Patients and samples
The HCC tissues were collected from surgical resections of patients without preoperative treatment at Eastern Hepatobiliary Surgery Hospital (Shanghai, China). A group of 30 HCC specimens were used for analyzing the correlation between miR-96 and SOX6 mRNA expression. Three HCC specimens were used for isolating CD133 and EpCAM positive liver T-ICs. Four specimens were used for patient-derived xenograft (PDX) model. Patient informed consent was obtained and the procedure of human sample collection was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital.
Cell lines and cell culture
Patient-derived primary HCC cultures of tumor cells were obtained from fresh tumor specimens of HCC patients as previously described. The human primary hepatoma cells were isolated by collagenase perfusion and centrifugation. Briefly, the liver cancer tissues were washed several times in pre-cooled sterile PBS buffer containing Red Blood Cell Lysis Buffer and 0.5% collagenase IV to remove blood and connective tissue; GBSS mixed enzyme solution was used for digestion. The cells were centrifuged, and the supernatant was discarded. Cell activity was detected by Trypan Blue staining, and cultured in a bottle containing complete medium heavy suspension at 37℃ and 5% CO2 environment culture. During this process the cell morphology was identified.
The HCC cell line HCCLM3 were purchased form the Chinese Academy of Sciences (Shanghai, China). The HCC cell line CSQT-2 was obtained from professor Shuqun Chen. The HCC cells were cultured with Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, and 25 µg/ml of gentamicin and maintained at 37°C in 5% CO2 incubator. The cultured cells were digested with 0.5% trypsin and moved to a new plate twice a week. miR-96 mimic or miR-96 sponge lentivirus and their control lentivirus were purchased from Shanghai RiboBio (Guangzhou, China). The HCC cells were infected with lentivirus and then screened by puromycin as described before (14). The SXO6 siRNA and its control were obtained from GenePharma (Shanghai, China).
RNA interference
Small interference RNAs (siRNAs) against SOX6 and NC (NC, negative control) siRNA were synthetized by Genepharma (Shanghai, China). The siRNAs were transfected into the hepatoma cells at a final concentration of 200 nM using siRNA transfection reagent according to the manufacturer’s instructions (Polyplus, Illkirch, France). The cells were harvested or subjected to further downstream experiments 24-72 hours after transfection. Gene knockdown was validated by western blotting.
Flow-cytometry analysis
For CD133+ and EpCAM+ cells sorting, primary HCC patients’ cells and hepatoma cells were incubated with the primary anti–CD133 (Cat. no. 372806, Biolegend, Inc., San Diego, CA) or anti-EpCAM (Cat. no. ab8666; Abcam, USA) for 30 minutes at room temperature. The cells were then subjected to flow cytometry using a MoFlo XDP cell sorter from Beckman Coulter (Indianapolis, IN, USA) according to the manufacturer’s instructions. The sorted cells from three independent experiments were subjected to Real-time PCR assay.
miR-96 mimic or miR-96 sponge and control hepatoma cells were incubated with the primary anti-EpCAM for 30 minutes at room temperature. The flow-cytometry analysis was performed using a MoFlo XDP from Beckman Coulter according to the manufacturer’s instructions.
Spheroid formation assay
miR-96 mimic or miR-96 sponge and their control hepatoma cells were cultured in a 96-well ultra-low attachment (300 cells per well) and cultured in DMEM/F12 (Gibco) media, supplemented with 1% FBS, 20 ng/mL bFGF and 20 ng/mL EGF for seven days. The total number of spheres was counted under the microscope (Olympus).
In vitro limiting dilution assay
Various numbers of miR-96 mimic or miR-96 sponge and their control hepatoma cells (2, 4, 8, 16, 32, 64 cells per well, n=16) were seeded into 96-well ultra-low attachment and cultured in DMEM/F12 (Gibco) supplemented with 1% FBS, 20 ng/mL bFGF and 20 ng/mL EGF for seven days. The CSC proportions were analyzed using Poisson distribution statistics and the L-Calc Version 1.1 software program (Stem Cell Technologies, Inc., Vancouver, Canada) as previously described (15).
In vivo limiting dilution assay
For the in vivo limiting dilution assay, miR-96 mimic or miR-96 sponge and their control hepatoma cells were mixed with Matrigel (BD) at a ratio of 1:1 and injected subcutaneously at indicated cell doses per NOD-SICD mouse (n=8). After 8 months, tumors formation was evaluated.
Real-time PCR
For detection of mature miR-96, total RNA was subjected to reverse transcription using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR analysis of miR-96 expression was carried out using TaqMan MicroRNA assay kits (Applied Biosystems). Results were normalized to U6 snRNA using the comparative threshold cycle (Ct) method.
The total RNA was extracted by using Trizol reagent (Invitrogen, 15596-018). Total cDNAs were synthesized by ThermoScript TM RT-PCR system (Invitrogen, 11146-057). The total mRNA amount present in the cells was measured by RT-PCR using the ABI PRISM 7300 sequence detector (Applied Biosystems). PCR conditions included 1 cycle at 94 °C for 5 minutes, followed by up to 40 cycles of 94 °C for 15 seconds (denaturation), 60 °C for 30 seconds (annealing) and 72 °C for 30 seconds (extension). The sequences of primers used was listed in Supplementary Table 1.
Western blotting assay
Thirty micrograms of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated with the primary antibody overnight. The protein band, specifically bound to the primary antibody, was detected using an IRDye 800CW-conjugated secondary antibody and LI-COR imaging system (LI-COR Biosciences, Lincoln, NE, USA). The primary antibodies used were listed in supplementary Table 2.
Luciferase reporter assay
Wild type and mutated SOX6 3’UTR was cloned to psiCHECK-2 Vector (Promega, Madison, WI, USA) to construct the psiCHECK-SOX6 3’UTR-wt and psiCHECK-SOX6 3’UTR-mut vectors with Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). miR-96 sponge and its control hepatoma cells were transfected with SOX6 WT or SOX6 mutant 3’UTR plasmids for 48h. The luciferase activity was measured using a Synergy 2 Multidetection Microplate Reader (BioTek Instruments, Inc.). The data were normalized for transfection efficiency by dividing firefly luciferase activity by Renillaluciferase activity.
Apoptosis Assay
miR-96 mimic or miR-96 sponge and their control hepatoma cells were treated with sorafenib (10 μM) for 48 hours, followed by staining with Annexin V and 7-AAD for 15 minutes at 48C in the dark. Apoptotic cells were determined by an Annexin VFITC Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA) and detected by flow cytometry according to the manufacturer’s instructions.
Patient-derived xenograft
For the patient-derived xenograft (PDX) model, primary tumor samples were obtained for xenograft establishment as described previously. The mice with xenografts were given sorafenib (60 mg/kg) or vehicle daily orally for 24 days (n=5 for each group). Tumor volumes were measured at the end time points. All procedures and protocols were approved by the Institutional Review Board of Eastern Hepatobiliary Surgery Hospital.
Statistical analysis
GraphPad Prism (GraphPad Software, Inc. La Jolla, USA) was used for all statistical analyses. Statistical analysis was carried out using t test or Bonferroni Multiple Comparisons Test: *p<0.05. A p value of less than 0.05 was considered significant.