All liver tissue samples in this study were collected from the Second Affiliated Hospital of Naval Medical University, Shanghai, China. Proper amounts of samples were collected for formalin fixation, paraffin embedding and HE staining. All the samples were identified by two professional pathologists (supplementary Fig. 1). The staging of liver fibrosis was determined based on the Metavir semi-quantitative evaluation system. 6 human liver fibrosis tissues were used for transcriptome sequencing (RNA-seq). In addition, 22 hepatic fibrosis tissues were used for further detection of the expression of hsa_circ_0008494. All tissue samples were stored in liquid nitrogen until use. The research was approved by the Research Ethics Committee of Second Affiliated Hospital of Naval Medical University, Shanghai, China. Informed written consent was obtained from each sampled patient.
The human HSC cell line LX-2 was cultured in Dulbecco’s modified Eagle’s medium, supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin (Beyotime, Shanghai, China) and 10% fetal bovine serum (Gibco, New York, NY, USA) at 37 ºC in an atmosphere of 5% CO2. Recombinant human TGF-β1 (PeproTech, NJ, USA) was used to activate LX-2 cells.
Male Balb/c mice（6~8 weeks old) were purchased from the Chinese Academy of Sciences (Shanghai, China). The mice were fed in SPF facilities and the methods were approved by the Research Ethics Committee of Second Affiliated Hospital of Naval Medical University, Shanghai, China. For the dimethyl nitrosamine (DMN)-induced hepatic fibrosis model, 20 mice (body weight of 20 ± 3g) received 0.08％DMN on every Monday, Tuesday and Wednesday morning for three consecutive weeks. The control group was given saline injections. The DMN-induced hepatic fibrosis animal model was identified and evaluated by a senior pathologist.
Fluorescence in situ hybridization
The expression level of hsa_circ_0008494 in liver tissues was evaluated by fluorescence in situ hybridization (FISH) assay using a specific Cy3-labeled-circ_0008494 probe (circ103134, Ruibo Bio, Guangzhou, China) and Fluorescent in Situ Hybridization Kit (C10910，Ruibo Bio, Guangzhou, China). After prehybridization, tissue sections were hybridized in hybridization buffer with Cy3-labeled-circ_0008494 probe overnight at 37ºC according to the manufacturer’s procedures. After cleansing with hybrid wash buffer I, II, III and counterstaining by 4′,6-Diamidino-2-phenylindole (DAPI), the images were acquired under the fluorescent microscope (Zeiss, Thornwood, USA).
Six liver fibrosis tissues were used for RNA sequencing (RNA-seq) detection. The clinical information of patients is presented in Table1. All RNA samples for RNA-seq were of high-quality with a 28S/18S ratio ≥ 1.0 and RIN ≥ 7.0 according to the RNA quality assessment. Sequencing and data analysis were performed using the Illumina HiSeq system (Illumina, San Diego, CA, USA). The experimental process was carried out according to the standard procedures provided by Illumina, including the preparation of library and sequencing experiment. After the quality inspection, ribosomal RNA (rRNA) was removed through epicenter ribo zeroTMkit produced by the Illumina company. The remaining RNA was purified, recovered and randomly broken into small pieces by fragmentation buffer for RNA library construction. Illumina hiseq4000 was used for RNA sequencing. The reading length is 2 × 150 bp (PF150).
Mice tissues were fixed in 10% formalin and embedded in paraffin wax before 4 μL serial sections were cut. The sections were then deparaffinized and rehydrated routinely. The slides were incubated with the following antibodies: anti-α-SMA, anti- Col1a1(CST, MA, USA) for 30 minutes at room temperature, and maintained overnight at 4°C. Afterwards, the slides were visualized using diaminobenzidine and hematoxylin on the second day.
Actinomycin D and Ranse R treatment assay
Transcription blocking assay was performed through the addition of 2 mg/mL actinomycin D (Med Chem Express, NJ, USA) for 4 h, 8 h, 12 h, and 24 h. To detect the expression level of circ_0008494 and its line isoform ARID1A mRNA, the same amount of RNA was utilized for reverse transcription and quantitative real-time PCR analysis. The RNase R treatment assay was used to verify the stability of circ_0008494. In short, 1 μg of total RNA was incubated with RNase R or DEPC-treated water in 10×reaction system (Epicenter Technologies, Madison, WI, USA) for 30 minutes at room temperature. For the RNase R treatment group, 0.15 μL RNase R （20 U/μL）and 1.5 μL reaction buffer were added. As for the control group, 0.15 μL DEPC-treated water and 1.5 μL reaction buffer were added.
Quantitative real-time PCR (qRT-PCR)
Detection of mRNAs were performed using a qPCR-RT Kit （RR036A, Takara, Tokyo, Japan）and a SYBR Premix Ex TaqTM II Kit （RR820A, Takara, Tokyo, Japan. Detection of circRNAs was performed using Reverse transcription and qRT-PCR kits (R11088.2, Ruibo Bio, Guangzhouou, China). Detection of miRNAs was performed using Reverse transcription and qRT-PCR kits (R10031.7, Ruibo Bio, Guangzhou, China). The primers used for detection of mRNAs and circRNAs are shown in Table2 and supplementary Table1.MiR-185-3p primer, miRNA mimic, miRNA inhibitor and miRNA NC were designed by Ruibo Bio (Guangzhou, China). The mRNA and circRNA levels were normalized to total GAPDH. The miRNA level was normalized to U6.
Western blot analysis
RIPA-PMSF (100:1) buffer (Beyotime, Shanghai, China) was used for cell lysis, and the lysates were separated by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (Millipore, CA, USA) at 350 mA for 2 h. The membranes were incubated overnight using the primary antibodies at 4° C. After incubation with secondary antibodies (Sigma, CA, USA) for 2 h, the membranes were subjected to chemiluminescence exposure and photographed using a Tannon 3500 imager (Tannon, Shanghai, China). The primary antibodies worked in this paper included anti-α-SMA, Col1a1, FGF5, BRD4 and GAPDH （CST, MA, USA）. The protein levels were normalized to total GAPDH.
Establishment of stable cell line
Three siRNAs targeting the junction site of hsa_circ_0008494 and a negatively controlled siRNA were designed and packaged into a lentiviral vector (No. Gv493, element sequence hu6-mcs-cbh-gcgfp-ires-puromycin). The negatively controlled scramble sequence was TTCTCCGAACGTGTCACGT. LX-2 cells were infected with lentiviral constructs, including LV-circ_0008494-KD1, LV-circ_0008494-KD2, LV-circ_0008494-KD3 and LV-NC. The GFP labeling of the recombinant viruses was confirmed using an inverted fluorescent microscope (Zeiss, Thornwood, USA). The knockdown efficiency of circ_0008494 was confirmed by RT-qPCR assays. To generate a stable circ_0008494-interfering cell line, LX-2 cells infected with LV-circ_0008494-KD3 and LV-NC viruses were selected with 5 μg/mL puromycin. Clones resistant to puromycin were collected and expanded in a culture dish.
miRNA products and transfection reagent were purchased from Ruibo Bio (Guangzhou, China). Transfection was carried out according to the manufacturer’s instructions. The final transfection concentration was 125nM. For cell function detection or rescue assays, miRNA products were respectively transfected into LX-2 cells, stable LV-circ_0008494-KD LX-2 cells or LV-NC cells. 48 hours after transfection, the cells of each experimental group were re-suspended, collected and subjected to downstream experiments.
LX-2 cells of each experimental group were seeded in 96-well plates overnight at a density of 1 × 103/ mL. After 1, 2, 3, 4 or 5 days, 10 μL CCK-8 (Dojindo, Kumamoto, Japan) was added to each well and incubated for 4 hours at room temperature. The absorbance was measured with a microplate reader at 450 nm.
Trans-well migration assay
LX-2 cells of each experimental group were resuspended and seeded in upper trans-well chambers for the migration assay (Corning, NY, USA) at a density of 1 × 106 cells/mL. The cells were allowed to cross the chamber for 48 hours. The penetrated cells were fixed, stained with 1% crystal violet, and counted under an inverted microscope at a magnification 100× (Zeiss, Thornwood, USA).
Cell apoptosis was quantified using an Annexin V-APD Single Staining Kit (eBioscience, San Diego, CA, USA). The experimental grouping was carried out as above. Each Falcon tube was added with Annexin V-APC (10 μL) and incubated at 37ºC in the dark for 10~15 minutes. Next, each tube was added with 400 μL 1× Binding Buffer. Apoptosis was detected within 1 hour in a flow cytometer (Beckman Coulter, CA, USA).
Luciferase reporter assay
To detect the binding of hsa_circ_0008494 and miR-185-3p, wild-type of circ_0008494 or its complementary or mutant sequences were inserted into a psiCHECK vector. To detect the binding of Col1a1 and miR-185-3p, the Col1a1 3'UTR and its mutant sequences were inserted into a pMIR vector. Lipofectamine 2000 (Invitrogen, CA, USA) was used to co-transfect the miR-185-3p, which was mimicked with psiCHECK-circ_0008494-WT, psiCHECK-circ_0008494-complementary and psiCHECK-circ_0008494-MUTANT vectors, into 293T cells. Additionally, miR-185-3p inhibitor and inhibitor NC were co-transfected with pMIR-Col1a1-WT 3'UTR and pMIR-Col1a1-MUTANT 3'UTR vectors. 48 hours since the transfection, the relative luciferase absorbance value of each group was examined with a Dual-Luciferase Reporter Assay System (Promega, Madison, USA) and normalized to Renilla luciferase absorbance values.
A Simple ChipTM Enzymatic Chromatin IP Kit (CST, MA, USA) was used for RNA immunoprecipitation (RIP) experiments. 1 × 107 LX-2 cells were pelleted and re-suspended with an equal pellet volume of RIP Lysis Buffer. The cell lysates (100 μl) were incubated with 5 μg of anti‐argonaute‐2 (AGO2) antibody (CST, MA, USA) or an isotype-matched IgG (CST, MA, USA) coated beads at 4 ºC overnight, respectively. With the treatment of proteinase K, the immunoprecipitated RNAs were extracted by RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany). The abundance of circ_0008494 or miR-185-3p was detected by qRT–PCR assay.
Biotin-coupled miRNA capture
A total of 1 × 107LX-2 cells were harvested, lysed and sonicated. The miR-185-3p probe and miRNA NC probe were incubated with the C-1 magnetic beads (Life Technologies, Guilford, CT) at 25 ºC for 2 hours to generate probe-coated beads. The cell lysates were incubated with miR-185-3p probe and miRNA NC probe at 4 ºC overnight. After cleansing with the wash buffer, the RNA complexes bound to the beads were eluted and extracted by RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) for qRT–PCR assay. Biltinylated-miR-185-3p probe was designed and synthesized by Ruibo Bio (Guangzhou, China).
Statistical analyses were performed using SPSS software 25.0 and GraphPad Prism 8.0. Statistically significant differences were calculated using Student’s t-test. Data were expressed as the mean ± SD of three independent experiments and p<0.05 was considered to indicate a significant difference. The asterisks *, **and*** stand for p<0.05, p<0.01and p<0.001 respectively.