Patients
On institutional review board approval, we identified 90 patients with hepatocellular carcinoma (HCC) treated with radical cystectomy between 2011 and 2019 at Renmin Hospital of Wuhan University and Tongji Hospital of Huazhong University of Science and Technology. None of the patients received adjuvant therapy. Data collected from each patient included gender, age at diagnosis, grade, stage, and overall survival time. Pairs of cancer tissues and adjacent epithelium tissues of the same HCC patients were obtained by radical cystectomy. The study was approved by the medical ethics committee of each participating institute. Informed consent (written or verbal) was obtained from the patients within this study. All the samples were anonymous.
Cell lines
Human colorectal cancer cell lines HepG2, HCC-1973, Hep3B, ZR-75-30, MCF-7, MDA-MB-231, T-47D, SMMC-7721 and HEK293T cell were grown in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; ScienCell, Carlsbad, CA). Cells were maintained in cell incubator with 5% CO2 at 37°C. All cell lines were purchased from ATCC. Microsart® Mycoplasma Kit (Sartorius Inc., Gottingen, Germany) was used to monitor cells for Mycoplasma contamination routinely.
Antibodies
Primary antibody against AGO2 (ab186733) and Survivin (ab469) were purchased from Abcam Inc. (Cambridge, MA). Antibodies for detecting Snail (#3895) and Vimentin (#5741) were purchase from Cell Signaling Technology, Inc. (Danvers, MA). Primary antibody for GAPDH (sc-25778), and secondary antibodies including anti-rabbit IgG (sc-2004) and anti-mouse IgG (sc-2005), were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).
Construction of AGO2 knock-out cell line
We knocked out the AGO2 in SMMC-7721 cells using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 method. sgRNA (5’-GCCACCATGTACTCGGGAGC, TSINGKE Inc., Beijing, China) was designed to target at genomic AGO2 exon and cloned into plasmid lenti-CRISPR-v2 (Addgene plasmid # 52961). And then the construct was transfected into HEK293T cells with psPAX2 and psMD.2 (Addgene plasmid # ) using Lipofectamine 2000 (Invitrogen). At 72 hours post transfection, the lentivirus was harvested and infected SMMC-7721 cells. At 48 hours post infection, the stable cell lines were generated by selection in 2 μg/ml puromycin for one week. Then, monoclonal cell was culture in 96-well plate until indicated cell population. Cells were harvested for genome DNA sequencing and western blot to identify AGO2 knocked out.
CCK8 assay
For the cell growth experiment, 2000 cells were cultured at each of the 96-well plate. And then we test the absorbance at 450 nm using CCK8 kit every day (100 μl fresh DMEM medium and 10 μl CCK8 solution were added into each 96-well plate. Subsequently, incubated at 37℃ for 2 hours). The absorbance was tested at 450nm using CCK8 kit as described above.
Cell focus colony formation and migration assay
In the colony formation assay, SMMC-7721 cells were grow in 6-well culture plates at a density of 600 cells per well. Then, the colonies were fixed by methanol and stained by crystal violet (Sigma–Aldrich, St. Louis, MO, USA) until the colonies were visible. The number of cells formed into a clone was calculated.
In the cell migration assay, 1×105 suspended SMMC-7721 cells in 600 μl with serum-free medium placed in the upper compartment of 8-μm transwells (Corning Inc., Acton, MA) in a 24-well plate. Additional 600 μl medium containing 10% FBS was added to the bottom wells. At 12 hours post culture, cells in bottom transwells were fixed by methanol and stained by crystal violet. The number of migration cells into bottom transwells was counted.
Mice and xenograft tumor model
BALB/c nude mice (4-6 weeks old female) were purchased from Beijing HFK Bioscience Co. Ltd. Laboratory Animal Center. The mice were housed under specific pathogen-free (SPF) conditions in separated ventilated cages. Mice were housed as 5 per cage and in a climate-controlled room (25 °C, 55% humidity and 12-h light/darkness cycle). All procedures involving mice and experimental protocols were approved by the ethics committee of Tongji Hospital of Tongji Medical College and in compliance with the NIH guidelines.
Mice were randomly assigned as control group and model group (n = 10 mice per group). In model group, SMMC-7721 cells (Control and AGO2 knock out) were subcutaneously injected in the flank of BALB/c nude mice with 2×106 cells. Mice in control group only received sterile PBS injection. About 7 days post injection, the tumor size was measured every other day. The tumor size was calculated as (length×width2)/2. At the indicated time, all mice were sacrificed with CO2 inhalation and the tumors were photographed.
Reverse Transcription Reaction and Quantitative Real Time PCR
Total RNA was extracted with Trizol reagent (Invitrogen) and reverse transcribed into cDNA using the M-MLV reverse transcriptase (Promega). GAPDH was used as an internal control to normalize the amount of total mRNA in each sample.
Real time PCR was performed with a standard SYBR Green PCR kit (Takara Shuzo Co. Ltd, Kyoto, Japan) according to manufactor’s instructions in a real-time PCR system (Applied Biosystems7500, Foster City, CA) as follows: 95°C for 3 min followed by 40 cycles of 95°C for 5 sec, 60°C for 20 sec and 72°C for 30 sec and then 94°C for 1 min, 60°C for 1 min, with addition of a cycle for every 0.5°C. The primers used in the study are shown as follow: AGO2 forward: 5’-CCTGTATGAGAACCCAATGTC; reverse: 5’-CAGCTAGTTTGAGCCCATCA. GAPDH forward: 5’-AAGGCTGTGGGCAAGG; GAPDH reverse: 5’-TGGAGGAGTGGGTGTCG.
Western Blot
Cells were lysed with radioimmunoprecipitation assay buffer (RIPA Buffer) containing protease inhibitors and centrifuged at 13000×g and 4ºC for 5 minutes. The protein concentration of the supernatants was determined with the BCA kit (Thermo Scientific). 100 μg of cell lysatea were resolved by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham, Piscataway, NY). After blocking with 5% non-fat milk in TBST containing 0.05% (V/V) Tween-20 at room temperature for 1 hour, membranes were incubated overnight at 4ºC with the appropriate primary antibody. Blots were then incubated at room temperature for 1 hour with a horseradish peroxidase (HRP) conjugated secondary antibody and the peroxidase activity was detected with a chemiluminescent HRP substrate (Millipore, Billerica, MA) and imaged by a chemiluminescence system (Fujifilm LAS-4000, Tokyo, Japan).
Statistical Analysis
Survival curves were plotted using the method of Kaplan-Meier and the significance of observed differences was calculated with log-rank test. All other comparisons were determined by the Student’s t test. All reporter assays were repeated for at least three times. Data are shown as average values (mean) ± SD (standard deviation) from one representative experiment. The P value < 0.05 was considered statistically significant.