubated for 10-12 days. Then, colonies were stained with 0.1% crystal violet (Sigma) and counted 22.
Cell cycle analysis
HCT116 and SW480 cells were washed with cold PBS and then fixed with ice-cold 70% ethanol at 4°C overnight. On the second day, the cells were washed with PBS, and intracellular DNA was labeled with propidium iodide (PI, Sigma-Aldrich; Merck KGaA) at 4°C for 30 min and analyzed using BD FACSCalibur flow cytometry (BD Bioscience, San Jose, CA, USA). ModFit software (Verity Software House Inc., Topsham, ME, USA) was used to analyze the proportions of cells in the GO/G1, S, and G2/M phases 21.
Wound healing assay
Cells (1x10^5/well) were seeded into 6-well plates, and before starting the assay, cells were starved in fetal bovine serum (FBS)-free culture medium overnight. Then, a wound was made using a 200 µl pipette tip. Next, the cells were incubated with 2% FBS medium. The wound was imaged at 0 h and 36 h 22.
Transwell invasion assay
The Transwell chamber (8-μm pore size, Corning, Cambridge, MA, USA) was used to perform the invasion assay. Cells (2x10^5/well) were cultured in the upper chamber with Matrigel (BD Biosciences), and complete medium containing 20% FBS was added to the lower chamber. After incubation for 36 h at 37°C, cells adhering to the lower surface of the Transwell membrane were fixed in 20% methanol and stained with 0.1% crystal violet. The number of invaded cells was analyzed 22.
Tumorigenesis assay in vivo
For the subcutaneous xenograft assay, 5x105 cells infected with sh-Ctrl or sh-LINC01124 were subcutaneously inoculated into the flanks of 5-week-old male athymic nude BALB/c mice. The tumor volumes were examined by using calipers every three days. After 5 weeks, the mice were sacrificed by euthanasia. The tumors were then removed and weighed 21. All animal studies were performed in strict accordance with the recommendations in the guidelines for the Animal Care and Use Committee of Traditional Chinese Medicine University of Guangzhou. Permit number: 20190228035.
Luciferase reporter assay
The binding sequences of miR-654-5p and LINC01124 were predicted through online websites (http://www.mircode.org/ and https://cm.jefferson.edu/ran22/Interacti-
ve/). The potential binding sequences of miR-654-5p and LINC01124 (WT) and the mutant (Mut) miR-654-5p binding sequences in LINC01124 were inserted into a pmirGL3-basic vector (Promega Corporation, Madison, USA) to construct dual luciferase reporter plasmid. Then, cells were cotransfected with the WT (or Mut) plasmid and a Ctrl mimic or miR-654-5p mimic. After 48 h, luciferase activity was detected using a Dual Luciferase Reporter Gene Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol. The relative luciferase activity was normalized to Renilla luciferase activity 22.
Protein samples were isolated with lysis buffer (RIPA) containing protease inhibitors. After quantification with a BCA kit (Beyotime, China), total protein (25 µg) was separated by 8-15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skimmed milk for 60 min, the membranes were incubated with the primary antibodies anti-HAX-1 (ab137613, 1:500) and anti-GAPDH (#5174, CST, 1:1000), overnight, followed by incubation with fluorescence-conjugated secondary antibodies (1:1,000) for 30 min. Bands were detected by a two-color infrared laser imaging system (Odyssey; Li-Cor, Lincoln, NE, USA) 22.
All data in this study were obtained from experiments repeated at least three times and are presented as the mean ± standard deviation (SD). Two independent sample t-tests or one-way ANOVA for multiple comparisons were performed using SPSS v22.0 (IBM, Armonk, NY, USA). **p < 0.01 or *p < 0.05 was considered to indicate a statistically significant difference 22.