In our research, HCC tissues (n=46) and adjacent normal tissues (n=46) were acquired from patients who underwent surgery at Laiyang Central Hospital Of Yantai City. These tissues were harvested and timely frozen in liquid nitrogen, and then preserved at -80℃ until the experiments were performed. These subjects did not receive any treatment and provided informed consents.
Cell culture and transfection
Human HCC cell lines (Hep3B and Huh7) and human normal liver cell line (THLE-2) were bought from COBIOER (Nanjing, China). These cells were grown in Dulbecco’s modified eagle medium (DMEM; Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) in a moist atmosphere with 5% carbon dioxide at 37℃.
Small interfering RNA against circ_LRIG3 (si-circ_LRIG3) and its matched control (si-NC), MAP2K6 overexpression plasmid (MAP2K6) and its matched control (pcDNA) were synthesized by RIBOBIO (Guangzhou, China). Mimic or inhibitor of miR-223-3p (miR-223-3p or anti-miR-223-3p) and mimic or inhibitor negative control (miR-NC or anti-miR-NC) were provided by GenePharma (Jiangsu, China). Lentivirus-mediated shRNA interference targeting circ_LRIG3 (sh-circ_LRIG3) and its matched control (sh-NC) were bought from Genechem (Shanghai, China). Lipofectamine 3000(Invitrogen, Carlsbad, CA, USA) was used for cell transfection.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Trizol reagent (Invitrogen) was utilized to obtain total RNA from tissue samples and cells. For detecting genes expression, Prime Script RT reagent Kit (Takara, Dalian, China) and TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) were used to synthesize the first strand of complementary DNA (cDNA). All reactions were performed on the ABI 7300 system (Thermo Fisher Scientific) using SYBR Green PCR kit (Thermo Fisher Scientific). Primers for circ_LRIG3, LRIG3, miR-223-3p, MAP2K6, U6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were exhibited as followed: circ_LRIG3 forward (F, 5’- TCACTGGTTTGGATGCATTG-3’; R, 5’-AAGGTGGCTCATGGAACTTG-3’), LRIG3 (F, 5’-CACATCAATGGAACCTGGGTATTTTGAC-3’; R, 5’- GTTTCGGTTCAATTCGAGATGTTGCAGTT-3’), miR-223-3p (F, 5’- AGCTGGTGTTGTGAATCAGGCCG-3’; R, 5’-TGGTGTCGTGGAGTCG-3’), MAP2K6, (F, 5’-ATTTGGAGTCTGGGCATCAC-3’; R, 5’- ACTTGTCTGCTGGGAGTTGTG-3’), GAPDH (F, 5’-CGCTCTCTGCTCCTCCTGTTC-3’; R, 5’-ATCCGTTGACTCCGACCTTCAC-3’), U6 (F, 5’-CTCGCTTCGGCAGCACATATACT-3’; R, 5’-ACGCTTCACGAATTTGCGTGTC-3’). The circ_LRIG3, LRIG3, MAP2K6, or miR-223-3p expression was assessed using the 2-ΔΔCt method and standardized by GAPDH or U6, respectively.
RNase R and Actinomycin D treatment
To assess the stability of circ_LRIG3 and its linear isoform (LRIG3), dimethyl sulfoxide solution (DMSO) or actinomycin D (2 mg/mL) was added to cultured medium. RNase R (3 U/μg, Epicentre Technologies, Madison, WI, USA) was utilized to incubate the total RNA (2 μg) at 37℃ for 30 min. After treatment with RNase R or Actinomycin D, these cells were collected and then subjected to qRT-PCR for detecting the expression levels of circ_LRIG3 and LRIG3.
Subcellular fractionation location
PARIS Kit (Life Technologies Corp., Grand Island, NY, USA) was employed to isolate cytosolic and nuclear fractions. In brief, Hep3B and Huh7 cells were carefully washed by phosphate-buffered saline (PBS) and placed on the ice. Subsequently, these cells were re-suspended in fractionation buffer and centrifuged at 500 × g at 4℃ for 5 min. Subsequently, the cytoplasmic fraction would be separated from the nuclear pellet. After that, the remaining nuclear pellet was again lysed by cell disruption buffer as nuclear fraction. Lastly, the abundance of U6, GAPDH and circ_LRIG3 was examined by qRT-PCR in the nuclear and cytoplasmic fractions. GAPDH and U6 were served as controls for the cytoplasmic and nuclear, respectively.
Cell cycle assay
Hep3B and Huh7 cells were collected following transfection for 48 h, and fixed by ice‐cold ethanol (70%) at −20℃ for 24 h. Afterward, these cells were centrifuged and washed with PBS, followed by staining with 25 μg/mL propidium iodide (PI ) solution in PBS supplemented with Triton X‐100 (0.2%) and RNase A(50 μg/mL) for 20 min in the dark. Lastly, flow cytometry (Guava Technologies, Hayward, CA, USA) was employed to examine the distribution of cell cycle.
Cell proliferation assay
Cell proliferation ability was evaluated using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. In short, Hep3B and Huh7 cells were placed in the 96-well plates overnight and then transfected with si-NC, si-circ_LRIG3, si-circ_LRIG3 + anti-miR-NC, si-circ_LRIG3 + anti-miR-223-3p, si-circ_LRIG3 + pcDNA, or si-circ_LRIG3 + MAP2K6. MTT solution (20 μL, 5 mg/mL, Sangon Biotech, Shanghai, China) was added to per well following transfection for 0 h, 24 h, 48 h, or 72 h. Following incubation for 4 h at 37℃, DMSO (150 μL) was added to per well after removing the cultured medium. The absorbance of per well was examined with the microplate reader (Bio-Teck, Winooski, VT, USA) at 490 nm.
Cell apoptosis assay
Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (Sangon Biotech) was applied to detect cell apoptosis according to the recommendations. In short, Hep3B and Huh7 cells were harvested and double stained by Annexin V-FITC and PI for 20 min in the darkness. Afterward, apoptotic cells were detected using a flow cytometer.
Transwell chambers (pore size 8 μm) (Corning Incorporation, Corning, NY, USA) coated without and with Matrigel (BD Biosciences, San Jose, CA, USA) were utilized to assess Hep3B and Huh7 cell migration and invasion abilities, respectively. In brief, cells were suspended in serum-free medium (DMEM, 100 µL) and then placed in the top surface of the chamber, and DMEM mixed with FBS (10%) was placed in the bottom surface of the chamber. Non-migrated or non-invaded cells from the top surface were gently wiped off using a cotton wool after incubation for 24 h. After that, the migrated or invaded cells were fixed using paraformaldehyde (4%) and stained using crystal violet (0.1%). Lastly, a microscope (Olympus, Tokyo, Japan) was utilized to photograph and count the migrated and invaded cells.
Western blot assay
To extract the total protein, tissues or transfected cells were lysed by RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing 1mM phenylmethylsulphonyi fluoride (PMSF; Sigma-Aldrich). After quantification by using BCA protein assay kit (Thermo Fisher Scientific), protein (about 40 μg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, polyvinylidene fluoride (PVDF; Beyotime, Shanghai, China) membranes were applied to transfer the protein. Next, membranes would be blocked using 5% skim milk (Yili, Beijing, China) for 1 h, and then the membranes were probed with speciﬁc primary antibody against snail (1:500, ab180714, Abcam, Cambridge, MA, USA), E-cadherin (1:500, ab15148, Abcam), MAP2K6 (1:2000, ab154901, Abcam), mitogen-activated protein kinase (MAPK) (1:1000, ab236738, Abcam), phospho-MAPK (p-MAPK) (1:500, ab47363, Abcam), extracellular signal-regulated kinases (ERKs) (1:1000, ab17942, Abcam) phosphor-ERKs (p-ERKs) (1:1000, ab47339, Abcam), or GAPDH (1:2000, ab37168, Abcam) overnight at 4℃. After that, these membranes were incubated by horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) G (1:4000, ab205718, Abcam). Finally, all protein bands were observed with the enhanced chemiluminescence reagent (Tanon, Shanghai, China). Relative protein expression was quantified by ImageJ software, followed by normalizing to GAPDH expression.
Dual-luciferase reporter assay
Circinteractome or TargetScan software online was utilized to predict the potential binding sites of miR-223-3p and circ_LRIG3 or MAP2K6. The circ_LRIG3 or MAP2K6 3’UTR fragments containing wild-type (WT; containing the specific binding site with miR-223-3p) or mutant (MUT; harboring the mutational binding sits with miR-223-3p) were amplified and cloned into pmirGlO luciferase reporter vector (Promega, Madison, WI, USA), namely WT vectors (circ_LRIG3-wt, MAP2K6-wt) or MUT vectors (circ_LRIG3-mut, MAP2K6-mut). Hep3B and Huh7 cells were co-transfected with those reporter vectors and miR-223-3p (or miR-NC) for 48 h. Lastly, dual-luciferase assay system (Promega) was utilized for detecting the luciferase activity, followed by normalizing to Renilla luciferase activity.
In vivo tumor model
The sh-circ_LRIG3 or sh-NC was transfected into Huh7 cells. Stably transfected cells (2×106) were injected subcutaneously into BALB/c nude mice (n=6/group, male, six-week-old, Shanghai Experimental Animal Center, Shanghai, China). From the 7th day, tumor length and width were examined with a caliper every week and tumor volumes (length × width2 ×0.5.) were calculated. After injection for 4 weeks, these mice would be sacrificed and tumor specimens were weighed and collected for further analysis.
In this study, all data from at least three independently experiment were displayed as mean ± standard deviation (SD). The significance of differences between groups was analyzed with Student’s t-test (for 2 groups) or a one-way analysis of variance (ANOVA; for more than 2 groups). Correlation between miR-223-3p and circ_LRIG3 or MAP2K6 was detected by Spearman rank correlation. Statistical analyses were performed by Graphpad Prism version 6.0 software (GraphPad Software, San Diego California, USA). P<0.05 was considered to be a statistically significant difference.