Chemicals and reagents
Highly pure (>98%) baicalein and sorafenib (Raf-1 and B-Raf multi-kinase inhibitor, first-line drugs approved by the Food and Drug Administration for liver cancer) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The structures of baicalein and sorafenib are shown in Fig. 2k.
Clinical tissue specimens
A total of 36 pairs of human hepatocellular carcinoma tissues and adjacent normal tissues were collected from the Second Affiliated Hospital of Xi’ an Jiaotong University (Shaanxi, China) between Jan 2015 to May 2021 with histologically confirmed. Patients who were neoadjuvant chemo- or radio therapy naïve were selected. The study was conducted with the written informed consents of all patients and the approval of the Ethics Committee of the Second Affiliated Hospital of Xi’ an Jiaotong University. The clinical data was collected in Supplemental Table S1. Tissue sections are frozen and stored at -80°C until use.
Cell culture and transfection
7 different human HCC cell lines including Bel-7402, Bel-7404, SMMC-7721, MHCC-97H, HepG2, Hep3B, Huh7 and human L-O2 normal liver cell line was purchased from the American Type Culture Collection (Manassas, USA) and were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Carlsbad, CA, USA). All cells were cultured at 37°C with 5% CO2.
The human HDAC10 cDNA clone expression plasmid was synthesized by Sino Biological Inc. (Beijing, China). miR-3178 mimic, inhibitor and their negative controls were synthesized by Ribobio Co., Ltd (Guangzhou, China). All the sequences were listed in Supplementary Table S2. Primers for miR-3178, U6, HDAC10 and GAPDH were synthesized by GENEWIZ Biotech Co., Ltd (Suzhou, China) and the sequences were listed in Supplementary Table S3. Transfection was performed with Lipofectamine 3000 and supplied with Serum-free Opti-MEM® medium (Invitrogen, Waltham, USA) following the protocols of manufacturer.
Total RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) assay
Total RNA including miRNA was extracted from clinical samples and HCC cells by TRIzol reagent (Invitrogen, USA). The extracted RNA was reverse-transcribed to be cDNA by RT reagent Kit (#RR047A, TaKaRa, Japan) and to be cDNA for miRNA using miRNA First Strand Synthesis Kit (#638313, Takara, Japan). Then, qRT-PCR was implemented by SYBR® Premix Ex Taq II Kit (RR820A, Takara, Japan) on an ABI-7500 fast system (Applied Biosystems, Foster City, USA). 2-ΔΔCt method was used and data were expressed as the fold change of the control group. U6 and GAPDH were used as internal normalization controls for miR-3178 and HDAC10, respectively. All sequences of specific primers were described in Supplementary Table S3. Experiments were implemented in triplicate.
Luciferase reporter assay
To verify the interaction between miR-3178 and HDAC10, wild type 3’UTR of HDAC10 containing the putative miR-3178 binding site (Site: 204-210, termed HDAC10 wt 3’UTR) and its mutant (termed HDAC10 mut 3’UTR) were cloned into PsiCheck-2 luciferase reporter vector (Genepharma Biotechnology Co., Shanghai, China). Cells were seeded in 24-well plates at a density of 5×104 cells per well overnight. Then, cells were co-transfected with luciferase reporter vector with either 50 nmol/L miRNA mimics negative control (termed miR-NC) or 50 nmol/L miR-3178 mimics (termed miR-3178) (RiboBio Co., Guangzhou, China) for 48 hours. Renilla luciferase reporter plasmid (Promega, Madison, USA) was co-transfected as an internal control. After incubation, a dual-luciferase reporter assay kit (#E1910, Promega, Madison, USA) was used to examine the luciferase activities. Experiments were implemented in triplicate and renilla luciferase activity was used to normalize the firefly luciferase activity.
Cell counting kit-8 (CCK8) and colony formation assay
For CCK8 assay, cells were incubated at a density of 1.5×103 cells per well after transfection and cultured for 1-5 days at 37˚C in 96-well plates. At the indicated time points, cell viability was then measured by incubating cells with 10 μl of CCK-8 solution (1 mg/ml, Dojindo, Kumamoto, Japan) for 2 h. The absorbance was determined at 450 nm by using the Microplate reader (M20 pro, TECAN, Switzerland).
For colony formation assay, Transfected MHCC-97H or SMMC-7721 cells were counted and implanted into 6-well plates at 1500 cells/well. After cultured at 37˚C with 5% CO2 for 7-14 days until visible colony formation can be observed, cells were fixed with paraformaldehyde (4%) and dyed with crystal violet solution (0.1%) for 15 min. The colonies were photographed and counted when they contain 50 cells.
Apoptotic rate detection and cell cycle assay
As for apoptotic rate analysis, the Annexin V-FITC Apoptosis Detection Kit (Invitrogen, USA) was applied. Briefly, after transfection and/or baicalein treatment for 48h in 6-well plates, cells were harvested and stained with Annexin V-FITC conjugate (5 μl) and propidium iodide (PI, 5 μl) solution in the dark at room temperature for 20 min. Following the incubation, apoptotic rate was investigated via flow cytometer (BD Biosciences, San Jose, USA).
To determine cell cycle arrest, cells was detected by PI staining. Briefly, after transfection and/or baicalein treatment for 48h in 6-well plates, cells were harvested and fixed with 70% (v/v) ethanol at -20˚C overnight. Cells were then washed and stained with cold PBS containing propidium iodide (PI, 10 μg/ml) and RNase A (1 mg/ml) in the dark at room temperature for 30 min. The cell cycle distribution was then investigated using flow cytometer (BD Biosciences, San Jose, USA) and quantified based on the DNA content of the cells using FlowJo software (Version 7.6, BD, USA).
Western blotting
Cells were lysed using cold RIPA lysis buffer with protease and phosphatase inhibitor (#04693159001 and #04906837001, Roche, USA) and quantified by Pierce BCA Protein Assay Kit (Thermo, USA). 40 μg of proteins were loaded and isolated from 10% sodium dodecyl sulfate-polyacrylamide gel and blotted on polyvinylidene difluoride membranes (Millipore, Bedford, USA). After blocking non-specific proteins with 5% non-fat milk for 2 h at 37 °C, the membranes were probed with primary antibodies at 4 °C overnight. After washed, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at 37 °C. Finally, after washing, the fluorescence of proteins were catalyzed by enhanced chemiluminescence (ECL) substrate (Millipore, USA) and relative fluorescence intensities were determined by Image Lab System (Bio-Rad, USA). The antibodies used in this study were shown in Supplementary Table S4.
Immunohistochemical (IHC) staining
The paraffin-embedded tumors from xenografts were cutted at the thickness of 4 μm, deparaffinized and rehydrated. After blocking endogenous peroxidase activity in 0.3% H2O2 and blocking nonspecific immunoglobulin binding sites with goat serum, sections were immune-stained with primary antibodies at 4 ◦C overnight. Then, the slides were incubated with biotinylated secondary antibodies at 37 ◦C for 30 min. After that, protein expression was examined by 3,3-diaminobezidine tetra hydrochloride (DAB) staining. Hematoxylin was also applied to counterstain the nucleus.
Animal experiments
All animal experiments were reviewed and performed with the approval of the Ethics Committee of the Second Affiliated Hospital of Xi’ an Jiaotong University and the methods were in compliance with the animal welfare guidelines.
Male BALB/c nude mice (3-4 weeks old, weighing 14-18 g) were fed in Xi'an Jiaotong University Experimental Animal Center. For baicalein treatment xenograft model, mice were randomly subdivided into 2 groups. 5 × 106 untreated MHCC-97H cells (in 100 μL of normal saline) were subcutaneously injected in the armpits of nude mice (n = 6). Tumor formation was monitored every two days with calipers and the volumes were calculated using formula V (mm3) = (Length × Width × Width /2). When tumors reached an average volume of 50-100 mm3, mice were intraperitoneally injected with baicalein (80 mg/kg, dissolved in normal saline) or normal saline (100 μL) as controls every two days. Mice were euthanized at day 28 after first baicalein injection. For miR-3178 overexpression xenograft model, mice were randomized into 2 groups. 5 × 106 MHCC-97H cells stably transfected with LV-miR-3178 or LV-miR-NC were subcutaneously injected in the armpits of nude mice (5 mice per condition). Tumor sizes were neatly excised every two days. Mice were euthanized at day 20 after cell inoculation. Tumors were stripped and tumor weights were excisely measured. A portion of tumor tissue was fixed in 10% formalin solution and then embedded in paraffin for subsequent histological examination, and other parts were subjected to total RNA or protein isolation, or froze in liquid nitrogen for later use.
Data and statistical analysis
Values were expressed as the mean ± standard deviation and were analyzed via Graphpad Prism 8.0.2 Software (San Diego, USA). A two-tailed Student’s t-test was applied to analyze statistically differences between two groups. For multiple comparisons, a one-way analysis of variance (ANOVA) was used followed by Tukey's post hoc test. Pearson correlation analysis was used to explore the correlations between expression of HDAC10 and miR-3178 in tissues or cell lines. P-values less than 0.05 were thought as statistically significant (‘*’ denotes P < 0.05). All experiments were separately repeated at least three times.