Ethics statement
All animal experiments were in line with the Guide for the Care and Use of Laboratory Animal of the National Institutes of Health. The protocol was permitted by the Committee on the Ethics of Animal Experiments of Xinxiang Central Hospital.
Cell culture and sorting of CC stem cells (CD133+CaSki)
Human CC cell line CaSki was purchased from the Cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences, cultured in dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and placed in a saturated humidity incubator with 37 ℃, 5% CO2. The cells were detached with 0.25% trypsin and sub-cultured. CaSki cells in logarithmic growth phase were detached with trypsin, the cell concentration was adjusted to 1 × 106 cells/mL. And then, cells were hatched with CD133 antibody avoiding light for 20 min and the proportion of CD133+CaSki cells was detected by flow cytometry. The proportion of CD133+CaSki cells in CaSki cells before and after sorting was 2.99% ± 0.47% and 95.42% ± 6.30%, respectively, suggesting the successful sorting.
Extraction, identification, labeling and grouping of exosomes
Exosomes extraction: CC CaSki cells in logarithmic growth phase were placed in DMEM (Invitrogen, Carlsbad, California, USA) containing 10% FBS without exosomes, and cultured in an incubator for 3 d. The cell supernatant was gathered and centrifuged at 4 ℃ (2000×g) for 20 min and the dead cells were removed. And then cells were centrifuged at 4 ℃ (10,000×g) for 30 min, and the cell fragments were removed. Next, cells were centrifuged at 4 ℃ (100,000×g) for 70 min, and the adherent exosomes were re-suspended and precipitated by PBS, and centrifuged at 4 ℃ (100,000×g) again for 70 min. The pellets were re-suspended and precipitated by PBS, centrifuged at ultra-high speed for 2 h, re-suspended and precipitated by PBS, filtered by 0.22 μm filter and then saved at −80℃.
Exosomes identification: (1) CaSki-Exo suspension (20 μL) was placed on the sample copper net for 5 min and dripped with 3% phosphotungstic acid (20 μL) for 5-min staining. Exosomes were dried under a white lamp at 65℃ for 15 min, and the morphology of exosomes was observed under a transmission electron microscope (Hitachi High-technologies Corporation, Tokyo, Japan). (2) The expression of CD63 and CD81 was tested by western blot analysis and the marker protein in exosomes was identified. (3) The size distribution and concentration of exosomes were evaluated by nanoparticle tracking analysis (NTA).
The uptake of CaSki-Exo by CD133+CaSki cells was tested by fluorescence labeling: CaSki-Exo suspension (20 μL) was mixed with 1 mL Diluent C solution to prepare for CaSki-Exo working solution which was combined with PKH26 dyeing solution (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 5 min, added with 2 mL PBS containing 0.1% bovine serum albumin, centrifuged with ultra-high speed (11,000×g) for 1 h at 4 ℃ and stored at −80 ℃. Stained CaSki-Exo (10 μL) was added to CD133+CaSki culture system for 24 h, fixed with 4% paraformaldehyde, dyed with nuclear 4'-6-diamidino-2-phenylindole (DAPI), observed and photographed under the inverted laser confocal microscope (Olympus, Tokyo, Japan).
Grouping: cells were cultured conventionally in a 5% CO2 incubator at 37℃ with 10% FBS-DMEM. The CD133+CaSki cells in the logarithmic growth phase were inoculated in the well plate. The control group and CaSki-Exo group (added with 200 μg/mL exosomes) were set, and 5 parallel wells were set up in each group.
UCA1 and miR-122-5p shuttle experiment
The synthesized sequence siRNA-negative control (NC), siRNA-UCA1, mimic-NC, miR-122-5p mimic, pcDNA-UCA1 + mimic-NC, and pcDNA-UCA1 + miR-122-5p mimic (all purchased from Shanghai GenePharma Co, Ltd, Shanghai, China) were transfected into CaSki cells. After 24 h, the CaSki-Exo were extracted and co-cultured with CD133+CaSki cells. A Transwell chamber (0.4 μm) was put in the six-well plates and seeded with 1 × 105 CaSki cells, and 3 × 105 CD133+CaSki cells were seeded into the plate for 7 d to establish a co-cultured system.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The RNA of cells was extracted by RNA extraction kit (Invitrogen). UCA1, miR-122-5p, SOX2, U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were designed by TaKaRa Biotechnology Co. Ltd (Liaoning, China) (Table 1). The RNA was reversely transcribed into cDNA using the PrimeScript RT kit, the reverse transcription system was 10 μL. Fluorescent quantitative PCR was operated in the light of the procedure of the SYBR® Premix Ex Taq™ II. The relative transcriptional levels of target genes were computed by 2−△△Ct method.
Western blot analysis
The total protein was extracted from the cells and the protein concentration was determined by bicinchoninic acid kit (AmyJet Scientific, Wuhan, Hubei, China). The extracted protein was mixed with the loading buffer and centrifuged after boiling at 95 ℃ for 10 min, separated with 10% polyacrylamide gel electrophoresis, and transferred to membrane. The membrane was sealed with 5% skim milk in tris-buffered saline with tween 20 (TBST) for 1 h, added with primary antibody against SOX2 (1: 1000, Abcam, Cambridge, MA, USA), OCT4 (1: 1000), Nanog (1: 1000) (Cell Signaling Technology, Beverly, MA, USA), CD63 (1: 200), CD81 (1: 200) and GAPDH (1: 1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 ℃. The protein was added with the corresponding secondary antibody (1: 2000, Abcam, Cambridge, MA, USA) for 1 h at 37 ℃ and developed by chemiluminescence reagent. The protein imprinted image was analyzed with ImageJ2x Software (National institutes of health, Maryland, USA).
Scratch test
CD133+CaSki cells (5 × 107 cells/mL) in the logarithmic phase were seeded in the 24-well plates (300 μL/well) with four parallel wells in each group. A sterilized 100 μL pipette tip was adopted for scratching in the 24-well plate and cells were photographed at 0 h and 24 h, respectively to measure the migration distance.
Invasion and migration experiment
Migration experiment: serum-free DMEM (100 μL) was added to a Transwell upper chamber, and incubated in a 5% CO2 incubator for 1 h to activate a polycarbonate membrane. CD133+CaSki cells (2 × 105 cells/mL, 100 μL) in the logarithmic growth phase, together with serum-free DMEM were added to the upper chamber, and 600 μL DMEM containing 20% FBS without exosomes was added to the lower chamber, and the cells were incubated for 24 h. The cells were fixed with methanol for 10 min, dyed with 1% crystal violet staining solution for 10 min, pictured under the microscope in eight visual fields and counted.
Invasion test: all steps were the same as the above migration experiment, except that 100 μL serum-free DMEM was changed to 50 mg/L Matrigel (1: 40, 100 μL).
Flow cytometry
AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used to analyze cell apoptosis with the Annexin V-FITC Apoptosis Detection Kit I (556547, BD Biosciences, Franklin Lakes, NJ, USA). The cells in the logarithmic growth phase were seeded on the 6-well plate with 2 × 105 cells/well, cultured for 72 h and amassed. The cells were suspended in the 500 μL binding, mixed with 5 μL FITC and 5 μL PI and incubated for 15 min, and the apoptosis was analyzed by flow cytometry.
Cell counting kit (CCK)-8 assay
When the cell confluence reached about 80%, the cells were made to a single cell suspension, and 3000 cells/100 μL per well were seeded in 96-well plates and hatched in an incubator. CCK-8 reagent (10 μL, Sigma, St. Louis, MO, USA) was added to each well at 24 h, 48 h and 72 h, respectively. Then the cells were continually cultured for 2 h and then the optical density (OD) value at 450 nm of each well was read by a microplate reader (Beijing Potenov Technology Co., Ltd, Beijing, China). The cell viability curve was drawn with the time point as the transverse coordinate and the OD value as the longitudinal coordinate.
Tumor xenografts in nude mice
Sixty female mice (SJA Laboratory Animal Co., Ltd., Shanghai, China) aged 4 w and weighed 80–90 g were selected. Mice were fed in a clean laminar flow rack of specific pathogen-free grade barrier system, the temperature was (25 ± 1) ℃, and the relative humidity was 40–60%. Matrigel was diluted with serum-free medium to 50%, and mixed with CD133+CaSki cell suspension with a proportion of 1: 1. Cells (2 × 106 cells/200 μL) were injected subcutaneously in nude mice. The general status of the nude mice was observed. The tumor volume (V) was monitored every 5 days. Tumor volume was calculated by measuring tumor length (a) and width (b): V = ab2/2. The average volume of subcutaneous tumor was calculated, and the tumor growth curve was drawn. Thirty days after injection, the nude mice were euthanized, the tumor was carefully peeled off, pictured and weighed.
Fluorescence in situ hybridization (FISH) assay
The subcellular localization of UCA1 in cells was identified by FISH. According to the instructions of Ribo™ lncRNA FISH Probe Mix (Red) (RiboBio Co., Ltd, Guangdong, China), the specific methods were as follows: the slide was put into the 24-well culture plate, and the cells were seeded at 6 × 104 cells/well and grown to 80% confluence. The slide was taken out, the cells were fixed with 1 mL 4% paraformaldehyde after cleaning with PBS. After being treated with protease K, glycine and phthalylation reagent, the cells were added with 250 μL pre-hybrid solution and incubated at 42 ℃ for 1 h. The pre-hybrid solution was absorbed, cells were added with 250 μL UCA1 (300 ng/mL) hybrid solution containing probe and hybridized overnight at 42 ℃. The nucleus was stained with phosphate-buffered saline with Tween (PBST)-diluted DAPI (ab104139, 1:100, Abcam, Shanghai, China), added to the 24-well culture plate, and stained for 5 min. The cells were sealed with anti-fluorescence quenching agent, observed and photographed under a fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan).
Dual luciferase reporter gene assay
The target relationship between UCA1 and miR-122-5p or miR-122-5p and SOX2 and the binding site between miR-122-5p and UCA1 3’untranslated region (3’UTR) or SOX2 3’UTR were analyzed by bioinformatics software https://cm.jefferson.edu/rna22/Precomputed/. The sequence of UCA1 3’UTR or SOX2 3’UTR promoter region containing miR-122-5p binding site was composed to construct UCA1 3’UTR wild-type (WT) plasmid (UCA1-WT) or SOX2 3’UTR WT plasmid (SOX2-WT). The UCA1 3’UTR mutant type (MUT) plasmid (UCA1-MUT) or SOX2 3’UTR MUT plasmid (SOX2-MUT) was constructed by mutation binding site. The CaSki cells with 70% confluence were transfected with UCA1-WT/UCA1-MUT or SOX2-WT/SOX2-MUT with mimic-NC or miR-122-5p mimic by Lipofectamine 2000. The cells were gathered and lysed 48 h after transfection, and luciferase activity was verified by luciferase detection kit (RG005, Shanghai Beyotime Biotech Co., Ltd., Shanghai, China).
RNA-pull down assay
The biotin labeled miR-122-5p WT plasmid and miR-122-5p MUT plasmid (50 nM each) were transfected into the cells, respectively. After 48 h, the cells were gathered and cleaned with PBS and incubated with specific cell lysate (Ambion, Austin, Texas, USA) for 10 min. And then, 50 mL sample cell lysate was divided into two groups. The residual lysate was incubated with M-280 streptavidin magnetic beads (Sigma, St. Louis, MO, USA) which pre-coated with RNase-free and yeast tRNA (Sigma, St. Louis, MO, USA) for 3 h at 4 ℃. Then the cells were washed twice with cold lysate, three times with low salt buffer, and once with high salt buffer. An antagonistic miR-122-5p probe was set up as a NC. The total RNA was extracted by Trizol and the expression of UCA1 was tested by RT-qPCR.
Statistical analysis
All data were analyzed by SPSS 21.0 software (IBM Corp. Armonk, NY, USA). The measurement data were represented by mean ± standard deviation. Comparisons between two groups were conducted by t-test, while comparisons among multiple groups were assessed by one-way analysis of variance (ANOVA). P value < 0.05 was indicative of statistically significant difference.
