Cell lines and cell culture
The HEK-293, HEK-293T, and T24 cell lines used in this study were purchased from American Type Culture Collection (ATCC). Normal human primary fibroblasts derived from the epidermis were kindly provided by Dr. T. Chen (Shantou University, Shantou, China). All cell lines were grown in DMEM medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in the presence of 5% CO2. Cell cultures were also confirmed to be free of Mycoplasma contamination by using 16S rRNA-based Mycoplasma group-specific polymerase chain reaction (PCR).
Construction of plasmids
The backbone plasmid expressing human codon-optimized CasRx protein was purchased from Addgene (# 109049). The shRNA-expressing plasmids were purchased from Syngentech Co., Ltd. (Beijing, China). crRNA sequences and crRNA-ribozymes that exactly match the corresponding shRNA sequences were designed. The cDNA sequences for crRNA, ribozyme, crRNA-ribozyme, and shRNA were then synthesized and inserted into corresponding backbone plasmids digested with restriction endonucleases. All vectors were transformed into One Shot TOP10 Chemically Competent E. coli cells. The desired expression clones were identified using PCR amplification and electrophoresis and then confirmed with Sanger sequencing. The sequence information of the related elements is shown in Additional file 2: Table S1.
DNA transfection into cells
HEK-293, HEK-293T, and T24 cells were cultured in plates until they reached 70% confluence. Cells were transiently transfected with the constructed plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.
Dual-luciferase reporter assay
Both the Renilla luciferase and firefly luciferase activities were measured in a 1.5-ml Eppendorf tube using the Promega Dual-Luciferase Reporter Assay kit (Promega E1980) according to the manufacturer’s protocol 48 h after DNA transfection. The relative luciferase activity was calculated as the Renilla luciferase value normalized to that of the firefly luciferase value. The assays were performed in triplicate and experiments were repeated five times.
RNA extraction and real-time quantitative PCR
Total RNAs were isolated from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The concentration and purity of total RNA were measured using UV spectrophotometric analysis at 260 nm. cDNAs were synthesized using the Superscript III First Strand Synthesis System (Invitrogen). Real-time quantitative reverse transcription PCR (qRT-PCR) was performed using a SYBR Green PCR Master Mix (Invitrogen). GAPDH was chosen as the endogenous control. PCR mixtures were prepared according to the manufacturer’s protocol and amplification was performed under the following PCR parameters on an ABI PRISM 7300 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA): 40 cycles of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 70 °C. All primer sequences are shown in Additional file 3: Table S2. Expression fold changes were calculated using the 2−△△ct method.
RNA-denaturing gel electrophoresis
Total RNAs were obtained using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. One gram of agarose was heated in 72 ml water until dissolved and then cooled to 60 °C. Ten milliliters of 10X MOPS running buffer and 18 ml 37% formaldehyde (12.3 M) were then added to prepare the gel. The gel was prerun for 10 min at 80 V, and 5 μg of total RNA was then loaded and separated until the bromophenol blue migrated 3 cm into the gel. The gel was visualized on a UV transilluminator.
Analysis of the specificity of CCRS
Total RNA was isolated, mixed with oligo (dT)25 Dynabeads, and mRNA was purified according to the manufacturers’ instructions (Invitrogen). The purified mRNA was first fragmented by using divalent cations before being subjected to library preparation. Adapters were ligated to fragmented RNAs by using truncated T4 RNA ligase 2 (NEB) according to the manufacturer’s instructions, and RNA was reverse transcribed to DNA by using SuperScript® III (Thermo Scientific, Scotts Valley, CA, USA) and circularized by using Circligase™ (EpiBio/Illumina Madison, WI, USA). Barcodes were added by PCR using Phusion® polymerase (Thermo Scientific). DNA libraries were sequenced on an Illumina HiSeq 2500 (Illumina). Reads were processed by Cufflinks v2.1.1 and fold changes were then calculated according to fragments per kilobase of transcript per million mapped reads (FPKM) values.
AAV packaging, purification and titer detection
The pAAV packaging plasmid, pHelper plasmid, and pAAV plasmid were co-transfected into HEK-293T cells using Lipofectamine 3000. The culture supernatants were collected at 48 hours after plasmid transfection, concentrated, and used as virus stocks for subsequent AAV infection experiments. The AAV titer was calculated by qPCR using 2× EvaGreen Master Mix (Syngentech).
Cell proliferation assay
Cell proliferation was assayed using the Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) according to the manufacturer’s instructions. At various time points (0, 24, 48, or 72 h) post-transduction, 10 μl of CCK-8 reagent was added to each well of the 96-well plates, and the cells were incubated for an hour. Absorbance was read at a wavelength of 450 nm using a microplate reader (Bio-Rad, Hercules, CA). The assays were performed in triplicate and experiments were repeated five times.
Cell apoptosis assay
According to the manufacturer’s protocol, cell apoptosis was determined by the caspase-3/ELISA (enzyme-linked immunosorbent assay) assay (Cusabio, China). Caspase-3 is a marker for inflammation and apoptosis signaling. The experiment for each sample was repeated five times independently.
Cell migration assay
Cell migration was determined by the wound healing assay. Briefly, cells were seeded into 12-well plates at equal density and cultured to 80% confluency. Artificial gaps were generated using a sterile pipette tip. Areas of wound were marked and photographed using a digital camera system. Cell migration distance (mm) was calculated using the software program HMIAS-2000. Each experiment was repeated five times.
Tumor xenografts
The mice were housed under standard laboratory conditions. BALB/c-Nude mice were randomly assigned into either the experimental group or control groups (five mice for each group). Bladder cancer T24 cells (5 × 107) were hypodermically injected on the backs of BALB/c-Nude mice. Next, intratumoral injection of AAV (100 μl, 2 × 1011 vp/ml) was performed. Tumor volumes were calculated using the formula: V = L × W2/2, where L is the length and W is the width of the tumor. The mice were sacrificed and tumors were removed at the end of the experiment.
For in vivo imaging experiments, BALB/c-Nude mice were randomly assigned into either the experimental group or control groups (three mice for each group). Bladder cancer T24 cells (5 × 107) were hypodermically injected on the backs of BALB/c -Nude mice, and then a single injection of AAV (100 μL, 2 × 1011 vp/mL) via tail vein was conducted 10 days after inoculation. Four weeks later, mice were anaesthetized with isoflurane and injected with D-luciferin sodium salt (150 mg/kg) intraperitoneally. Subcutaneous tumor was then monitored by an in vivo imaging system, Xenogen IVIS (PerkinElmer, MA, USA).
Experimental metastasis mouse model
T24 bladder cancer cells stably expressing luciferase (1 × 105) were suspended in 200 μL PBS and injected into the lateral tail veins of 5-week-old male B-NDG mice (BIOCYTOGEN, Beijing, China). Four weeks later, mice were anaesthetized with isoflurane, and D-luciferin sodium salt (150 mg/kg) was injected intraperitoneally. Bladder cancer cells were detected with an in vivo imaging system, Xenogen IVIS (PerkinElmer, MA, USA). The total flux in photons per second was calculated for the lung region using Living Image 4.3.1 (PerkinElmer/Caliper).
H&E staining of lung tissues
Mouse lung tissues were fixed in 10% formalin and dehydrated in ethanol. Paraffin embedding, sectioning, and hematoxylin and eosin staining were performed according to the manufacturer’s procedures, and then slides were imaged on a Nikon Ci-L bright field microscope.
Statistical analyses
Data were expressed as the mean ± standard deviation (SD). Significance tests were performed using the SPSS version 20.0 software (SPSS, Chicago, IL, USA). Statistical significance was determined using Student’s t-test or ANOVA and assigned at p < 0.05.