Cell lines, cell culture, and materials
The bladder cancer (T24) cells were purchased from the ATCC (Manassas, VA, USA) and cultured using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. 4′,6-diamidino-2-phenylindole (DAPI) was acquired from Molecular Probes (Eugene, OR, USA). Dojindo Laboratories (Kumamoto, Japan) supplied the cell counting kit 8 (CCK-8), and BD PharMingen (Heidelberg, Germany) supplied the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit. Genepharma (Shanghai, China) synthesized and supplied the siRNAs. The circPRMT5-targeting siRNA consisted of a sense strand 5′-AUCUUCCGGCUCCUCAAGUUCTT-3′ and an antisense strand 5′-GAACUUGAGGAGCCGGAAGAUTT-3′. The scrambled negative control siRNA (NC)-siRNA also consisted of a sense strand 5′-UUCUCCGAACGUGUCACGUUU-3′ and an antisense strand 5′-ACGUGACACGUUCGGAGAAUU-3′. The sequence of the FAM and Cy5-labeled (fluorescent) NC-siRNAs was the same, and the dye was attached to the antisense strand.
Preparation of SCNTs-siRNA and characterization
Chrysotile preparation: Using tetraethoxysilane (Si (OCH3)4) as the silicon source and magnesium chloride hexahydrate (MgCl2·6H2O) as magnesium source, MgCl2·6H2O was dissolved in deionized water at the ratio of Mg:Si = 3:2 to form a solution, then Si (OCH3)4 was added and stirred for 15 min to prepare a 0.1 M solution in 50 mL. The pH of the solution was adjusted to 13.0 by dropping 1 mol/L and 4 mol/L NaOH solution and then stirring for 20 min. The solution was poured into a reactor for hydrothermal synthesis. The synthetic chrysotile sample was obtained by constant temperature reaction at 240 °C for 72 h, repeated filtration and drying in an oven at 60 °C for 6 h.
SiRNA loading: Chrysotile was put into an autoclave for high temperature sterilization, kept at 121 °C for 20 min, cooled to 60–70 °C for sampling. After centrifuging siRNA at 3000 rpm for 5 min, and injecting DEPC water and siRNA into alcohol, the experiment was carried out on the sterile operation platform. Add 62.5 μL of DEPC water into the siRNA NC tube (containing 0.5 OD siRNA), and repeatedly wash with a pipette gun for 2 to 3 times, mix evenly, and place all samples in the same centrifuge tube. Then, 12 μL siRNA and 30 μL chrysotile solution and blank DEPC water were mixed evenly, sealed with sealing film, and then pricked with syringe needle to ensure that the pressure was below 0.8 and vacuum impregnated for 3 h to obtain RNA loaded chrysotile.
The ability of SCNTs to bind to the siRNA was assessed using agarose gel electrophoresis. Agarose gels (0.5%, run at 90 V for 60 min) were used to separate 15 μL of SCNTs/siRNA nanoparticles with different mass ratios (1–80), with a control comprising free siRNA. Ethidium bromide staining was used to visualize the nucleic acids on the gel.
Cellular uptake assay
BD FACSCalibur flow cytometry (BD Biosciences San Jose, CA, USA) was used to quantify the uptake of SCNTs/FAM-si-circPRMT5 nanoparticles by cells. T24 cells (2 × 105 cells/well in 2 mL of DMEM) were added to the wells of six-well plates and grown for 1 d. Phosphate-buffered saline (PBS) was used to rinse the cells, which were then incubated with SCNTs/FAM-si-circPRMT5 nanoparticles for 2 h. Thereafter, cold PBS was used to rinse the cells three times, and then the cells were centrifuged, resuspended, and assessed using flow cytometry.
Next, T24 cells (5 × 104 cells per well) were seeded in chambered coverslips and cultured for 24 h. Subsequently, SCNTs/FAM-si-circPRMT5 nanoparticles at a 20:1 mass ratio was added and incubated for 4 h, after which the culture medium was discarded. The cells were washed twice using PBS and then fixed using 4% paraformaldehyde (v/v). DAPI was used to stain the cell nuclei. Mounting medium was used to seal the cells, which were examined via confocal laser scanning microscopy (CLSM; Nikon, Tokyo, Japan).
To study the SCNTs/FAM-si-circPRMT5 nanogel’s endosomal escape properties, we seeded 5 × 105 T24 cells (per well) into CLSM dishes and grew them for 1 d at 37 °C. The cells were then incubated with 100 nM SCNTs/FAM-si-circPRMT5 for 2 h. Afterwards, the SCNTs/FAM-si-circPRMT5-containing medium was discarded and fresh medium was added to the cells and incubated for 3 and 6 h, respectively. The cells were then washed with PBS three times and incubated for 30 min with 250 nM LysoTracker Red. The cells were then observed immediately using CLSM.
Confocal imaging of pathways related to endocytosis
In glass bottom confocal dishes, 5 × 104 T24 cells per well were and incubated overnight. Next day, Alexa Fluor 555-labeled endocytic markers (5 μg/mL Cyclotraxin B (CTX-B), 25 μg/mL transferrin, and 25 μg/mL dextran) and SCNTs/FAM-si-circPRMT5 were added to the cells and incubated for 2 h at 37 °C. During the last 15 min of incubation, Hoechst 33342 at 2 μg/mL was added. At the end of the 2 h incubation, the cells were washed and subjected to confocal microscopy visualization.
Quantitative real-time reverse transcription PCR (qRT-PCR)
The Trizol reagent was used to extract total RNA from cells or tissues. Total RNA (5 μg) was subjected to reverse transcription using a first-strand cDNA synthesis kit (Beyotime Institute of Biotechnology, Jiangsu China) following the supplier’s protocols. The resultant cDNA was then used as the template for qPCR. The qPCR primers were as follows: circPRMT5 sense: 5′-CCACTGTACTCCTCTGTGTGT-3′ and circPRMT5 anti-sense: 5′- CCACTGTACTCCTCTGTGTGT-3′; miR-30c sense: 5′- ACCATGCTGTAGTGTGTGTAAACA-3′ and miR-30c anti-sense: 5′- TCCATGGCAGAAGGAGTAAA-3′; GAPDH sense: 5′- TGCACCACCAACTGCTTAGC-3′ and GAPDH anti-sense: 5′- GGCATGGACTGTGGTCATGAG-3′.
Western blotting analysis
Total proteins were obtained from tissues and transfected cells by incubation for 30 min on ice with Radioimmunoprecipitation assay (RIPA) cell lysis buffer containing protease inhibitors, with gentle shaking. After centrifugation of the lysates at 12000 rpm for 10 min, the supernatant was retained and the protein concentration was measured using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). 8% SDS-PAGE was used to separate the proteins, which were then electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and then were incubated with primary antibodies at overnight at 4 °C: Anti-SNAIL1 (1:1000 dilution, Proteintech, Chicago, IL, USA), anti-E-cadherin (1:1000 dilution, Abcam, Cambridge, MA, USA), or anti-GAPDH (1:5000 dilutions, Proteintech). Next, the membranes were reacted for 2 h with goat anti-rabbit IgG-horseradish peroxidase (HRP) secondary antibodies. The immunoreactive proteins’ fluorescent signals were assessed using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA).
Staining via immunohistochemistry (IHC)
Proteins were stained using primary antibodies and their expression was revealed utilizing a Dako Real Envision Kit (Dako, Carpentaria, CA, USA) following the supplier’s instructions. The staining intensity was scored manually by two experience pathologists independently. To evaluate IHC staining, we used semiquantitative scoring criteria, incorporating the staining intensity and positive areas. A staining index (scores 0–12), obtained as the intensity of SNAIL1, E-cadherin, or Ki67 positive staining (negative = 0, weak = 1, moderate = 2, or strong = 3 scores) and the proportion of immunopositive cells of interest (< 10% = 1, 10–50% = 2, > 50% and < 80% = 3, ≥ 80% = 4), was calculated. In the case of heterogeneous staining, the percentage of different staining intensities was determined individually in each area and the total sum was calculated. The primary antibodies used comprised: Anti-SNAIL1 (1:100 dilution, Proteintech), anti-E-cadherin (1:200 dilution, Abcam), anti-Ki67 (1:30 dilutions, Cell Signaling Technology, Danvers, MA, USA).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity analysis
We performed MTT assays on T24 cell. Cells at 5000 cells/well were seeded into 96-well plates 24 h prior to the assay. Cells were treated for 48 h, after which they were washed three times with PBS, and 100 μL of fresh medium was added to each well. Next we assed 20 μL of MTT solution (5 mg/mL in deionized water) to the cells and incubated them for 4 h. The medium was discareded, acid SDS solution (100 μL of 10% SDS, 5% isopropanol, 0.012 mol/L HCl) was added to every well and mixed with the purple formazan crystals using transfer-pipettes, and then incubated for 12 h. A microplate reader (Bio-Rad, Hercules, CA, USA) read the optical densities at 570 nm.
Colony formation assay
In a six-well plate, 1 × 104 cells in 0.4% Seaplague agar were spread on a base of 0.6% agar and grown. Three weeks later, colonies comprising > 80 cells were counted and the results were expressed as the means ± SD of triplicate wells in the same experiment.
Cell cycle and apoptosis assays
First, T24 cells were grown to 80% confluence in six-well plates, and then treated with PBS, SCNTs, SCNTs/siNC, and SCNTs/si-circPRMT5 for 1 or 2 d. The cells were then assayed following the protocols of the Annexin V-FITC Apoptosis Detection Kit and Cell Cycle and Apoptosis Analysis Kit (Beyotime), followed by flow cytometry.
Terminal deoxynucleotidyl transferase-mediated (dUTP) nick end labeling (TUNEL) assays
Following the various treatments, the mice were killed humanely. Tumor tissues were surgically removed and fixed using 4% paraformaldehyde. The tissues were sectioned (10 μm thick), stained employing an ApopTag® red in situ apoptosis detection kit (Merck Millipore, Darmstadt, Germany), and viewed using confocal microscopy (Carl Zeiss, Jena, Germany).
Tumor cell motility in vitro
Migration assay: T24 cells were grown in six-well plates to 80% confluency, and then the cell surface was scratched using a 10 μL pipette tip. After two washes with PBS, the medium was swapped with fresh low serum medium (2% FBS). Images were obtained at 0 h and 48 h under a fluorescent microscope (IX51, Olympus, Tokyo, Japan) and compared to determine the scratch width.
Invasion assay: T24 cells (1 × 106 cells/mL; 200 μL) were added into the top chamber of a 24-well Transwell plate (pore size = 8 μm; Corning Star, Cambridge, MA, USA). Complete medium with 10% FBS (500 μL) was placed in the bottom chamber. The chambers were cultured for 1 and 2 days at 37 °C in 5% CO2. The unpenetrated cells were wiped off, and the cells in bottom chamber were fixed using 4% paraformaldehyde, stained with Giemsa, and viewed under a microscope. We counted the membrane-penetrating cells in five randomly selected fields of view.
In vivo distribution assay
Female BALB/c mice (4 weeks old; 18–22 g in weight), were raised under specific pathogen-free (SPF) conditions with free access to water and food. To investigate the SCNTs nanoparticle biodelivery of si-circPRMT5, we injected Cy5-siRNA and the Cy5 fluorescence into the mice, followed by detection using IVIS.
The injection of 0.1 mL of T24 cells (1 × 107 cells) into BALB/c nude male mice right flanks to establish the xenograft tumor model. When the tumor volume reached 200 to 300 mm3, the mice were divided randomly into four groups (n = 5) and injected intratumorally with SCNTs/Cy5-si-circPRMT5 complexes, SCNTs, naked Cy5-si-circPRMT5, or PBS. A Bio-Real Quick View 3000 imaging system (Bio-Real Sciences, Salzburg, Austria) was then used to scan the mice at 2 and 8 h after injection, using a 1 s exposure time for each image; images were analyzed using Living Imaging software (Bio-Real Sciences). For the tissue distribution study, tail veins were injected with PBS, naked Cy5-si-circPRMT5, SCNTs, and SCNTs/Cy5-si-circPRMT5 complexes. At 2 h and 8 h after intravenous tail vein injection, the mice were sacrificed. Living Imaging software was utilized for analysis of the excised tissues (tumor, spleen, kidney, lung, liver, and heart).
Assessment of in vivo antitumor effects using a subcutaneous injection model
1 × 106 T24 cells per mouse were injected subcutaneously into the flank region of each mouse to establish the xenograft tumor model. When the T24 tumors reached 100 mm3 on the right flank, the mice were divided randomly into four groups (n = 5 per group) and treated with PBS, si-circPRMT5, SCNTs, or SCNTs/si-circPRMT5, separately. In each group, treatments were delivered via intratumoral injection once per week for five weeks. Every four days, the mice were weighed and calipers were used to measure the tumors. The tumor volume calculation was: (length × width2 / 2). In addition, the mouse survival rate was recorded. At 24 h after the last injection, the mice were sacrificed, and the tumors were excised and photographed. Levels Ki67, SNAIL1, and E-cadherin proteins were detected using western blotting and IHC. TUNEL staining was carried out to assess apoptosis. An EVOS XL Core microscope was used to observe the stained sections.
In vivo antitumor effects by tail vein injection
To investigate the antitumor activity of SCNTs/si-circPRMT5 in vivo, a SCID mouse tail vein injection model of lung metastasis was constructed using implanted T24 cells. T24 cells (100 μL of cold PBS containing 2.5 × 106 cells) were injected into tail veins to create tumor-bearing BALB/c nude mice. Four weeks after injection, mice were then divided randomly into the following four groups: (1) PBS control, (2) si-circPRMT5s only, (3) free SCNTs, and (4) SCNTs/si-circPRMT5. The treatments were injected weekly for 5 consecutive weeks through the tail vein. After 5 weeks of treatment, CO2 euthanasia was carried out and lung tumor tissues were excised and subjected to histological examination.
In vivo antitumor effects by intravesical instillation
To determine the antitumor activity of SCNTs/si-circPRMT5 in vivo, an in situ model of bladder cancer was constructed. Ether inhalation was used to anesthetize female SD rats, whose bladders were then infused using 0.2 ml N-methyl-Nitrosurea (MNU) (10 mg/mL; Sigma, St. Louis, MO, USA,) employing a 22-gage angiocatheter once every 14 d for five times. For the avoidance of spontaneous micturition, the catheterized rats were kept under anesthesia for about 45 min [25, 26].
After the successful induction of tumors, 40 rats were divided into 4 groups comprising 10 rats per group. The rats were anesthetized and their bladders were instilled with 500 μL of SCNTs/si-circPRMT5, si-circPRMT5 only, an equivalent dose of free SCNTs, or PBS. For the avoidance of spontaneous micturition, the catheterized rats were kept under anesthesia for about 45 min. These treatments were then delivered once weekly for 5 weeks. At 2 days after termination of therapy, the rats were sacrificed humanely, their bladders were removed, weighed, fixed for 1 d in 4% paraformaldehyde, paraffin-embedded, and examined histopathologically. Transverse sections cut from the midportion of the bladder were subjected to hematoxylin and eosin (H&E) staining.
To investigate SCNTs/si-circPRMT5 toxicity, healthy normal mice received i.v. injections of SCNTs/si-circPRMT5, an equal dose of si-circPRMT5 only, an equal dose of free SCNTs, or PBS (n = 5 per group). Twenty-four hours later, blood was collected for routine blood tests. Renal and hepatic damage was assessed according to blood serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), total protein (TP), blood urea nitrogen (BUN), and creatinine (CREA). To assay immunotoxicity, enzyme-linked immunosorbent assay (ELISA) kits (Abcam) were employed to quantify the levels of serum cytokines (interleukin (IL)-1β, IL-6, interferon alpha (IFN-α), and tumor necrosis factor alpha (TNF-α)). The mice were sacrificed, their organs (heart, liver, spleen lung, and kidney) were removed, sectioned, and subjected to H&E staining.
GraphPad Prism 5 software (GraphPad Inc., La Jolla, CA, USA) was employed to carry out the statistical analysis. The mean ± SEM or the mean ± SD were utilized to present the numerical data. One-way ANOVA and Tukey’s test together were utilized for the statistical analyses and statistical significance was indicated by a P-value < 0.05.