Cell line and culture
Mouse mononuclear macrophage cell line RAW264.7 was purchased from the American Type Culture Collection (ATCC, VA, USA). The cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 and maintained in RPMI-1640 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, UT, USA), 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen, CA, USA).
circRNA library construction and sequencing
RAW264.7 cells were prepared as described previously [6]. RT (20 ng/mL) was used to treat cells for 8 h. Total RNA was extracted using TRIzol reagent (Takara, Tokyo, Japan) following the manufacturer’s instructions. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 10 ug of total RNA representing a specific adipose type was used to deplete ribosomal RNA according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA). Following purification, the poly(A)- or poly(A)+ RNA fractions is fragmented into small pieces using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the RNA-Seq sample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina Hiseq4000 at the (LC Sciences, USA) following the vendor’s recommended protocol. CircRNA expressions from different samples or groups were calculated by scripts in house. Only the comparisons with P < 0.05 were regarded as showing differential expression.
RNA extraction, reverse transcription, and qRT-PCR analysis
Total RNA was extracted from cultured cells and lung tissue using TRIzol reagent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. gDNA was extracted using Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). Reverse transcription was performed using Oligo (dT) primer for mRNA into cDNA with M-MLV Reverse Transcriptase, RNase H- (Takara, Tokyo, Japan). A Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) was used to verify the existence of circRNAs. The qRT-PCR was conducted using Applied Biosystems QuantStudio 3 Real-Time PCR Systems (Thermo Fisher Scientific, MA, USA) with SYBR Green PCR Master Mix (Takara, Tokyo, Japan). The expression levels of miRNA were determined using an All-in-One miRNA qPCR Kit (GeneCopoeia, MD, USA). β-actin was measured as an endogenous control for mRNA and circRNA, and U6 was used as a control for miRNA. The relative fold-change in expression with respect to a control sample was calculated by the 2-ΔΔCt method. Relative primers are shown in Table S1.
RNase R treatment
2 μg of total RNA was incubated with or without 5 U/μg RNase R at 37°C for 30 min in the buffer provided with the kit (Epicentre Technologies, Wisconsin, USA), and subsequently purified by RNeasy MinElute Cleaning Kit (Qiagen), then analyzed via RT-PCR.
Nucleocytoplasmic separation and RNA isolation
PARIS Kit (Invitrogen, CA, USA) was used to separately isolate nuclear and cytoplasmic RNA from cultured cells, according to the manufacturer’s protocol.
Actinomycin D assay
RAW264.7 cells were exposed to 2 μg/ml actinomycin D (Sigma Aldrich, St. Louis, MO, USA) at indicated time point. Then the cells were harvested, and total RNA was extracted. The stability of circEpc1 and Epc1 mRNA was analyzed using qRT-PCR.
Lentivirus vector constructs and cell transfection
For in vitro studies, the lentivirus vectors were constructed using the pHBLV-CMV-Circ-MCS-EF1-ZsGreen-T2A-PURO vector purchased from Hanbio Biotechnology (Shanghai, China), and further confirmation was obtained by Sanger Sequencing. The circEpc1 overexpressing lentivirus vector, two shRNAs (sh-circEpc1#1 and sh-circEpc1#2) targeting circEpc1, and miR-5114 mimics were also designed and synthesized by HanBio. Cell transfection was performed as we described previously [6]. Then, 5 μg/mL of Puromycin (MedChemExpress, NJ, USA) was used to select the stably infected cells. The transfection efficiency was determined by qRT-PCR.
Dual-Luciferase reporter assay
Luciferase assays were performed using the DLR Assay System (Promega, WI, USA) according to the manufacturer’s instructions. Briefly, the HEK293T cells were co-transfected by Lipofectamine 3000 (Invitrogen, CA, USA) with miR-5114 mimics or nonsense control (NC) mimics (Sequence: 5′-AGAACGUCGAAGGCAGAGGUCA-3′) and luciferase reporter constructs containing the wild-type or mutant 3′-UTR of circEpc1. The mimics and luciferase reporter constructs were purchased from Hanbio (Shanghai, China). The cells were lysed 24 h after transfection, then Renilla luciferase (RLuc) and Firefly luciferase (Fluc) activities were measured on a Synergy 2 luminometer (BioTek, USA). Rluc signals were normalized to the intraplasmid Fluc transfection control.
Fluorescence in situ hybridization (FISH)
For FISH, the RT-treated RAW264.7 cells were incubated using specific probes of circEpc1 and miR-5114 according to user manual of RNA FISH Kit (GenePharma, Shanghai, China). The cells and fluorescence-labeled probes were hybridized in a hybridization buffer and hybridized overnight at 37°C. The next day, after stringent washing with SSC buffer, the nucleus were counterstained with DAPI. Images were acquired using a LSM-780 confocal laser scanning microscope (Carl Zeiss, Germany) and digitized with a software program Zen Light Edition.
Animal experiments
Male BALB/c mice aged 6-8 weeks, weighting 20-25 g, were purchased from the Liaoning Changsheng biotechnology co., Ltd (Benxi, China). To explore the regulatory function of circEpc1 in vivo, the adeno-associated virus (AAV) circEpc1 overexpressing, shRNA targeting circEpc1, and negative control (NC) were constructed and packaged by HanBio. The mice were randomly divided into three groups (NC, circEpc1-overexpression, and sh-circEpc1) with eight mice in each group. One week after the mice have acclimated to the environment, a total of 50 µL solution containing above virus or RT was slowly injected into trachea by means of liquid aerosol lung delivery. All mice were sacrificed three weeks after the injection, and the lungs were cut out for histopathological analysis.
Ex vivo fluorescence imaging
In order to detect the transfection effect of AAVs in vivo, we carried out Ex vivo tissue fluorescence imaging experiment. Then, under anesthesia, three weeks after AAVs transfection, the imaging system was used for fluorescence imaging of lung tissue in mice. Maestro software was used to remove the mouse background fluorescence.
Histopathological analysis
Histopathological analysis was performed according to the manufacturer’s protocol. After injection, the mouse lungs were cut out and immersed in 4% paraformaldehyde overnight, embedded in paraffin and then sections 4 μm thick were cut. The sections were dehydrated in xylene and ethanol successively. Hematoxylin-eosin (H&E) staining was performed to evaluate the morphological variation in lung and inflammatory cell infiltration. Sections were microscopically examined using Nikon (Eclipse 80i; Tokyo, Japan), and the images were acquired on the imaging system (digital sight DS-FI2, Nikon, Japan).
Western blot analysis
Protein of RT-treated RAW264.7 cells and lung tissue were homogenized in RIPA lysis and extraction buffer (Thermo Fisher Scientific, MA, USA) supplemented with PMSF. A BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to quantify the concentration of protein samples in the cell lysates. Then, the proteins were separated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Following blocking the PVDF membranes with 5% nonfat milk (BD Biosciences, Franklin Lakes, NJ, USA) in 0.1% Tween-20 TBST buffer at room temperature for 1 h, the membranes incubated with antibodies included NOD2 (1:2000, proteintech, 66710), RIP2 (1:1000, Abcam, ab8428), p65 (1:2000, proteintech, 66535), IκB-α (1:4000, Abcam, ab32518), P-IκB-α (1:1000, CST, 28595), p38 (1:2500, BD Biosciences, 612280), p-p38 (1:5000, BD Biosciences, 612168), ERK (1:4000, BD Biosciences, 610030), p-ERK (1:1000, BD Biosciences, 612358), JNK (1:250, BD Biosciences, 612540), p-JNK (1:250, BD Biosciences, 610627) overnight at 4°C. β-actin (1:10000, proteintech, 66009) was used as the internal protein loading control. After being washed 5 times in TBST for 50 min, membranes were incubated with HRP-labelled secondary antibody at room temperature for 1 h. Finally, immunodetection was performed using an enhanced chemiluminescence (ECL) detection system with Chemiluminescence HRP Substrate (GE Healthcare, Buckinghamshire, UK).
Cytokine assays
After RT treated, mice lungs were irrigated three times with 1.0 mL PBS. The collected bronchoalveolar lavage fluid (BALF) was immediately centrifuged to separate the cells and supernatant. Measurement of cytokine in the supernatants of BALF and RT-treated RAW264.7 cells was carried out using Mouse TNF-α ELISA kit (Dakewe, Shenzhen, China) and Mouse IL-6 ELISA kit (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions.
Statistics
We performed our experiments in triplicate, and the results are presented as mean ± standard deviation of the mean. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA), and consisted of analysis of variance followed by Student’s t-test when comparing two experimental groups. P < 0.05 was considered statistically significant, P < 0.01 and P < 0.001 were considered indicative of highly significant difference.