Bioinformatics
TargetScan Human (http://www.targetscan.org/vert_72/) was used to identify the target miRNAs of DCLK-1. And we extracted candidate miRNAs via Ingenuity Pathway Analysis’ microRNA Target Filter.
Cell lines and cell culture
Human CRC cell lines DLD-1, HT29, HCT116, human pancreatic adenocarcinoma cell line Panc-1, and human non-tumor cell line HEK293 were purchased from the American Type Culture Collection (Rockville, MD, USA). These cell lines were authenticated by morphological inspection, short tandem repeat profiling, and mycoplasma testing. DLD-1, HT29, and Panc-1 cells were cultured in RPMI 1640 medium, and HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in the humidified incubator at 37°C and 5% CO2.
Transduction of the degron reporter
The degron sequence of ornithine decarboxylase (ODC) is recognized directly by proteasomes, which leads to the immediate destruction of the involved protein. The retroviral expression vector pQCXIN-ZsGreen-cODC, containing green fluorescence ZsGreen-labeled degron ODC (Gdeg), was kindly provided by Dr. Frank Pajonk (UCLA’s Jonsson Comprehensive Cancer Center, CA, USA). The vector was transfected into Platinum retroviral packaging cells, and the retrovirus collected from the supernatant was used to infect pancreatic cancer Panc-1 cells. Stable transfectants were selected with G418 solution (Roche, Germany). ZsGreen+ cells were sorted by the flow cytometry (Cell Sorter SH800, SONY, Japan).
MiRNA and plasmid transfection
Mimic-hsa-miR-1291 (miR-1291):
sense (5’-UGGCCCUGACUGAAGACCAGCAGU-3’) and antisense (5’-ACUGCUGGUCUUCAGUCAGGGCCA-3’), and negative control miR (NC): sense (5’-AUCCGCGCGAUAGUACGUA-3’) and antisense (5’-UACGUACUAUCGCGCGGAU-3’), mimic-hsa-miR-34a-5p (miR-34a): sense (5’- UGGCAGUGUCUUAGCUGGUUGU-3’) and antisense (5’- ACAACCAGCUAAGACACUGCCA-3’) were synthesized by Gene Design (Osaka, Japan). Cells were transfected with miRNAs and plasmids using Lipofectamine2000 (Thermo Fisher Scientific, Madison, USA) or Lipofectamine RNAiMAX (Thermo Fisher Scientific).
pmirGLO plasmid vector construction
The 3’ UTR of DCLK-1 mRNA was amplified by PCR using the following primer sequences (amplified product size 211 bp): Forward 5’-GCTCGCTAGCCTCGAGCTAGTGTACTGAGCCTGCGG-3’, Reverse 5’-ATGCCTGCAGGTCGACTGACTGGTCACATTCCACTG-3’. The amplified products were subcloned and ligated into the multicloning site between Sal I and Xho I in the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) using the In-Fusion HD Cloning Kit (Clontech, Mountain View, CA). The entire sequence (insert and vector) was confirmed by Sanger sequencing.
Luciferase reporter assay
Cells were seeded in 96-well plates at a density of 10,000 cells per well and co-transfected with 50 ng of the pmirGLO plasmid vector and 50 nM of either miR-NC or miR-1291. After 24 hours of transfection, cells were assayed for both firefly and renilla luciferase using the Dual-Luciferase Reporter Assay System (Promega).
RNA isolation
Total RNA, including miRNA, was isolated from cell lines using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA concentration and purity were measured using a NanoDrop one spectrophotometer (Thermo Fisher Scientific).
Quantitative real-time PCR analysis of miRNA
We used the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) to synthesize the complementary DNA from miRNA according to the manufacturer’s protocol. TaqMan Universal PCR Master Mix, No AmpErase UNG (Thermo Fisher Scientific), and a LightCycler 480 Real-Time PCR system (Roche Diagnostics, Mannheim, Germany) was used to quantify miRNA, and RNU6B was used as the endogenous control. Relative expression was quantified with the 2-ΔΔCt method.
Quantitative real-time PCR analysis of messenger RNA
A High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to synthesize the complementary DNA from mRNA according to the manufacturer’s protocol. qRT-PCR was amplified using oligonucleotide primers and the LightCycler 480 Real-Time PCR system (Roche). The amplification products were detected using the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan), and the level of target gene expression was calculated. The expression of the target gene was normalized to endogenous GAPDH expression. Relative expression was quantified by the 2-ΔΔCt method. The PCR primers are listed in Table S1.
MiRNA expression
TaqMan miRNA analysis (Applied Biosystems) was used to measure miRNA expression. The reverse transcription reaction was performed with the TaqMan MicroRNA RT Kit (Applied Biosystems) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using the 7900 HT Sequence Detection System (Applied Biosystems). Amplification data were normalized to endogenous RNU6B expression. The relative expression level was quantified by the 2-ΔΔCt method.
Proliferation assay
Cells were seeded in 96-well plates at a density of 4000-8000 per well and were transfected with miR-NC or miR-1291 at a final concentration of 30 nM the second day after seeding. Twenty-four, 48, and 72 hours after transfection,10 µl of Cell Counting Kit-8 (DOJINDO Molecular technologies, Inc., Kumamoto, Japan) was added to each well, and the 96-well plates were shaded for 2 hours. After that, the absorbance was detected by Multiskan Go (Thermo Fisher Scientific) to determine cell number.
Matrigel invasion assay
Cells were seeded in BD BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA) at a density of 50,000-100,000 cells per chamber. The cells were transfected with the miRNAs at a final concentration of 50 nM. After 48-72 h of transfection, invaded cells were stained with hematoxylin.
Wound healing assay
Cells were seeded in ibidi culture 2-well inserts (ibidi, Gräfelfing, Germany) in 24-well plates at a confluent density. The inserts were removed after 24 hours to create wounds. The miRNAs were transfected at a final concentration of 30 nM. The areas of the wounds were measured at 0-48 hours using ImageJ software.
Colony formation assay
Cells were transfected with miR-NC or miR-1291 at a final concentration of 30 nM for 8 hours and then seeded in 6-well plates at a density of 500 cells per well. After 10 days, the cells were stained by crystal violet and counted.
Cell cycle assay
Cells were starved in serum-free medium for 48 hours. Twenty-four hours before the end of starvation, miR-NC or miR-1291 was transfected at a final concentration of 30 nM (Fig. S1). Cells were collected at the indicated times (0, 12, 24, 48 hours), fixed in 70% ethanol for 30 minutes at 4°C. After fixation, cells were washed twice with PBS and incubated with RNase (Sigma Aldrich, St. Louis, MO, USA) for 20 minutes at 37°C. Cells were treated with PI (Dojindo) for 20 minutes on ice and analyzed by flow cytometry (Spectral Analyzer SA3800, Sony Biotechnology, Inc., Tokyo, Japan).
Western blot analysis
Cells were seeded in 6-well plates at a density of 100,000-200,000 per well and then transfected with miR-NC or miR-1291 at a final concentration of 30 nM the next day. After 48 and 72 hours, cells were lysed with RIPA buffer (0.05 M Tris-HCl pH 7.6, 0.15 M NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS) with 1% proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The protein samples were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride transfer membrane (PVDF, Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies, including anti-β-actin ((13E5) Rabbit mAb #4970, Cell Signaling Technology, Danvers, MA, USA), and anti-DCLK-1 (ab31704, Abcam), anti-Bmi1 (10832-1-AP, Thermo Fisher Scientific), anti-p21WAF1/CIP1 (ab80633, Abcam, Cambridge, UK), anti-p27KIP1 (sc-528, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CDC25A (#3652, Cell Signaling Technology), anti-CDC25B (#9525, Cell Signaling Technology), anti-CDC25C ((5H9) Rabbit mAb #4688, Cell Signaling Technology), anti-CDK4 (MAB8879, Merck Millipore, Burlington, MA, USA), anti-CDK6 (SAB4300596, Sigma-Aldrich), anti-Cyclin D1 (#2922, Cell Signaling Technology), anti-Cyclin E1 (sc-247, Santa Cruz Biotechnology), anti-Rb (ab24, Abcam). HRP anti-mouse IgG antibody (NA931, GE Health Care, Little Chalfont, UK) and anti-rabbit IgG antibody (NA934, GE Health Care) were used as secondary antibodies. The bands were visualized by the ECL Detection System (GE Health Care).
Sphere formation assay
Single cells were seeded 24 hours after transfection of miR-negative control or miR-1291 in 96-Well Clear Ultra Low Attachment Microplates (Corning Inc., USA) at the density of 1000 cells per well. And the cells were cultured in DMEM/F-12 serum-free medium (Invitrogen, USA) supplemented with 20 ng/ml epithelial growth factor, 10 ng/ml basic fibroblast growth factor-2 (PeproTech, USA), and 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in the humidified incubator at 37°C and 5% CO2. The number of spheres ≥40 µm was counted 4 days after seeding.
Flow cytometric analysis for CD133 marker expression
Suspensions of HCT116 single cells were stained with antibodies against human CD133 (APC-conjugated, No. 130-113-106, Miltenyi Biotec, Bergisch Gladbach, Germany). Dead cells were excluded by utilizing forward and side scatter. One million cells were incubated with antibodies on ice for 20 minutes in the dark, centrifuged, and washed twice with PBS containing 2% FBS. Spectral Analyzer (SA3800, Sony Biotechnology, Inc.) was used for flow cytometric analysis.
Statistical analysis
Data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 (San Diego, CA, USA) and Microsoft Excel. The statistical differences between the miR-NC and miR-1291 groups were analyzed by student’s t-test (two-tailed). Comparisons among more than two groups were performed by one-way analysis of variance (ANOVA). P<0.05 was considered significant.