We collected six fresh NSCLC tissues along with paired adjacent noncancerous lung tissues. We obtained consent from patients in Renji Hospital at Shanghai Jiaotong University. We evaluated pathological and histological diagnostics of NSCLC based upon Revised International System for Staging Lung Cancer. Our lab snap-froze samples in liquid nitrogen and maintained them at -80°C before RNA extraction. Ethics Committee of Shanghai University of Medicine and Health Sciences supervised this study.
Strand-specific RNA-Seq library preparation and next-generation RNA-Seq
Total RNA from 3 paired NSCLC tissues along with adjacent noncancerous lung tissue was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Our team prepared ~3 μg total RNA from each sample using VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co. Ltd., Nanjing, China) to erase ribosomal RNA retaining other classes of RNA such as ncRNA and mRNA. Our lab dealt RNA with RNase R (Epicenter, 40 U, 37°C for 3 h), followed by TRIzol purification. Technician made RNA-seq libraries through KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) and subjected them to deep sequencing leveraging the Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China.
We purchased BALB/c nude mice with four weeks old weighing 15~20 g from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Ethics Committee in Shanghai University of Medicine and Health Sciences of Shanghai supervised the procedures and our team conducted the experiments following their guidelines. Our lab performed all surgical procedures under anesthesia and tried to eliminate suffering. Our team anesthetized the mice with an intraperitoneal injection of 30 mg/kg sodium pentobarbital.
Our lab obtained NSCLC human pulmonary epithelial cell lines H1299, PC9, H1650, A549 and H1975 and control non-tumorigenic bronchial epithelium BEAS-2B cell line from American Type Culture Collection (Manassas, VA, USA). We cultured cell lines in Dulbecco’s Modified Eagle’s medium (Gibco, Grant Island, NY, USA) which was supplied with 10% fetal bovine serum in humidified atmosphere with 5% CO2 at 37°C.
RNA interference and overexpression
miR-532-5p inhibitors and siRNA against circ-PSMB6 (si-circ-PSMB6) were obtained from GenePharma (Shanghai, China). We transferred cells to 6-well culture plates and transfected them via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the protocol. To induce Enhancer of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) overexpression, we transfected cells with a pCDNA3.0 vector. For xenograft experiments, a lentiviral-mediated circ-PSMB6-silencing vector was transfected into PC9 cells (sh-circ-PSMB6).
Total RNA isolation and RT-qPCR
Our lab employed TRIzol reagent to obtain total RNA from tumor cells and tissues following manufacturer’s protocol. Our lab measured RNA sample concentration and purity at absorbances of 230, 260, and 280 nm with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). We regarded OD260/280 and OD260/230 ratios in 1.8~2.1 and >1.8 as acceptable.
We used SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) to reverse-transcribe RNA (1 μg) into cDNA. RT-qPCR was performed with an ABI 7300 Real-Time System (Applied Biosystems, Foster City, CA, USA) using primer pairs synthesized by GenePharma specific for glutathione synthetase (GSS), glutathione peroxidase 4 (GPX4) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and TaqMan Universal PCR Master Mix (Thermo Fisher Scientific). Technician quantified gene expression by 2−ΔΔCt method. Primers applied to assay circ-PSMB6 expression were forward, 5ʹ-GAGATGCCCTGTACTGATGC-3ʹ and reverse, 5ʹ-CTTTCTCCTCCGGTACTGC-3ʹ. The miR-532-5p primers were forward, 5ʹ-CGGCCATGCCTTGAGTGTA-3ʹ and reverse, 5ʹ-GCAGGGTCCGAGGTATTC-3ʹ. The EZH1 primers were forward, 5ʹ-ATGCGACTTCGACAACTTAAACG-3ʹ and reverse, 5ʹ-GGCTTCATTGACTGAACAGGTT-3ʹ. U6 primers were forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ and reverse, 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ. GAPDH primers were forward, 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ and reverse, 5ʹ-GGATCTCGCTCCTGGAAGATG-3ʹ.
Fluorescence in situ hybridization (FISH)
A probe specific to circ-PSMB6 (Dig-5′-GTTTGTCACAGAGAAACAGTTGCTTTGAG-3′-Dig) was made by Geneseed Biotech (Guangzhou, China). Our lab detected signals via Cy3-conjugated anti-digoxin and anti-biotin antibodies (Jackson ImmunoResearch Inc., West Grove, USA). Our team counterstained nuclei with 4,6-diamidino-2-phenylindole (DAPI) and captured figures with Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany).
We predicted the correlations between circRNA, miRNA and mRNA using the websites at http://starbase.sysu.edu.cn/, http://www.circbank.cn/ and https://circinteractome.nia.nih.gov/.
Transwell migration assay
Our lab assessed cell migration through Costar® Transwell® cell culture inserts (Corning Inc., Corning, NY, USA) following instructions. After incubating the cells for 1 d, our lab erased cells on Transwell chamber upper surfaces via cotton swabs. Technician fixed cells on lower surfaces in methanol for 10 minutes and stained them with Crystal Violet. Our team counted and photographed cells in 5 fields that randomly selected.
Plate colony formation assay
PC9 and A549 cells after different treatments were resuspended and counted, and we seeded 200 cells/well in plates with six wells. Our lab incubated cells for 2 weeks, with the medium changed every 3 days. We took photographs with a fluorescence microscope before ending the experiment, then washed the cells twice with PBS. The cells were stained in 500 µL Giemsa dye for 10–20 min, washed three times with ddH2O and photographed with a digital camera.
We determined proliferation rates 1, 2, and 3 days after transfection using Cell Counting Kit-8 (CCK-8) assays following instructions. Technician incubated cells in 10% CCK-8 solution diluted in normal culture medium at 37°C until visual color conversion occurred. We read every well absorbance using microplate reader at 570 nm.
Dual-luciferase reporter assay
Putative miR-532-5p binding sites in target gene EZH1 and in circ-PSMB6 were cloned into the psi-CHECK vector (Promega, Madison, Wisconsin, USA). Wild type (Wt) or mutated (Mut) 3'-UTR sequences from EZH1 or Wt and Mut miR-532-5p binding sites from circ-PSMB6 were inserted downstream from firefly luciferase as the primary luciferase signal and were designated EZH1-Wt/circ-PSMB6-Wt and EZH1-Mut/circ-PSMB6-Mut. psi-CHECK vector itself supplied a Renilla luciferase signal as a normalization factor to compensate for the differences between transfection and harvested efficiencies. Our team then transfected 293T cells with these constructs using Lipofectamine 2000 and detected Renilla and firefly luciferase activities 1 d after transfection with Dual-Luciferase Reporter Assay System (Promega) utilizing luminometer (Molecular Devices, U.S.A.). We processed relative Renilla luciferase activity following the manufacturer instructions.
Tumor sphere formation assays
PC9 and A549 cells were harvested and resuspended as single cells in serum-free medium. Following cell counting, our lab seeded 200 cells/well in 200 μL serum-free medium in plates with 96 wells, 10 wells/group, altering medium every 2 d. Our lab obtained five images from randomly selected regions in each well group using camera-equipped microplate reader (Leica, Wetzlar, Germany) and computed sphere percent as number of spheres/200.
Cell cycle detection
Our team dissociated cells in logarithmic growth phase with 0.25% trypsin, resuspended them in PBS and fixed them in ice-cold 70% ethyl alcohol overnight at 4°C. Our team centrifuged cells at 1000 rpm for five minutes, resuspended them in 50 μL RNase A and incubated them at 37°C for half of an hour. We added 400 μL propidium iodide (PI) to suspension for 0.5 h, followed by flow cytometry (BD Bioscience, CA, USA).
To detect the circ-PSMB6 role in lung cancer metastasis model, we intravenously injected 1×106 stable lentiviral-mediated circ-PSMB6-silenced PC9 cells (sh-circ-PSMB6) or negative control (NC) PC9 cells into male nude mice (Chinese Science Academy, Shanghai, China) via tail vein. After one month, our team analyzed PC9 cell metastasis through bioluminescence imaging following intravenous luciferin injection (150 mg luciferin/kg body weight) into tails.
For xenograft assays, our team subcutaneously injected 1×106 modified or control PC9 cells (sh-circ-PSMB6 or Wt,) into the male nude mice right flank. Tumor volumes were calculated as 0.5 × length × width2 at time points indicated, and technician excised all tumors 4 w after injection. All animals were treated according to instructions from Animal Care Committee in Shanghai University of Medicine and Health Sciences.
Our team fixed tumor tissue samples in 10% formalin solution, embedded them in paraffin and sliced them into 5-μm-thick sections, which we stained with antibodies against Ki67 to validate cell proliferation, using AxioPhot light microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with digital camera
Statistician compared differences between any 2 groups by unpaired or paired 2-tailed t-test. Bioinformatician leveraged Pearson’s correlation coefficient to characterize correlations between groups. Bioinformatician denoted data as means ± standard deviation. Our team considered probability (p) values < 0.05 statistically significant. We employed GraphPad Prism (GraphPad Software, La Jolla, CA, USA) for statistical analyses.