Reagents and Antibodies
DMSO and crystal violet were purchased from Sigma-Aldrich (St. Louis, MO, USA), Sorafenib was purchased from Selleck (Shanghai, P.R. China). Protease Inhibitor Cocktail (100X) was obtained from Cell Signaling (Danvers, MA, USA). Propidium Iodide (PI) was purchased from Meilunbio (Dalian, P.R. China). The antibodies were used as following: anti-SETD1A (Bethyl, Cat#A300-289A, 1:1000 for WB), anti-Cyclin D1 (Abcam, Cat#ab134175, 1:1000 for WB), anti-PCNA (Cell Signaling, Cat#2586, 1:1000 for WB), anti-Cleaved-PARP (Cell Signaling, Cat#5625, 1:1,000 for WB), anti-Cleaved Caspase-3 (Cell Signaling, Cat#9664 1:1,000 for WB), anti-Phospho-YAP (Ser127) (Cell Signaling, Cat#13008, 1:1,000 for WB), anti-YAP (Cell Signaling, Cat#14074, 1:1,000 for WB), anti-CYR61 (Cell Signaling, Cat#14479, 1:1,000 for WB), anti-CTGF (Cell Signaling, Cat#86641, 1:1,000 for WB) and anti-β-actin (Sigma, Cat#A5316, 1:2000 for WB).
Bioinformatic Analysis Of Clinical Data
HCC data were obtained from The Cancer Genome Atlas (TCGA). The expression of SETD1A was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/)[16]. The survival of HCC patients was analyzed by KM plotter: Kaplan-Meier Plotter (http://kmplot.com/analysis/)[17].
Cell Culture
Normal human hepatic cell line LO2, human hepatocellular carcinoma cell lines SMMC-7721, SK-HEP1-1, HLE, HepG2 and Hep3B, and HEK293T were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) medium containing 10% fetal bovine serum (FBS, Gibco, Cat#10099141) and 1% penicillin-streptomycin (Gibco, Cat#15140122) at 37 °C with 5% CO2.
Western Blot
Western blot assay was used to measure protein levels. Briefly, after treatment, cells were lysed in RIPA buffer with Protease Inhibitor Cocktail. The concentration of total proteins was measured by the BCA assay (Pierce, Rockford, IL, USA), and 20 µg proteins of each sample were separated by SDS-PAGE gel. Proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA) and probed with primary antibodies followed by incubation with an HRP conjugated secondary antibody. The primary and second antibody complexes were determined with the ECL Western blot kit (Pierce).
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNAs were isolated by using TRIzol (Invitrogen) and Reverse transcription was performed with 2 µg of total RNA using by gDNA Erase and PrimeScript RT reagent kits (TAKARA Biotechnology, Dalian, China) following with the manufacturer’s instructions, respectively. SYBR Green PCR master mix was employed for mRNA quantification. GAPDH was used as a control gene. The primer sequences are as follows: SETD1A: forward, 5’-TTGCCATGTCAGGTCCAAAAA-3’, reverse, 5’-CGTACTTACGGCACATATCCTTC-3’; CYR61: forward, 5’- GATCTGCAGAGCTCAGTCAG-3’, reverse, 5’-GCACTGCCCGGTAACTTTGA-3’; CTGF: forward, 5’-TGCCCTCGCGGCTTACCGAC-3’, reverse, 5’- TGCAGGAGGCGTTGTCATTG-3’; β-actin: forward, 5’-AGCGAGCATCCCCCAAAGTT-3’, reverse, 5’-GGGCACGAAGGCTCATCATT-3’.
Establishment Of Stable SETD1A-knockdown Cell Lines
Two special SETD1A short hairpin RNAs (shRNAs) were generated by inserting human SETD1A specific targeting sequences shSETD1A-1, 5’-GGAAAGAGCCATCGGAAATTT-3’; shSETD1A-2, 5’-GACAACAACGAATGAAATATT-3’ into pll3.7 puro vector plasmid. HEK293T cells were transfected with lentivirus constructs using PolyJet transfection reagent (SignaGen Laboratories, Ljamsville, MD, USA) following the manufacturer’s instructions. Lentiviral supernatants were harvested during the 48–72-h after transfection and centrifuged at 2,500 rpm for 30 min to remove contaminating cells. SMMC-7721 and MLE were transfected with the viral supernatant in the presence of 10 µg/ml of polybrene (Sigma-Aldrich). Puromycin (1 µg/ml) was used to select stable SETD1A-knockdown cell lines. The knockdown efficiency was confirmed by Western blot.
CCK-8 (cell Counting Kit-8) Assay
CCK-8 assays were used to detect cell growth. A total of 2000 cells of each well were plated into 96-well plates. After treatment, CCK-8 (10 µl) was added into each well. The plates were incubated at 37 °C for 1 h. OD values at 450 nm wavelength of 450 nm were measured.
Clone Formation Assay
Control and SETD1A knockdown SMMC-7721 and HLE cells (500 cells per well) were plated onto 6-well plates overnight and then treated with sorafenib (10 µM) for another 2 weeks. The culture medium was changed every 3 days and sorafenib treatment was maintained. Cells were fixed and stained with 0.1% crystal violet.
Cell Death Assay
Control and SETD1A knockdown SMMC-7721 and HLE cells were plated onto 6-well plates overnight and then treated with sorafenib (10 µM) for 24 h. Cell death was determined by PI and Trypan Blue staining. For PI staining, cells were harvested and resuspended in PBS with PI at 1 µg/ml. Cell death was performed by flow cytometry. For Trypan Blue staining, 0.9 ml of cells were mixed together with 0.1 ml of 0.4% Trypan Blue and maintained for 5 min at room temperature. Cells were counted.
Statistical analysis
Data were shown as mean ± SD of three or more independent experiments, and analyzed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Statistical differences were analyzed by student t-test, two-way ANOVA and Pearson r, p < 0.05 was considered statistically significant.