Clinical CRC tissues
A total of 65 CRC tissues and corresponding adjacent nontumor tissues were obtained from surgical resection at Affiliated Hospital of Nantong University (Nantong, China) between June 2017 and July 2018. All enrolled CRC patients were diagnosed based on histopathological examination and received no previous antitumor treatment, including radiotherapy or chemotherapy. The present study was approved by the clinical Research Ethics Committee of the Affiliated Hospital of Nantong University.
Cell lines and culture
Human normal colonic epithelial cells (NCM460), and CRC cell lines (DLD1, SW480, SW620, HCT8, CACO2), were purchased from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). All these cell lines were cultured in DMEM medium (Corning, USA) which contained 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin in a humid condition with 5% CO2 at 37℃.
RNA isolation and quantitative real-time RT-PCR
Total RNA of CRC tissues and cells was extracted using Trizol reagent (Invitrogen, CA, USA). The concentration and purity of RNA was determined with micro-spectrophotometer (Thermo Scientific, USA). Whole RNA (3ug) was reversed transcribed into cDNA using the Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA). The expression level of ABALON was quantified using SYBR Green Master Mix (Roche, GER) on Roche LightCycler 480 (Roche, Switzerland) according to the manufacturer’s protocol. GADPH were used as internal control. Relative amounts were calculated using the 2−ΔΔCt method. The reaction conditions of quantitative real-time RT-PCR (qPCR) were as follows: 95℃ for 10 minutes, then 40 cycles at 95℃ for 15 seconds and 60℃ for 30 seconds, finally 72℃ seconds for 30 seconds. The primer sequences of ABALON and GADPH were shown in Table 1.
Nuclear/Cytosol Fractionation
Cells were inoculated into 10 cm cell culture dishes until cells fusion rate reached 90%. Cells were treated according to the nuclear/cytosol fractionation kit (Invitrogen) for detecting the expression of lncRNA in the nucleus and cytoplasm, respectively.
Cell transfection
DLD1 and SW480 cells (1×10^5 cells) were seeded into 6-well plated in DMEM medium without penicillin-streptomycin for 16 hours. Next, cells in 6-well plates were transfected with 5nmol/L ABALON shRNA, pcDNA ABALON, siPINK1 (santa, sc44598) via 0.2% Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
CCK-8
The CCK8 method was used to detect the cell viability and the effect of 5-FU on cytotoxicity. DLD1 and SW480 transfected cells (3×10^3 cells/well) were seeded into 96-well plated and 10ul CCK-8 solution (Dojindo) was added to each well. Subsequently, the absorbance at 450nm was measured with a microplate reader every 24 hours. All experiments are repeated 3 times.
Colony formation
For the colony formation assay, a total of 1×10^3 cells were seeded into six-well plates in DMEM medium and cultured for 14 days. After two weeks, cells were washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet for 15 minutes.
Transwell and invasion assay
DLD1 and SW480 cells was collecting after transfection 48 hours, adjusting the cell density to 5×105cells/ml with DMEM medium. The matrix glue for the upper of transwell chamber (Corning, USA) was paved in advance, and 100ul of cell suspension was added. The lower chamber was covered with 600ul of DMEM medium containing 20% FBS. After incubation with 48 hours, cells were fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet for 15 minutes. Wiping off the cells that have not passed through the upper chamber with a cotton swab, and observing the cell metastasis and invasion with microscope.
EDU (5-Ethynyl-2’-deoxyuridine) proliferation
DLD1 and SW480 cells were collected and inoculated with 2×104 cells per well in 96 well plate for 24 hours after transfection 48 hours. Subsequently, EDU labeling was performed with 30uM of EDU solution (RiboBio) for 2 hours. 4% paraformaldehyde was fixed for 20 minutes and 0.5% Triton X-100 was infiltrated for 10 minutes. 1×Apollo solution was incubated in dark at room temperature for 30 minutes, and 1×Hoechst 33342 solution was used for DNA staining for 30 minutes. Finally, the staining intensity was observed using confocal microscope.
JC-1 staining and ROS detection
JC-1 probe (Fcmacs) was applied to examine the mitochondrial membrane potential. 1×JC-1 was adding into six-well plate, following the working concentration of 10uM and incubating at 37 ° C for 20 minutes. The red and green fluorescence intensities was observed with a fluorescent microscope. DCFH-DA (Beyotime) was used to detect the effect of ABALON on the production of reactive oxygen species (ROS). DCFH-DA was diluted with 1:1000 in DMEM medium, with a final concentration of 10uM. 1ml of diluted DCFH-DA was added to each well of the six-well plate, incubated at 37 ℃ for 20 minutes, and washed three times with PBS to fully remove DCFH-DA that didn’t enter the cell. The green fluorescence intensity was also observed with a fluorescent microscope.
Immunofluorescence
Cells were seeded into 24-well plates covered with small circular discs and incubated for 24 hours. Cells were fixed with 4% paraformaldehyde for 30 minutes, and permeated with 0.5% Triton X-100 for 20 minutes. 5% goat serum was utilized to seal for 30 minutes. Then, first antibodies (TOMM20, Cell Signaling Technology, #42406) and (LC3B, ImmunoWay, YM3642) were incubated at 4℃ overnight. Next day, fluorescent second antibodies (Coralite488-conjugated Affinipure Goat, Proteintech, SA00013-2) and (Coralite594-conjugated Goat Anti-Mouse, Proteintech, SA00013-3) were incubated at room temperature for 2 hours. Finally, the staining intensity was observed using confocal microscope.
FISH
Cells were cultured in a 24-well plate covered with small circular discs for 24 hours. 4% paraformaldehyde and 0.5% Triton X-100 were used for fixing and permeating, respectively. Subsequently, cells were treated with 200ul pre-hybridizing solution for 30 minutes, and 20uM hybridizing solution (RiboBio) overnight. Finally, the staining intensity was observed using confocal microscope.
Apoptosis
For cell apoptosis detection, the transfected cells were centrifuged for 5 minutes at 1500rpm, and the supernatant was discarded. Binding buffer was added to re-suspend the cells (1×106/ml). Then, 5ul Annexin V-FITC was added and mixed gently. After adding 5ul propidium iodide (PI) staining solution, cells were incubated at 37℃ for 15min. Data analysis was performed using cell quest software using a flow cytometer (FACScan; BD, USA).
Western blotting analysis
Proteins were isolated from DLD1 and SW480 cell lines using Native lysis buffer (Solarbio, Beijing Solarbio Science and Technology, Beijing China).
Protein concentrations were measured by BCA kits. Equal amounts of cell protein lysates (20ug) were separated by electrophoresis on 15% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto polyvinylidene difluoride membranes (PVDF, MA, USA) and blocked with 5% skimmed milk for 2 hours. Subsequently, the membranes were incubated with primary antibodies at 4℃ for 24 hours and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 2 hours. Specific bands were detected by Immunoblots visualized by ECL detection system (Quantity One software, BioRad). The specific antibody information was presented as followed: TOMM20 (Cell Signaling Technology #42406), LC3B (Cell Signaling Technology #2775), Parkin (Proteintech 14060-1-AP), PINK1 (Proteintech 23274-1-AP), β-actin (Cell Signaling Technology #4970).
Statistical analysis
The results were presented as the mean ± SD. Student’s t test was used to analyze the relative expression of CRC tissues between two groups, and one-way ANOVA was used to analyze multiple groups. Pearson correlation test was employed for clinicopathological feature correlation analysis and Kaplan-Meier method was used for prognostic analysis. Significant differences were considered where P < 0.05 represented as * and P < 0.01 represented as **. The figures were drawn using Graphpad prism 7.0.