Clinical tissues and cell culture
Ten paired HCC tissues and corresponding background livers (BLs) were obtained from HCC patients undergoing partial hepatectomy between January 2019 and December 2020. After surgical resection, the specimens were immediately snap-frozen in liquid nitrogen. RNA-later (#AM7201, Thermofisher, USA) was added to the cryogenic vials to prevent RNA degradation for subsequent extraction. The collection and usage of these samples were followed ethical and institutional guidelines and implement it after organizing informed consent from donors. The study was approved by the local Research Ethics Committee at Zhongnan Hospital of Wuhan University (Approval No.2018078). Cell lines (L02, Huh7, HepG2, HCCL-M3) involved in this experiment were purchased from Stem Cell Bank, Chinese Academy of Sciences. Mixture of Dulbecco’s Modified Eagle’s Medium (Gibco, USA) and 10% fetal bovine serum (Gibco, Thermo Fisher, USA) and 1% penicillin-streptomycin (Gibco, USA) was used to culture cells. All cells were grown in humidified 5% CO2 at 37℃.
MiRNA microarray analysis
The microarray data adopted for analysis have been described in detail before (19). Data have been uploaded in NCBI’s Gene Expression Omnibus (GEO) public database http://www.ncbi.nlm.nih.gov/geo/, accession number, GSE20077).
RNA extraction, Reverse transcription and quantitative real-time PCR
Total RNA extraction from tissues and cells were using Triol Reagent (Invitrogen, USA) following the instructions. The quality and concentration of RNA were detected by Nanodrop 2000 (ThermoFisher, USA). cDNA was obtained through a reverse transcription reaction with kits (Toyobo, Japan). The expression of miRNAs and genes was examined using UltraSYBR Mixture (CWbio, Beijing, China) on the CFX96Touch Real-Time PCR system (Biorad, USA). The mRNA levels of miR-21-3p and genes were normalized to U6 (Ribobio, Guangzhou, China) and GAPDH, respectively. The relative expression ratio was calculated with the 2-ΔΔCq method. Primers used listed here:
Smad7: 5’-TTCAGGACCAAACGATCTGCG-3’(sense);
5’-GATGGTGGTGACCTTTGGCAC-3’(antisense);
Yap1: 5’-GAACTCGGCTTCAGGTCCTC-3’(sense);
5’- GGTTCATGGCAAAACGAGGG-3’(antisense);
LATS2:5’-TGGAATGCCAACAATGTAGCG-3’ (sense);
5’-ATTATCACTCTCTCCAGGGGCG-3’ (antisense);
CTGF: 5’- AGAGGGCTGTCGGCG-3’ (sense)
5’-CACAGGAGCTGGTGTTCCAT-3’ (antisense);
GAPDH: 5’-AGAAGGCTGGGGCTCATTTG-3’(sense);
5’- GCAGGAGGCATTGCTGATGAT-3’(antisense).
MiRNA related reagents, plasmids and cell transfection
The precursor and inhibitor of miR-21-3p, mimic-NC and inhibitor-NC were obtained from Ribobio (Guangzhou, China). Inhibitors of miR-21-3p were the complementary strand of miR-21-3p and was dedicated to compete the binding sites between miR-21-3p and target genes, but would not decrease the mRNA expression level of miRNAs. The final concentration of these reagents was 50 nM. Smad7 expression vector (P-Smad7) was constructed using GV141 vector by Genechem (Shanghai, China) and plasmids-NC (P-NC) served as an experimental control. All transfection experiments were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the guidance.
Western blot
Proteins collection using lysis buffer (Byotime, China) containing protease inhibitor (PMSF, 1:100). Centrifuged at 15000rpm, 20min at 4℃ and then transferring supernatant to new tubes. Concentrations of protein were detected using a BCA protein kit (Byotime, China). Mixing lysis buffer with protein loading buffer, denaturing protein at 100℃ for 5min. An equal amount of proteins were separated by SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Blocking for 2h with 5% skimmed milk. The membrane was incubated with primary antibodies overnight at 4℃. Then reacted with second antibodies for 1.5h at room temperature. The protein bands were captured by enhanced chemiluminescence (ECL) kit (Servicebio, Wuhan, China) using GENESys ( Synoptics Ltd, China). Anti-Smad7 (#25840-1-AP), anti-Yap1 (#14074), anti-E-cad (#3195), anti-N-cad (#131161), anti-Vimentin (#5741), anti-LATS2 (#5888) and anti-CTGF (#86641) were purchased from CST (USA). Anti-Bcl2 (#ab182858), anti-Bax (#ab32503) and anti-GAPDH (#ab8245) were purchased from Abcam. Goat anti-mouse IgG-horseradish peroxidase (#A23220) and goat anti-mouse IgG-horseradish peroxidase (#A25012) were come from Abbkine.
Dual-luciferase reporter assay
PmiR-GLO-Smad7 vector was constructed, which consisted of predicted binding sites that were mutated and ligated between the PmeI and XbaI restriction enzymes sites of pmirGLO Dual-Luciferase miRNA Target Reporter Vector (#E1300, Promega, USA). Smad7 3’UTR region contains two putative binding sites for miR-21-3p with the seed regions at 1428-1434 and 1445-1451. The strand of mutant 3 (Mut 3) mutated two binding sites simultaneously. The wild-type (WT) and mutant Smad7 3’-UTR luciferase reporter plasmids (0.25μg/ well) were co-transfected into cell huh-7 with miR-21-3p mimics or miR-NC (50nM, 0.15μl/ well) respectively. Dual-Glo Luciferase Assay System (#E2920, Promega, USA) was employed to check firefly luciferase and renilla luciferase activity after 24h transfection, respectively. Firefly luciferase activity was normalized to renilla luciferase activity. Data were collected using Enspire 2300 (PerkinElmer). Six repetitions per group were calculated.
Cell apoptosis assay and flow cytometry
A total of 4×105 cells were transfected with miR-21-3p mimics with/or Smad7 plasmids. After 24h, collecting (including dead cells in the culture medium) and processing cells according to the instructions of Annexin V-FITC/PI Double stain apoptosis detection kit (#4101-2, Bestbio, Shanghai, China). Collecting the fluorescence intensity on the flow cytometer to check the apoptotic rate (Cytoflex Beckman, China) immediately.
Cell invasion and metastasis assay
The procedure for processing cell Huh-7 was the same as the cell apoptosis experiment. Huh-7 was harvested at 24h post-transfections. Suspending cells with 5% FBS culture medium and seeding 5×104 cells each upper chamber (6.5 mm. in diameter, 8.0 um pore size, Corning, USA). The lower chamber was filled with 600μl medium (15% FBS). Cells were fixed with 4% paraformaldehyde 20min at room temperature, stained with 0.1% crystal violet (Sigma-Aldrich, USA). The upper cells were wiped using cotton swabs. Selecting 100× and 200× fields and taking photos randomly under an inverted microscope (Olympus IX3). The difference between cell invasion and migration assay was that in the latter one upper chamber needed to be pretreated with 0.3% Matrigel matrix (#356234, Corning, USA), 37℃, 4h. Three independent experiments were performed.
Animal experiments
The establishment of Wistar rat models were detailed descripted before, and grouping of these rats were according to the Metavir score system (20,21). Here was brief introduction. Wistar rats ( male, 7-8 weeks old, 200-220g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal handling and experimental procedures were approved by the Animal Care and Use Committee of Wuhan University following the Animal Experiment/Animal Biosafety Level-III Laboratory Guidelines. Rats were injected 40% carbon tetrachloride (CCl4) dissolved in maize oil (1.5ml/kg) every twice a week. At 8, 14, 18 weeks, liver fibrosis, cirrhosis and liver cancer were formed, respectively. Hematoxylin-eosin (HE) staining and masson staing which is more conductive to observe the degree of fiber crosslinking of rat models were displayed in Figure S1A.
Immunohistochemistry (IHC)
Immunohistochemical staining of patient tissues and animal tissues was performed by Servicebio Company, Wuhan, China, and photographed by an inverted microscope (Olympus IX3). Positively stained cells were counted in at least five fields from each area with 400× magnifications. Primary antibodies listed here:
Anti-Smad7: (#25840-1-AP, Proteintech): 1:200; anti-Yap1 (#14074, CST): 1:400.
Immunofluorescence (IF)
The distribution of microfilaments and cytoskeleton was clearly shown through fluorescently labeled phalloidin staining. Cells were cultured in soft and still matrixes 24h. Fixed with 4% paraformaldehyde 20min at room temperature. Phalloidin dilution rate: 1:500. Incubation at 37℃ for 1h. Nuclear staining with DAPI for 5min. Images were processed with an inverted microscope (Olympus IX3).
Gene set variation analysis (GSVA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis with miR-21-3p/Smad7 expression
GSVA was a useful tool provided by R (3.5.2) to explore the potential biological process and enriched KEGG pathways of research objects according to its high or low expression. The results were presented in the form of intuitive volcano maps and specific heat maps. Gene terms with |logFC| ≥ 0.1 and P<0.05 were considered statistically significant. The KEGG gene sets (c2.cp.kegg.v6.2.symbols.gmt) downloaded from the Molecular Signatures Database–MsigDB (http://www.broad.mit.edu/gsea/msigdb/index.jsp) were used for enrichment analysis.
Bioinformatics data
MicroRNA Target Prediction Database (miRDB), PicTar and TargetScan databases were used for searching miRNA targets and predicting binding sites. Cohort data including 376 HCC patients RNA-seq and clinical characteristics were downloaded from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Data with incomplete clinical information were excluded during analysis.
Statistical analysis
Data were presented here as mean ± standard deviation (SD) and analyzed by the Student’s t-test or one-way ANOVA. Quantitative data were representative of three experiments. Wilcoxon signed-rank test and Wilcoxon rank-sum test was adopted to analyze gene expressions in paired and non-paired tissue samples. The relationships between disease stages and gene expressions was checked by Wilcoxon rank-sum test. Prognosis analysis was performed using Kaplan-Meier method and univariate Cox regression. All bioinformatics statistical analyses and plots were produced using R (v.3.5.2). P < 0.05 was considered statistically significant.