NSCLC tissue specimens (n = 30) and matched nearby normal tissues (n = 30) were obtained from NSCLC patients after surgical resection at the Central Hospital of Enshi Tujia and Miao Autonomous Prefectrue. All patients signed their written informed consents. With approval from the Research Ethics Committee of the Central Hospital of Enshi Tujia and Miao Autonomous Prefectrue, the current study was conducted.
Cell culture and transfection
NSCLC cells (A549 and H1299) and normal lung epithelial cells (BEAS-2B) were obtained from COBIOER (Nanjing, China). DMEM (Invitrogen, Carlsbad, CA, USA) that contained FBS (Invitrogen) was used for culturing BEAS-2B and NSCLC cells in standard conditions (5% CO2, 37 ℃). For treatment of propofol, cells were treated with propofol (Sigma-Aldrich, St. Louis, MO, USA) in a concentration gradient.
The siRNA against circ_0001727 (si-circ_0001727), miR-516b-5p inhibitor, miR-516b-5p mimic, LRRC1 overexpression plasmid (pc-LRRC1), and corresponding controls (si-NC, inhibitor NC, miRNA NC, and pc-NC) were all synthesized by RiboBio (Guangzhou, China). The oligonucleotide and plasmid were introduced into NSCLC cells (A549 and H1299) by Lipofectamine 3000 Reagent (Invitrogen).
Cell viability assay
Cell Counting Kit-8 (CCK-8) assay was used for analyzing cell viability. Briefly, cell suspension (100 μL) was inoculated into 96-well plates. Following treatment, CCK-8 (10 μL, Bimake, Shanghai, China) was placed into per well for 2–3 h, followed by detection of the optical density (OD) using a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm.
RNA isolation and qRT-PCR
After extraction of RNA using TRIzol (Invitrogen), RNA was subjected to reverse transcription with miScript II RT kit (for miRNA; Invitrogen) or PrimeScriptTM RT reagent Kit (for mRNA/circRNA; TaKaRa, Otsu, Japan). Thereafter, qRT-PCR reaction was manipulated using SYBR green PremixEx Taq II (Takara) on Bio-Rad CFX96 system (Bio-Rad). The information of primers was listed: circ_0001727 (F, 5′-CCCAGTCCCACTTCAAACAT-3′; R, 5′-TCAGGCTCCAGGAACTGACT-3′); ZKSCAN1 (F, 5′-CTCGGAGGAATCTCAGTAGGGA-3′; R, 5′-CGTGATGCTGAATCTTCCGAGG-3′); miR-516b-5p (F, 5′-GCCGAGATCTGGAGGTAAGAA-3′; R, 5′-CAGTGCGTGTCGTGGAGT-3′); LRRC1 (F, 5′-TCCTTACCAAAAGAGATCGG-3′; R, 5′-GGTAGATGCAGCAACCTGT-3′); GAPDH (F, 5′-GTCTCCTCTGACTTCAACAGCG-3′; R, 5′-ACCACCCTGTTGCTGTAGCCAA-3′); U6 (F, 5′-CTCGCTTCGGCAGCACATATACT-3′; R, 5′-ACGCTTCACGAATTTGCGTGTC-3′). The 2−ΔΔCt method was employed to analyze RNA levels. U6 (for miR-516b-5p) and GAPDH (for circ_0001727, ZKSCAN1, and LRRC1) were employed as internal controls.
RNase R treatment
RNase R treatment was applied for degrading linear RNA. In brief, total RNA was exposed to RNase R (Duma, Shanghai, China) for half an hour at 37 ℃. After that, circ_0001727 and ZKSCAN1 expression were examined via qRT-PCR.
Colony formation assay
NSCLC cells (A549 and H1299) were seeded into the 6-well plate, and cell culture medium was changed every 3–4 days. After culturing for 2 weeks, these wells were washed and fixed by paraformaldehyde (Beyotime, Jiangsu, China). After staining using crystal violet (Beyotime), these colonies were counted and photographed.
Transwell assay was used to detect cell migration and invasion. NSCLC cells (A549 and H1299) were suspended in FBS-free medium and plated in the top chamber precoated with (for detecting cell invasion) or without (for detecting cell migration) Matrigel. Meanwhile, completed medium was placed into the bottom chamber. 24 h later, the migrated and invasive cells were fixed in 4% paraformaldehyde (Beyotime) and stained with 0.1% crystal violet solution (Beyotime). Lastly, these cells were photographed by a microscope (× 100, Olympus, Tokyo, Japan).
Tube formation assay
Tube formation assay was carried out to evaluate angiogenesis activity in vitro. When NSCLC cells (A549 and H1299) reached 80% confluence, the supernatant was collected as conditioned medium (CM). Next, human umbilical vein endothelial cells (HUVECs; COBIOER) were inoculated in a 24-well plate coated with Matrigel and incubated for 0.5 h, and then seeded into wells under different CM. After incubation for 6 h in 37 ℃, tube formation was then observed and photographed using a microscope (Olympus). The number of branches in each well was calculated by ImageJ software.
Flow cytometry analysis
Annexin V-FITC&PI apoptosis detection kit purchased form Vazyme was utilized to measure apoptotic cells. Shortly, cells were seeded in 6-well plates and harvested after treatment, followed by resuspending in1 × binding buffer. After staining with Annexin V-FITC and PI for 0.5 h, cells were subjected to a flow cytometer to examine apoptotic cells.
Western blot (WB) assay
After extraction of total protein with RIPA lysis buffer (Vazyme), the total protein was denatured via heating for 3–5 min at 100 ℃. After measurement of protein concentration, total protein was separated by SDS-PAGE. Afterwards, these gels were transferred onto PVDF membranes (Beyotime). After blocking in 5% non-fat milk, these membranes were then probed with the following primary antibodies: vascular endothelial growth factor A (VEGFA; ab51745, 1:1500, Abcam, Cambridge, UK), Bcl-2 (ab194583, 1:1500, Abcam), LRRC1 (HPA031603, 1:1500, Sigma-Aldrich), or GAPDH (ab9485, 1:3000, Abcam) at 4 ℃ for 12–16 h, followed by incubation with secondary antibody (ab205718, 1:5000, Abcam). At last, ECL reagent (Abcam) was used for visualization of protein blots.
Tumor formation assay in vivo
For in vivo assay, we purchased BALB/c nude mice (male, n = 20) from Vital River (Beijing, China) to establish xenograft model and segmented into 2 groups. Short hairpin RNA targeting circ_0001727 (sh-circ_0001727) and sh-NC (as control) were provided by RiboBio. Briefly, mice were subcutaneously inoculated with un-transfected cells (A549) or transfected-cells (sh-circ_0001727 or sh-NC). After injection 7 days, these mice were randomly divided into 4 groups: Control (untreated), propofol, propofol + sh-NC, and propofol + sh-circ_0001727 (n = 5/group). In the propofol groups, propofol (45 mg/kg) was injected intraperitoneally twice a week. An external caliper was used to detect tumor volume. We calculated the volume of tumors according to the equation: 1/2 × length × width2. These mice were sacrificed 4 weeks later, and excised tissues were weighed, followed by collection of the tumor tissues. This study got approval from the Animal Care and Use Committee of the Central Hospital of Enshi Tujia and Miao Autonomous Prefectrue.
Tumor tissues were fixed with paraformaldehyde (4%), embedded in paraffin, and cut at a thickness of 4 μm slides. Next, these sections were incubated with the Ki67 (ab15580, 1:500, Abcam) antibody to measure Ki67 expression. After incubation with secondary antibody (ab171870, 1:2000, Abcam), these sections were then stained using diaminoaniline (DAB; Maxim, Fuzhou, China) and then counterstained using haematoxylin (Maxim). The image was captured using a microscope (Olympus).
Dual-luciferase reporter assay
Circular RNA interactome or starBase was employed to predict the association between miR-516b-5p and circ_0001727 or LRRC1. The fragments of circ_000172 or LRRC1 containing the putative bindings sites for miR-516b-5p were synthesized and cloned into the pmirGLO vectors (GenePharma, Shanghai, China), thereby constructing WT-circ_0001727 or WT-LRRC1-3′UTR. Meanwhile, binding sites for miR-516b-5p were mutated and cloned into the same vector to create MUT-circ_0001727 or MUT-LRRC1-3′UTR. NSCLC cells (A549 and H1299) were co-introduced with the recombinant plasmid and miRNA mimic or miR-516b-5p. 48 h later, the luciferase activities were measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA).
RNA immunoprecipitation (RIP) assay
RIP assay was conducted with EZ-Magna RIP Kit (Millipore, Billerica, MA, USA). After lysing using RIP buffer, cell lysate (0.1 mL) was incubated with magnetic beads previously binding to human anti-IgG (as the control) or anti-Ago2. The magnetic beads were digested using proteinase K for removing the protein. Lastly, the purified RNA used for measuring the expression of relevant RNAs via qRT-PCR.
Data from at least 3 independent experiments displayed as mean ± standard deviation and were analyzed by GraphPad Prism software (Version 7.0). Statistical significance was analyzed via Student’s t test or a one-way analysis of variance. P value < 0.05 was determined to be statistically different.