CircRNAs that were differently expressed in blood samples from AMI patients (n=3) and healthy subjects (n=3) were obtained from GEO database (accession: GSE160717; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE160717).
Human cardiac myocytes (AC16) were bought from BeNa cell bank (Beijing, China) and cultured in DMEM containing 10% FBS.
Myocardial injury model was established by exposing AC16 cells to OGD/R. In brief, AC16 cells were cultured in glucose-depleted DMEM and placed at an anaerobic and humidified chamber (Brinkmann Instruments, Westbury, NY, USA) containing a gas mixture (94% N2, 1% O2 and 5% CO2), at 37℃. At 4 h post-incubation, AC16 cells were incubated with normal high glucose DMEM containing 10% FBS in a 37℃ incubator containing 5% CO2 and 95% air. Cells were allowed to recover reoxygenation for 12 h.
Divergent primers and convergent primers of circCARM1 were provided by Geneseed (Guangzhou, China). These primer pairs were used to amplify cDNA and gDNA in AC16 cells.
Actinomycin D (CST, Danvers, MA, USA) was dissolved in DMSO (50 mg/mL) and added into the culture medium of AC16 cells. Cells were then maintained for 0, 6, 12 or 24 h and collected for RNA isolation. The expression of circCARM1 and GAPDH was examined by RT-qPCR.
RNase R (BioVision, Milpitas, CA, USA) was utilized to treat total RNA (3 U/μg) for 30 minutes at 37℃. Total RNA was synthesized into cDNA to examine the expression of circCARM1 and GAPDH using RT-qPCR.
To define the distribution of circCARM1, cytoplasm RNA and nucleus RNA were separately isolated from AC16 cells using the PARIS kit (Thermo Fisher, Waltham, MA, USA). Then, RNA in each fraction was subjected to RT-qPCR, with U6 as an internal reference in nucleus, GAPDH as an internal reference in cytoplasm.
CircCARM1-related oligos, such as its special small interference RNA (si-circCARM1) and negative control (si-NC), were synthesized by Geneseed (Guangzhou, China). MiR-338-3p-related oligos, including mimic (miR-338-3p), inhibitor (anti-miR-338-3p) and their negative controls (miR-NC and anti-NC), were purchased from Ribobio (Guangzhou, China). TRAF3 overexpression vector and pcDNA blank vector were constructed by Tiandz (Beijing, China). Cells were transfected with oligos or vector with the application of Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA).
Real-time quantitative PCR (RT-qPCR)
Firstly, total RNA was isolated from cells using a Trizol reagent (Cwbio, Beijing, China). Then, total RNA was quantified and used for reverse transcription with the use of SuperRT cDNA Synthesis Kit (Cwbio). Noticeably, miRNA cDNA synthesis was performed using miRNA cDNA Synthesis Kit (Cwbio). Afterwards, equal amount of cDNA was diluted and used for RT-qPCR using the UltraSYBR Mixture (Low ROX; Cwbio). GAPDH or U6 acted as an internal reference. We obtained the Ct values and then processed them using the 2-ΔΔCt method. Primer sequences were shown in Table 1. Three replicates were set for one sample in each test, and a total of three independent biological experiments were conducted.
Cells with treatments were seeded into a 96-well plate (2,000 cells/well) and next cultured in cell incubator for 48 h. Cells in each well were treated with 10 μL CCK-8 reagent (Elabscience, Wuhan, China) for another 2 h. Relative cell viability was assessed according to the absorbance at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Three replicates were set for one sample in each test, and a total of three independent biological experiments were conducted.
Cells with treatments were seeded into a 96-well plate (2,000 cells/well). After culturing for 24 h, EdU from a Cell-Light EdU Apollo567 in Vitro Kit (Ribobio) was added to culture cell for another 24 h. Then, cells were stained with Apollo and DAPI. The signal of EdU-positive cells was observed under a light microscope (Nikon, Tokyo, Japan). Three replicates were set for one sample in each test, and a total of three independent biological experiments were conducted.
Flow cytometry assay
With the use of FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA), cell apoptotic rate was assessed by a flow cytometer (BD Biosciences). In brief, cells (1×105) at 48 h post-transfection were collected in FITC-Annexin V binding buffer, and then cells were stained with FITC-Annexin V and propidium iodide. Finally, cells were examined by flow cytometry. Three replicates were set for one sample in each test, and a total of three independent biological experiments were conducted.
Malondialdehyde (MDA) and superoxide dismutase (SOD) assay
Cell supernatant was collected at 48 h post-transfection to examine MDA content and SOD activity, using MDA Assay Kit (Solarbio, Beijing, China) and SOD Activity Assay Kit (Solarbio). Three replicates were set for one sample in each test, and a total of three independent biological experiments were conducted.
Western blot assay
Firstly, cells (1×106) were collected at 48 h post-transfection and lysed in RIPA buffer (Solarbio). Protein extracts were quantified using a BCA kit (Solarbio). Then, proteins were separated by SDS-PAGE and loaded onto PVDF membranes. Next, protein-loaded membranes were blocked by non-fat milk and incubated with the primary antibodies (Abcam, Cambridge, MA, USA) at 4℃ overnight, such as NADPH oxidase 2 (NOX2; ab129068), SOD2 (ab68155), TRAF3 (ab239357), GAPDH (ab9485). Subsequently, membranes were probed with HRP-coupled secondary antibody for 1.5 h. Finally, we used the ECL reagent (Solarbio) to visualize the protein signals.
MiRNAs targeted by circCARM1 were predicted using circinteractome (https://circinteractome.nia.nih.gov/) and starbase (http://starbase.sysu.edu.cn/) tools. mRNAs targeted by miR-338-3p were predicted using Targetscan tool (http://www.targetscan.org/vert_72/).
Biotin-labeled RNA probe of circCARM1 (circCARM1 probe; Geneseed) could effectively enrich the abundance of circCARM1, with oligo probe as a control. Probes were cocultured with magnetic beads (Invitrogen) to generate probe-beads complex. Cells were lysed, and cell lysates were incubated with probe-beads complex. RNA compounds enriched in the beads were eluted and analyzed by RT-qPCR.
Dual-luciferase reporter assay
To verify the binding site between miR-338-3p and circCARM1 or TRAF3 3’UTR, circCARM1 and TRAF3 wild-type sequence containing miR-338-3p binding site, and circCARM1 and TRAF3 mutant-type sequence containing miR-338-3p mutated binding site were synthesized and cloned into pmirGLO vector (Promega, Madison, WI, USA). The recombinant plasmids, including WT-circCARM1, MUT-circCARM1, WT-TRAF3 3’UTR and MUT-TRAF3 3’UTR were respectively transfected with miR-338-3p or miR-NC into AC16 cells. After culturing for 48 h, luciferase activity was evaluated using a Dual-Luciferase Reporter Assay System (Promega). Three replicates were set for one sample in each test, and a total of three independent biological experiments were conducted.
Data were processed by Graphpad Prism 6.0 (GraphPad, La Jolla, CA, USA) and shown as the mean ± standard deviation. In terms of difference comparison in different groups, Student’s t-test was suitable for two groups, and analysis of variance was suitable for multiple groups. P-value less than 0.05 was defined to be statistically significant.