Chemicals and antibodies
RNase R was purchased from Epicentre (Farmingdale, NY, USA), actinomycin D purchased from RayStarBio (HangZhou, Zhejiang, China) was used to assess the half-life. The specific STAT3 small-molecule inhibitor C188–9 was obtained from StemMed, Ltd. (Monmouth, NJ, USA). And the STAT3 activator colivelin was purchased from Santa Cruz Biotechnology (sc-361153). The primary antibodies including anti-p-STAT3 (#9145), anti-STAT3 (#9139), anti-EZH2 (#5246), anti-GAPDH (#2118) and anti-Ki-67 (#9449) were all purchased from Cell Signaling Technology (Danvers, MA, USA).
HCC tissues, plasma and cell lines
A total of 130 paired surgically excised HCC and adjacent normal tissues from Henan Provincial People’s Hospital (Zhengzhou, China) were immediately collected into liquid nitrogen to preserve the integrity of the RNA. In addition, plasma samples from 36 HCC patients and 36 healthy controls were also collected to test the non-invasive diagnostic value of circ-LRIG3. None of the patients received preoperative radiotherapy or chemotherapy, and they all provided written informed consent. This study was conducted with the approval of the ethics committee of Henan Provincial People’s Hospital. Six HCC cell lines including SNU-423, HepG2, Hep3B, Huh7, SMMC-7721 and MHCC-97L and one normal LO2 hepatocytes were all purchased from ATCC and cultured in DMEM medium containing penicillin/streptomycin and 10% fetal bovine serum.
RNA isolation and qRT-PCR analysis
Total RNA was classically extracted by Trizol reagent (Invitrogen, CA, USA) and dissolved in DEPC water. Then, RNA was reverse transcripted into cDNA using All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia, MD, USA), followed by quantification using BlazeTaq™ SYBR Green qPCR Mix 2.0 (GeneCopoeia). The primer sequences are shown in Table S3. GAPDH was used as the internal reference. The relative expression was calculated using the comparative Ct (2-ΔΔCt) method. For determining the junction sequence of circ-LRIG3, the PCR product was purified and subjected to Sanger sequencing.
Fluorescence in situ hybridization (FISH)
The FISH assay was performed by using RNA FISH Kit (GenePharma, Shanghai, China) according to manufacturer’s protocol with FAM-labeled circ-LRIG3 probe against the junction site of circ-LRIG3 (GenePharma). The nucleus was stained with DAPI. The fluorescence signal was observed, photographed and merged using confocal laser microscope system.
Generation of circ-LRIG3-overexpressing and -silenced HCC cell lines
To overexpress circ-LRIG3, pLC5-ciR-GFP (#GS0108) vector containing one paired reverse complementary sequence (RCS) provided by Geneseed (Guangzhou, China) was used, in which the full-length of circ-LRIG3 (chr12: 59277301-59308117) was inserted into pLC5-ciR vector between EcoRI and BamHI restriction sites, followed by packaging into the lentivirus and infection into HepG2 cells. The stable circ-LRIG3-overexpressing cells were selected by 0.8μg/mL puromycin. To knock down circ-LRIG3, two antisense oligonucleotide (ASO) against circ-LRIG3 junction site were designed and synthesized by RiboBio (Guangzhou, China). Then, the ASO was transfected into SMMC-7721 cells using riboFECT™ CP transfection solution (RiboBio) based on manufacturer’s protocol.
Cell proliferation and apoptosis assays
The CCK-8 assay was used to assess cell viability. In brief, 1500 HCC cells were seeded into the 96-well plate. After 24, 48 and 72h, the medium was added with 10μL CCK-8 solution (Dojindo, Kumamoto, Japan) for 2.5h, followed by detection of the absorption value using a microplate spectrophotometer. Besides, the DNA synthesis rate was assessed by using EdU Staining Proliferation Kit provided by RiboBio based on manufacturer’s protocol. The positive cells were observed and photographed using confocal laser microscope system. For detecting cell apoptosis, the Annexin V-PE/7-ADD Apoptosis Detection Kit (BD Biosciences, CA, USA) was used, in which HCC cells were incubated with 5μL Annexin V-PE and 5μL 7-ADD in 500μL binding buffer, followed by analyzing apoptotic cells using a flow cytometer (BD Biosciences).
In vitro invasion and migration assays
The upper chamber of the trans-well insert with pore size of was coated with (invasion) or without (migration) Matrigel (Corning) and was placed in a 37°C incubator for 2.5h. Then, HCC cells were added into the upper chamber, and the lower chamber was added with 600μL DMEM complete medium. 20h later, the trans-well was fixed with formaldehyde for 5 min, permeabilized by methanol for 5 min, and was then stained by 0.5 % crystal violet for 10 min. The invaded/migratory cells were counted under a light microscope at least 5 random fields.
mRNA sequencing
SMMC-7721 cells were transfected with control and two ASOs (ASO-circ-LRIG3#1 and #2) for 48h, then cells were washed and total RNA was extracted by TRIzol and purified by DNase I. After that, the oligo-dT magnetic beads were used to separate mRNA from total RNA for construction of cDNA libraries (GeneSky, Shanghai, China). Lastly, the differentially expressed genes (fold change ≥ 2 and P < 0.05) were determined and subjected to Gene Set Enrichment Analysis (GSEA). The RNA sequencing data was deposited in the Gene Expression Omnibus database under accession code GSE154021.
Western blot, Co-IP and Immunohistochemistry (IHC)
For Western blot, whole cell extract was prepared by lysing cells in NP-40 lysis buffer. Then, proteins were resolved by 10% SDS-PAGE gel and transferred onto PVDF membrane. After incubating with corresponding primary and secondary antibodies, the membrane was strictly washed using TBST, and the chemiluminescent signals were developed using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA). The Co-IP assay was carried out as previously described.[18] The IHC assay was performed by using Histostain-Plus IHC Kit (NeoBioscience, Guangzhou, China) as per Manufacturer's instructions.
RNA pull-down and RIP assays
For the RNA pull-down assay, the biotinylated probe against circ-LRIG3 junction site was designed and synthesized by RiboBio. Then, the indicated HCC cell lysates were prepared using the NP-40 lysis buffer and incubated with above probe at 26°C for 2h with agitation. 50μL streptavidin magnetic beads (Invitrogen) were pre-washed with 20mM Tris (pH=7.5) twice and added into above complex, incubating at 26°C for 30min with agitation. After strict washing, the proteins enriched by circ-LRIG3 were eluted for Western blot analysis. The RIP assay was performed using MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) based on the manufacturer’s instructions with additional UV crosslink step as previously described[18].
Chromatin immunoprecipitation (ChIP)
HCC cells were treated with formaldehyde for 10min, followed by addition with glycine for 5min. Then, cells were harvested and digested with a micrococcal nuclease for 20min at 37°C, making DNA break into 200~1000bp fragments. The indicated antibodies were incubated with above cell lysates at 4°C overnight with agitation. The second day, the protein A agarose (Santa Cruz Biotechnology) was prepared and added into above antibody-target protein-DNA complex, and incubated for 1h at 26°C with agitation. Lastly, the enriched DNA fragments were purified and collected for PCR analysis.
Luciferase reporter assay
The Cignal STAT3 Reporter (luc) Kit (SA Biosciences, CA, USA) was used to assess STAT3 transcriptional activity according to manufacturer’s instructions. For determining the effect of STAT3 on transcriptional activity of circ-LRIG3 promoter, the full-length of circ-LRIG3 promoter with wild-type or mutant STAT3 binding site was inserted into pGL3-basic vector (Promega, WI, USA), followed by transfection into HepG2 and SMMC-7721 cells treated with colivelin or c188-9. The Dual-Luciferase Reporter Assay System (Promega) was applied to detect luciferase luminescence.
Animal experiments
The stable circ-LRIG3-overexpresing HepG2 cells were subcutaneously or tail vein injected into age-matched male BALB/c mice, followed by growth for 5 weeks under specific-pathogen-free conditions. During this period, the mice were intraperitoneally injected with C188-9 (50mg/kg) daily for 3 weeks. At the end of observation, the mice were killed by cervical dislocation, and the subcutaneous tumors were weighed and subjected to hematoxylin&eosin (H&E), IHC and TUNEL (Beyotime, Beijing, China) staining. For mice injected with cells via the tail vein, the lung tissues were collected for counting surface metastases under a dissecting microscope, followed by H&E staining. All experimental procedures in our study were approved by the Institutional Animal Care and Use Committee of Henan Provincial People’s Hospital.
Statistics
Comparisons between two groups were analyzed by Student's t test or Chi-square test as appropriate. ROC and Kaplan Meier-plotter were used to determine the diagnostic and prognostic values of circ-LRIG3, respectively. The link between circ-LRIG3 and p-STAT3 expression in HCC tissues was analyzed by Pearson Correlation Coefficient. All P-values were two-sided unless otherwise specified. P<0.05 is indicative of statistical significance.