Yes-associated protein (YAP) serves an essential role in tumorigenesis. However, the potential role and the molecular mechanism underlying the effect of YAP on hepatocellular carcinoma (HCC) cells have not been elucidated. In the current study, it was revealed that YAP expression was increased significantly in HCC cancer tissues and its overexpression was associated with tumor differentiation. The silencing of YAP by small interferring RNA led to the inhibition of HCC cell growth, which was associated with the promotion of apoptosis. The silencing of YAP also decreased the invasive potential of HCC cells and the activity of the phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway. Furthermore, silencing of YAP increased the chemosensitivity of HCC cells to cisplatin (CDDP) through inactivation of the PI3K/AKT signaling pathway.
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-associated mortality worldwide (
Yes-associated protein (YAP), a key component of the Hippo signaling pathway, serves roles in development, growth, repair and homeostasis (
In the present study, the effects of YAP expression manipulation on proliferation, invasion, cisplatin (CDDP) resistance were investigated, and its mechanism in HCC cells was explored. It was demonstrated that YAP overexpression is associated with the tumor differentiation in HCC. Downregulation of YAP using small interfering (si)RNA inhibited the proliferation and invasion of HCC cells, and resulted in a significant decrease in the activity of the phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway. Furthermore, the knockdown of YAP increased the sensitivity of HCC cells to CDDP via inhibition of the PI3K/AKT signaling pathway. In a patient-derived tumor xenograft (PDTX) model, the knockdown of YAP inhibited the growth of HCC, and also increased the anti-tumor activity of CDDP. Therefore, the findings of the present study suggest a promoter role for YAP on HCC. Inhibition of YAP, alone or in combination with traditional chemotherapy, may effectively combat HCC.
The present study was approved and supervised by the Research Ethics Committee of Jiaxing University College of Medicine (Jiaxing, China). Written informed consent was obtained from all patients. Paired liver cancer and adjacent normal liver tissue were obtained between September 2015 and December 2017 from 80 patients who underwent primary surgical resection of liver cancer at The Second Affiliated Hospital of Jiaxing University College of Medicine. The patients included 36 women and 44 men, aged 36–81 years; 32 patients had WHO grade T1 disease, 28 patients had WHO grade T2 disease, and 16 patients had WHO grade T3, 4 patients had WHO grade T4 disease (
SMMC-7721 cells (derived from liver tissues of a male patient with HCC) and THLE-3 cells (a normal liver cell line) were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), where the cell line was tested and authenticated. These procedures include cross-species checks, DNA authentication and quarantine. Cells were placed in culture for <6 months and authenticated at The Second Affiliated Hospital of Jiaxing Medical College using morphology and growth rate assays. SMMC-7721 cells were maintained in Dulbecco's modified Eagle medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated at 37°C in a 5% CO2 atmosphere. THLE-3 cells were cultured in BEGM medium kit (Lonza/Clonetics Corporation, Walkersville) and incubated at 37°C in a 5% CO2 atmosphere.
For immunohistochemistry, paraffin-embedded sections of 5-µm thickness were deparaffinised in 100% xylene for 20 min at room temperature, and rehydrated in graded ethyl alcohol (100, 90, 70 and 50% ethyl alcohol) for 10 min at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in 100% methanol for 10 min at room temperature. Antigen retrieval was performed in sodium citrate buffer for 2 h at room temperature. The rabbit polyclonal antibody directed against YAP (cat. no. ab39361; 1:100 dilution; Abcam, Cambridge, MA, USA) was added to sections and incubated at 4°C overnight. A horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (cat. no. ab6721, 1:1,000 dilution, Abcam) was used according to the manufacturer's protocol. The slides were then incubated with DAB (Beyotime Institute of Biotechnology, Haimen, China) for 5 min at room temperature, and then YAP expression was visualised, followed by 0.5% hematoxylin (Beyotime Institute of Biotechnology) counterstaining for 10 min at room temperature. PBS was used as a negative control. The images were captured using a camera connected to an Olympus BH2 light microscope (Olympus Corporation, Tokyo, Japan) at magnifications, ×100 and ×200. Staining results were assessed independently by two pathologists blinded to the clinical data of patients. The intensity of YAP protein staining for each slide was scored and accessed according to the criterion based on the intensity of staining: 0, negative; 1+, low; 2+, medium; 3+, high. The percentage of stained cells was calculated as: 0, 0% stained; 1+, 1–10% stained; 2+, 11–49% stained; 3+, 50–100% stained. YAP IHC data were analyzed using X-tile 1.9 software program (The Rimm Lab at Yale University, New Haven, CT, USA;
Total RNA was extracted from cells or tumor samples using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), and 2 µg of total RNA from each sample was used for cDNA synthesis using a cDNA synthesis kit (Tiangen Biotech Co., Ltd., Beijing, China). The qPCR analysis was performed using PowerUp™ SYBR®Green Master Mix (cat. no. A25741; Thermo Fisher Scientific, Inc.) containing Dual-Lock Taq DNA Polymerase, according to the manufacturer's protocol. Data collection was conducted using an ABI 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 50°C for 2 min and 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The conditions for melt curve analysis were 95°C for 15 sec, 60°C for 1 min and 95°C for 15 min. PCR amplification was conducted using the following primers: YAP forward, 5′-CCCTCGTTTTGCCATGAACC-3′ and reverse, 5′-ATCTGTTGCTGCTGGTTGGA-3′; β-actin forward, 5′-GATGAGATTGGCATGGCTTT-3′ and reverse, 5′-GTCACCTTCACCGTTCCAGT-3′. The 2−ΔΔCq method (
Whole-cell extracts from cultured cells or tissues were prepared with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) and subjected to western blotting. Protein concentration was analyzed using the Pierce™ BCA Protein Assay kit (cat. no. 2322; Invitrogen; Thermo Fisher Scientific, Inc.). Total protein (40 µg/lane) was separated by 12% SDS-PAGE and transferred into polyvinylidene fluoride membranse (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with 5% skimmed milk powder for 1 h at room temperature. The membranes were then incubated with primary antibodies overnight at 4°C. All primary antibodies were purchased from Abcam (Cambridge, UK) as follows: Rabbit anti-YAP1 antibody (cat. no. ab39361; dilution, 1:1,000), mouse anti-β-actin antibody (cat. no. ab8226; dilution, 1:10,000), rabbit anti-pan-AKT antibody (cat. no. ab8805; dilution, 1:500), Rb anti-pan-AKT (phospho T308) antibody (cat. no. ab38449; dilution, 1:800), rabbit anti-p21 (cat. no. ab109199; dilution, 1:5,000), rabbit anti-Bax (cat. no. ab32503; dilution, 1:1,000), rabbit anti-active-Caspase-3 (cat. no. ab2302; dilution, 1:200) and rabbit anti-c-myc (cat. no. ab32072, dilution, 1:10,000). The membranes were then washed three times with PBS-Tween-20 for 10 min and then incubated with the HRP-conjugated goat anti-mouse IgG H&L (cat. no. ab6789; dilution, 1:5,000; Abcam) or goat anti-rabbit IgG H& L (HRP) (cat. no. ab6721; dilution, 1:10,000; Abcam) secondary antibodies for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence Western Blotting Detection system (Thermo Fisher Scientific, Inc.) and X-ray film. The band density was quantified using Image J v1.48 u software (National Institutes of Health, Bethesda, MD, USA).
pcDNA3.1 vector containing wild type YAP insert was provided by General Biosystems, Inc. (Morrisville, NC, USA). siRNA targeted against YAP (Shanghai GenePharma Co., Ltd., Shanghai, China) with the sequence, 5′-GGUGAUACUAUCAACCAAAdTdT-3′ was used to knock down YAP expression and the sequence of negative control siRNA was 5′-CAGUACUUUUGUGUAGUACAAdTdT-3′. Cells (5×105 cells/well) were cultured in 6-well plates until 60% confluent, and then transfected with plasmids (4 µg/well) or siRNAs (20 nM/well) using Lipofectamine 2000® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. LY294002 (Selleck Chemicals, Houston, TX, USA), dissolved in 1% DMSO, was added 24 h following transfection and used at 10 µM.
The invasion chambers with 0.8-mm pore size (BD Biosciences, Franklin Lakes, NJ, USA) was coated with Matrigel (cat. no. 354234; 1:3 dilution; BD Biosciences) at 37°C for 30 min.. A total of ~1×105 cells suspended in DMEM medium with 2% FBS were added to the upper chamber, and the medium containing 20% FBS was added to the lower chamber. After 24 h of incubation at 37°C in 5% CO2, cells were fixed with 100% ice-cold methanol for 30 min at room temperature, and stained with 5% crystal violet for 15 min at room temperature. Then, cells on the upper surface of the inner chamber were removed with cotton swabs. Invaded cells that adhered to the lower surface of the membrane were viewed through an optical microscope (TS100F, Nikon, Japan) at magnification, ×100, and counted using Image J v1.48u software.
An MTT assay was performed to measure cell viability. Briefly, cells (5×103 cells/well) were cultured in 96-well plates for 24 h, transfected with YAP siRNA or YAP expression plasmids, and cell viability was determined 48 h later. The siRNA-trasfected HCC cells were exposed to CDDP (cat. no. S1166, Selleck Chemicals, Houston, TX, USA) at 0, 1, 10, 20, 40 or 80 µM for 48 h or treated with 10 µM LY294002 for 12 h prior to being treated with CDDP. To determine the cell viability, MTT solution (5 mg/ml in PBS) was added to each well and incubated at 37°C for 4 h. The media was removed and 150 µl dimethyl sulfoxide was added to each well. The absorbance of each well was measured at 490 nm using an automated microplate reader.
Cell apoptosis was evaluated by flow cytometry using an Annexin-V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences) according to the manufacturer's protocol. Briefly, the cells were harvested at 160 × g for 5 min at room temperature and washed twice in PBS and resuspended in 500 µl of binding buffer. A volume of 5 µl of Annexin-V-FITC and 5 µl of propidium iodide was added and agitated gently, and the cells were stained in the dark for 15 min at room temperature. The cells were analyzed immediately by flow cytometry and analyzed using FlowJo 10.0 (Tree Star., Inc., Ashland, OR, USA).
Untreated cells, or cells transfected with plasmid or siRNA were washed thoroughly with medium and cultured in fresh DMEM containing 10% FBS and 10 µM BrdU (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at 37°C. Cells were then harvested at 160 × g for 5 min at room temperature or left to grow in BrdU-free medium 24 h prior to harvest. Cell pellets were washed with PBS, fixed in 70% ice-cold ethanol for 20 min at room temperature, and then were resuspended in 2N HCl and incubated for 30 min at room temperature. Following washing with PBS, cells were hybridized with a mouse monoclonal anti-BrdU antibody (Abcam, cat. no. ab8152) diluted at a ratio 1:100 in PBST (PBS containing 0.1% BSA and 0.2% Tween-20, pH 7.4) and incubated overnight at 4°C. Cells were then washed with PBST and incubated with FITC-conjugated goat anti-mouse immunoglobulin antibody (cat. no. 715-545-155; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) diluted at a ratio 1:400 in PBST for 2 h at room temperature in the dark. Cells were then washed with PBS and stained with DAPI solution for 10 min at room temperature prior to capturing images under a light microscope (BX43, Olympus, Japan).
To produce the liver PDTX model, 6–8 week old male nu/nu mice (Laboratory Animal Center of Jiaxing University) weighing 18–20 g were used. All mice experiments were performed in accordance with the guidelines and approved by the Institutional Animal Care and Use Committee of Jiaxing University. Fresh surgical tumor tissues (F0) were collected immediately following surgery and cut into 2–3 mm3-sized pieces in Penicillin-Streptomycin Solution (Beyotime Institute of Biotechnology)-containing DMEM. Tumor fragments were implanted into the right armpit of mice. When the tumor size reached 100–200 mm3, the samples (F1) were subsequently divided into pieces for passaging
The four groups were injected intravenously into the tail once a week with stroke-physiology saline solution, CDDP (5 mg/kg), YAP-shRNA lentivector (5 million infection units per 100 µl for animal injection; Obio Technology Corp, Ltd., Shanghai, China) respectively or CDDP in combination with YAP-shRNA lentivector. Tumor diameters were serially measured with a digital caliper every 5 days, and tumor volumes were calculated using the following formula: (L × W × W)/2, whereby; V, volume; L, length; and W, width. On day 25, mice were sacrificed and tumor tissues were collected.
Statistical analysis was performed using SPSS 17.0 software (IBM Corp., Armonk, NY, USA). Statistical analysis was performed using one-way analysis of variance followed Tukey's multiple comparisons test. Results are expressed as the mean ± standard deiviation. P<0.05 was considered to indicate a statistically significant difference.
To understand the role of YAP in HCC, the expression of YAP was examined in HCC tissues and adjacent normal tissues. The results of western blotting and RT-qPCR revealed that YAP expression in HCC tissues was significantly higher compared with peri-tumor tissues at the mRNA and protein levels (
In order to determine if YAP serves a functional role in HCC cell behavior
To investigate whether YAP affects the invasive capabilities of HCC cells, Matrigel invasion assays were performed. It was demonstrated that downregulation of YAP in SMMC-7721 cells significantly decreased cell invasion rates by 41.3% compared with control cells (
It has been well documented that the PI3K/AKT pathway is essential for the proliferation and invasion of HCC cells (
Accumulating evidence has indicated that YAP is involved in drug resistance in a variety of cancer cells (
A liver PDTX model was used to investigate the function of YAP
YAP has been proven to be upregulated in various types of cancer, including HCC (
In the present study, it was demonstrated that siRNA targeting of YAP in SMMC-7721 cells led to the efficient and specific inhibition of endogenous YAP mRNA and protein
Previous studies revealed that YAP is involved in the activation of numerous signaling pathways, including mitogen activated protein kinase (MAPK)-extracellular signal-regulated kinase signaling pathway (
A number of recent studies have demonstrated that the increased level of YAP was revealed to be associated with increased drug resistance in multiple lines of cancer cells (
In conclusion, the present study demonstrated that overexpression of YAP in HCC is associated with the tumor differentiation. Knockdown of YAP by siRNA
Not applicable.
The present study was supported by a project grant from the Technology Department of Jiaxing (grant no. 2014AY21031-1).
All data generated or analyzed during this study are included in this published article.
XGW was a major contributor in writing the manuscript. XGW and ZXZ collected and analyzed the experimental data. XGW and BW performed the histological examination of HCC tissues and performed the experiments of HCC cells
This study was approved and supervised by the Research Ethics Committee of Jiaxing University College of Medicine (Jiaxing, China). Written informed consent was obtained from all patients.
The patient, or parent, guardian or next of kin (in case of deceased patients) provided written informed consent for the publication of any associated data and accompanying images.
The authors declare that they have no competing interests.
Expression of YAP is increased in HCC. (A) Representative western blot analysis of YAP protein in HCC (T1-T4) and paired normal tissues (N1-N4) from 4 patients. The expression of β-actin used as a loading control to normalize the YAP protein levels in each sample. (B) Determination of YAP mRNA level in HCC tissues and paired normal tissues by reverse transcription-quantitative polymerase chain reaction. **P<0.01. (C) Expression of YAP was analyzed by immunohistochemistry in HCC of different differentiation degrees using an anti-YAP antibody. Scale bars of the left-hand panels, 200 µm; scale bars of the right-hand panels, 100 µm. YAP, Yes-associated protein; HCC, hepatocellular carcinoma.
YAP knockdown inhibits hepatocellular carcinoma cell proliferation. (A) YAP expression is determined in a normal liver cell line and a HCC cell line by western blot analysis. (B) YAP expression in SMMC-7721 cells following transfection with control siRNA (control), YAP siRNA (siYAP) or YAP siRNA and YAP cDNA (Rescue) was measured by western blot analysis. β-actin served as a loading control. (C and D) The effect of YAP siRNA on cell growth. SMMC-7721 cells transiently transfected with control siRNA, YAP siRNA or YAP siRNA and YAP cDNA were cultured for 72 h. (C) Cell viability was measured using an MTT assay and (D) cell proliferation was measured with a BrdU assay. Scale bar, 75 µm. (E) Flow cytometry analysis of SMMC-7721 cells. (F) Western blot analysis of c-myc, p21, caspase 3 and Bax protein expression levels in transfected cells. *P<0.05; **P<0.01. YAP, Yes-associated protein; siRNA/si, small interferring RNA; siYAP, siRNA against YAP; Bax, BCL2 associated X apoptosis regulator.
YAP knockdown inhibits hepatocellular carcinoma cell invasion. SMMC-7721 cells transfected with YAP siRNA or rescued with YAP cDNA as indicated were subjected to a Transwell invasion assay. The number of cells invading through the Matrigel are presented. Magnification, ×100. **P<0.01. YAP, Yes-associated protein; CDDP, cisplatin; siRNA/si, small interferring RNA; siYAP, siRNA against YAP.
Knockdown of YAP enhances sensitivity of hepatocellular carcinoma cells to CDDP through modulating phosphoinositide 3-kinase/AKT signaling. (A) SMMC-7721 cells were transfected with control siRNA or siRNA against YAP. Then, 48 h later proteins were extracted and subjected to western blot analysis using antibodies against AKT and p-AKT. (B) Untransfected and siRNA-transfected cells were seeded at 4×103 cells/well in 96-well plates and treated with different concentrations of CDPP for 48 h. Then, cell viability was determined using an MTT assay. (C) Western blot analysis of AKT and p-AKT in SMMC-7721 cells transfected with control siRNA or siYAP following treatment with CDDP for 48 h. (D) SMMC-7721 cells transfected with control siRNA or siYAP were treated with 10 µM LY294002 for 12 h prior to being treated with CDDP. Then, cell viability was determined using an MTT assay. *P<0.05; **P<0.01. YAP, Yes-associated protein; CDDP, cisplatin; siRNA/si, small interferring RNA; AKT, AKT serine/threonine kinase; p-, phosphorylated; siCtr, control siRNA; siYAP, siRNA against YAP; Untrans, untransfected.
Inhibitory effect of YAP knockdown on