Clinical sample collection
The colorectal cancer patients in our hospital were recruited as the research subjects, and the case detection results of the research subjects were all colorectal cancer. Cancer tissue and paracancerous tissue were collected during surgery. After sampling, the samples were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for subsequent operations. All subjects in the study had not received other radiotherapy and chemotherapy before the experiment. This study was approved by the ethics committee of our hospital, and all participating patients were signed the informed consent. All experimental manipulations involved were performed in compliance with the Declaration of Helsinki.
All cell lines were purchased from BFB Cell Culture Bank (Shanghai, China). The cell lines involved can be divided into two types: 1. NCM460 (normal colorectal cell line). 2. LoVo, HT29, HTC116 and SW480 (colorectal cancer cell line). These cell lines were cultured in DMEM (Waltham, MA, USA). The medium contained fetal bovine serum (10%), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo fisher Scientific, MA, USA). Constant temperature incubators were used for the cultivation of cell lines. Conditions were as follows: 37°C 5% CO2. Subsequent experiments were mainly performed on cells in logarithmic growth phase. Medium exchange rate: once every 3 days.
The online bioinformatics analysis website circinteractome was used to analyze the binding sites between circ_0052184 and miRNA. The possible binding sites between miR-604 and HOXA9 was used to analyze the bioinformatics analysis website Starbase (https://starbase.sysu.edu.cn/).
TRIzol (Invitrogen, California, US) was used to extract RNA from CRC tissues and cells. Then SuperScript IV reverse transcription kit (Thermo fisher scientific) was used to synthesize cDNA. LabScript Two-Steps SYBR Green qRT-PCR Kit is used for qRT-PCR detection. Primer list is detailed in Table 2.
GAPDH or U6 were used as controls, and the relative expression levels were normalized.
Reaction conditions: pre-denaturation at 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min for 30 s.
Relative expression levels of related genes were calculated by 2-△△CT method.
The successfully transfected Lovo and HT29 cell lines were collected. These cells were suspended in serum-free medium. Cell suspension was added to the upper chamber of the Transwell. The lower chamber of Transwell is medium supplemented with 20% fetal bovine serum. Remove the cell compartment from the incubator after 24 hours of culture.. 4% paraformaldehyde (Beyotime Biotechnology, Shanghai, China) fixed the migrated cells. After fixation, cells were stained with 0.1% crystal violet (Beyotime Biotechnology), rinsed, and photographed for counting after drying. In the invasion experiment, a layer of Millipore matrigel (MA, USA) was coated on the surface of the Transwell chamber. Other steps were equally the migration experiment steps.
The transfected cells in logarithmic growth phase were taken, and the concentration of the cell suspension was adjusted to about 3×10 4 cells/mL. Add 100 μL of the cell suspension to a 96-well plate, incubate at 37°C for about 24 hours until the cells grow to a monolayer, aspirate the supernatant, wash with PBS, add 20 μL MTT solution (5 mg/mL) and 80 μL serum-free medium , placed in a 37°C cell incubator for 4h. Add 100ul of Formanzan lysis solution to each well, and continue to incubate for about 4h in the cell culture incubator. The absorbance at 570 nm was read in a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Dual-luciferase reporter assay
The wild-type and mutant circ_0052184 and HOXA9 luciferase reporter vectors were constructed. After the colorectal cancer cell lines LoVo and HT29 were seeded in 96-well plates for a period of time, the Wt or Mut luciferase reporter plasmids and miR-604 mimic or NC were co-transfected into LoVo and HT29 cells by Lipofectamine TM 2000 (Invitrogen). Cells were cultured after 48h. The cellular luciferase activity of the different groups was detected by a Renilla-Firefly Luciferase Dual Assay Kit (MCE, Shanghai, China).
Targeting relationships between genes were examined using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit(Millipore, Massachusetts, US). After lysis of transfected cells with RIP lysis buffer, Incubate anti-ago2 antibody or negative control IgG-conjugated magnetic beads with RIP-added buffer. Next, added proteinase K to the mixture. and incubated with shaking. qRT-PCR verifies targeting relationships between genes in purified RNA.
The successfully transfected colorectal cancer cell lines were diluted and placed in 6-well plates for 24 hours. And then streaked with a sterilized pipette tip. Photographs were taken at 24h and 48h to record the wound closure. Relative cell migration ratio = (0h scratch area-48h scratch area)/0h scratch area.
Western blotting assay
RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract total protein from tissues and cell lines. SDS-PAGE and Polyvinylidene fluoride (PVDF) separate target proteins. Finally, the ECL kit (Beyotime) was used to detect total protein.
Graphpad Prism 8.0 (CA, USA) was used to analyze all data involved in this experiment. In order to better demonstrate the authenticity of the data, all experiments were performed at least three times. Mean ± standard deviation (X ± SD) represents measurement data. Two groups were compared by t-test or one-way ANOVA. Pearson correlation analysis was performed to analyze the correlation between genes. The difference was statistically significant with P<0.05.