The human LC cells, HepG2, Huh-6 and Huh-7, were purchased from the American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.) and maintained in a 5% CO₂ incubator at 37°C.

To generate CDDP-resistant LC cells, cells were treated with increasing concentrations of CDDP (Sigma-Aldrich; Merck KGaA) over 6 months, with a final concentration of 1 µM, as reported previously (17,18). The resistant cells were named HepG2/CDDP, Huh6/CDDP and Huh7/CDDP, respectively.

Cells were plated and cultured in 96-well plates in 100 µl medium at a density of 1×10³ cells/well. Following treatment with increasing concentrations (0, 0.5, 1, 5, 10, 20 and 50 µM) of CDDP for 48 h at room temperature, 10 µl Cell Counting Kit-8 (Abmole Bioscience Inc.) reagent was added to each well and incubated at 37°C for 2 h. In order to evaluate the effect of YAP, HepG2/CDDP and Huh-7/CDDP cells were pre-treated with or without 4 µM verteporfin (VP; Sigma-Aldrich; Merck KGaA; cat. no. SML0534) for 90 min at room temperature and then further treated with increasing concentrations of CDDP (0, 0.5, 1, 5, 10, 20 and 50 µM) for 48 h at room temperature. In order to investigate whether IL-6 and TGF-β were involved in YAP-regulated chemoresistance of LC cells, HepG2/CDDP cells were pre-treated with 100 ng/ml anti-IL-6 (cat. no. MAB206-SP; R&D Systems, Inc.) or anti-TGF-β (cat. no. BE0057; Bio X Cell) for 2 h at room temperature and then further treated with increasing concentrations of CDDP (0, 0.5, 1, 5, 10, 20 and 50 µM) for 48 h at room temperature. Additionally, HepG2/CDDP or Huh-7/CDDP cells were pre-treated with VP (4 µM) combined with recombinant (r)IL-6 (100 ng/ml; cat. no. 206-IL-010/CF; R&D Systems, Inc.) or rTGF-β (100 ng/ml; cat. no. 240-B-002/CF; R&D Systems, Inc.) for 2 h at room temperature, and then further treated with increasing concentrations of CDDP (0–20 µM) for 48 h at room temperature. The absorbance was measured at 450 nm using a microplate reader (ENSIGHT; PerkinElmer, Inc.) according to the manufacturer's protocol. The cell viability was calculated as the percentage of the viability of untreated control cells. Experiments were repeated ≥3 times.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and treated with DNase I (Promega Corporation) to remove the DNA contamination. RNA (1 µg) was reverse transcribed into cDNA using the cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Ltd.) according to the manufacturer's protocol. qPCR was subsequently performed using the SYBR Premix Ex Taq II kit (Takara Biotechnology Co., Ltd.) and a Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc.). The following primer sequences were used: YAP forward, 5′-GGCATACACCTACTCAACTACGG-3′ and reverse, 5′-TGGGCGGTGTAGAATCAGAGTC-3′; precursor-YAP forward, 5′-CCGGCTTGCTCTTATCAAAC-3′ and reverse, 5′-GTCATCGCTTCCCAAACATT-3′; IL-6 forward, 5′-ACTCACCTCTTCAGAACGAATTG-3′ and reverse, 5′-CCATCTTTGGAAGGTTCAGGTTG-3′; IL-10 forward, 5′-TCTCCGAGATGCCTTCAGCAGA-3′ and reverse, 5′-TCAGACAAGGCTTGGCAACCCA-3′; IL-12 forward, 5′-TGCCTTCACCACTCCCAAAACC-3′ and reverse, 5′-CAATCTCTTCAGAAGTGCAAGGG-3′; TNF-α forward, 5′-CTCTTCTGCCTGCTGCACTTTG-3′ and reverse, 5′-ATGGGCTACAGGCTTGTCACTC-3′; TGF-β forward, 5′-TACCTGAACCCGTGTTGCTCTC-3′ and reverse, 5′-GTTGCTGAGGTATCGCCAGGAA-3′; MALAT1 forward, 5′-AAAGCAAGGTCTCCCCACAAG-3′ and reverse, 5′-GGTCTGTGCTAGATCAAAAGGCA-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG 3′. The PCR cycling conditions were 15 min at 95°C, followed by 40 cycles for 10 sec at 95°C, 30 sec at 60°C and 1 sec at 72°C, and 1 cycle of cooling for 30 sec at 50°C.

To analyze the expression levels of miRNAs, the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to generate cDNA according to the manufacturer's protocol. The thermocycling conditions included an initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec. The forward primer is the exact sequence of the mature miRNA (http://www.mirbase.org/search.shtml). The forward primer for U6 was 5′-TGCGGGTGCTCGCTTCGCAGC-3′. The reverse primer was supplied by the aforementioned kit. GAPDH and U6 were used as the internal reference genes for the normalization of mRNA and miRNA, respectively. The gene expression levels were quantified using the 2^−ΔΔCq method (19). Each sample was analyzed in triplicate.

The cytoplasmic and nuclear fractions of cells were prepared using the PARIS™ kit (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The protein expression levels within the cytoplasmic and nuclear fractions were analyzed by western blotting. Aliquots of cytoplasmic and nuclear fractions were also subjected to RNA isolation and RT-qPCR, as aforementioned, to analyze the subcellular localization of YAP mRNA. Transcripts of the housekeeping gene GAPDH were used for normalization, while nuclear MALAT1 RNA was selected as endogenous control for the nuclear RNA.

Total protein was extracted from cells using 1X RIPA lysis buffer (50 mM Tris HCl, 150 mM NaCl and 1 mM EDTA) containing a protease inhibitor cocktail (Roche Diagnostics). Total protein was quantified using a bicinchoninic acid assay kit and 20 µg protein/lane was separated by 10% SDS-PAGE. The separated proteins were subsequently transferred onto a nitrocellulose membrane (EMD Millipore) using a wet transfer apparatus. The membranes were blocked with 5% skimmed milk at room temperature for 2 h. Following the incubation with the primary antibodies at 4°C overnight, the membranes were further incubated with the HRP-conjugated secondary antibody (cat. no. ab7090; Abcam; 1:10,000) diluted in 5% skimmed milk. Protein bands were then visualized in a gel imaging system (MG8600; Bio-Rad Laboratories, Inc.). The following primary antibodies (1:1,000; Abcam) were used: Anti-H2A.X (cat. no. ab229914), anti-YAP (cat. no. ab56701), anti-TAZ (cat. no. ab84927), anti-calpain (cat. no. ab39170) and anti-GAPDH (cat. no. ab229914). GAPDH was used as the loading control for normalization. The gray values were analyzed using ImageJ software (version 1.46; National Institutes of Health).

The small interfering RNA (siRNA/si) negative control (si-NC; 5′-GCACAACAAGCCGAAUACA-3′), si-YAP (siYAP-1, 5′-GCGUAGCCAGUUACCAACA-3′; siYAP-2, 5′-CAGUGGCACCUAUCACUCU-3′), miRNA control (miR, 5′-UUCUCCGAACGUGUCACGUTT-3′) and miR-375 mimics (5′-UUUGUUCGUUCGGCUCGCGUGA-3′) were synthesized by Shanghai GenePharma Co., Ltd.. Upon cells reaching 50–60% confluence, the transfection was performed using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions with 20 µM of each construct or siRNA. After transfection for 6 h at 37°C, the medium was replaced with fresh complete medium. To investigate the effect of YAP on chemosensitivity, HepG2/CDDP, Huh-6/CDDP and Huh-7/CDDP cells were transfected with si-NC or si-YAP-1 for 12 h and then further treated with increasing concentrations of CDDP (0, 0.5, 1, 5, 10, 20 and 50 µM) for 48 h.

To determine the mRNA stability, cells were treated with 5 µg/ml actinomycin D (Act-D; cat. no. A9415; Sigma-Aldrich; Merck KGaA) at 37°C for 0, 2, 4 or 8 h. Subsequently, total RNA was collected and the target mRNA was analyzed using RT-qPCR, as aforementioned. For the protein stability assay, cells were incubated with 100 µg/ml cycloheximide (CHX) at 37°C for 0, 2, 6 or 12 h and then protein expression was analyzed using western blotting, as aforementioned.

Cells cultured on coverslips were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After blocking with 3% BSA in PBS containing 0.3% Triton X-100 solution at 37°C for 1 h, cells were incubated with a primary antibody against YAP (cat. no. ab56701; 1:1,000; Abcam) overnight at 4°C and then treated with an anti-Alexa Fluor 594 secondary antibody (1:200; R&D Systems China Co., Ltd.; cat. no. IC1420T) for 1 h at room temperature. Then, DAPI solution (5 µg/ml) was added to stain the cell nuclei for 5 min at room temperature. The fluorescence signal was observed under a confocal microscope (TCS-SP5; Leica Microsystems GmbH; magnification, ×10).

Statistical analysis was performed using SPSS 17.0 software (SPSS Inc.) and presented as the mean ± SD. The comparisons between two groups were analyzed using an unpaired Student's t-test. All experiments were performed ≥3 times independently. P<0.05 was considered to indicate a statistically significant difference.

The CDDP sensitivity of both resistant and parental LC cells was investigated. The results revealed that the established CDDP-resistant cells were more resistant to CDDP treatment compared with their corresponding parental cells (Fig. 1). The IC₅₀ values of CDDP for HepG2/CDDP and HepG2 cells were 22.8 and 3.45 µM, respectively (Fig. 1A), those for Huh-6/CDDP and Huh-6 cells were 30.6 and 5.05 µM, respectively (Fig. 1B), while the IC₅₀ values of CDDP for Huh-7/CDDP and Huh-7 cells were 30.5 and 6.51 µM, respectively (Fig. 1C). The current data confirmed the successful establishment of LC/CDDP cells.

It has been previously reported that the Hippo signaling pathway regulates the progression of LC (20). Thus, the present study analyzed the expression levels of YAP and the transcriptional coactivator with PDZ-binding motif (TAZ), another important member of the Hippo signaling pathway (20), in both parental and CDDP-resistant LC cells. The protein expression levels of YAP, but not TAZ, were significantly upregulated in the HepG2/CDDP, Huh-6/CDDP and Huh-7/CDDP cells compared with in their corresponding parental cells (Fig. 2A). Furthermore, RT-qPCR analysis revealed that the mRNA expression levels of YAP were significantly upregulated in the CDDP-resistant LC cells compared with in their respective parental cells (Fig. 2B). In addition, the amount of YAP localized in both the cytosol and nucleus was increased in HepG2/CDDP cells compared with in HepG2 cells (Fig. 2C), which was confirmed by immunofluorescence staining (Fig. 2D).

To investigate whether YAP was involved in the resistance to CDDP in LC cells, the CDDP-resistant LC cells were transfected with si-YAP-1 and si-YAP-2 (Fig. 3A). si-YAP-1 was used for subsequent experiments since it displayed increased efficiency. The results revealed that si-YAP-1 markedly increased the CDDP sensitivity of HepG2/CDDP (Fig. 3B), Huh-6/CDDP (Fig. 3C) and Huh-7/CDDP (Fig. 3D) cells. Since the results revealed that YAP expression was markedly increased in HepG2/CDDP and Huh-7/CDDP cells, these cell lines were further treated with VP, a suppressor of the YAP-TEAD complex (21). VP increased the sensitivity of CDDP in HepG2/CDDP (Fig. 3E) and Huh-7/CDDP (Fig. 3F) cells.

It has been previously reported that YAP regulates the expression levels of various cytokines to regulate cancer progression (12–14). In the present study, an array of cytokines was analyzed, including IL-6, IL-10, IL-12, TNF-α and TGF-β, in si-YAP-1-transfected LC/CDDP cells. si-YAP-1 significantly downregulated the expression levels of IL-6 and TGF-β in both HepG2/CDDP (Fig. 4A) and Huh-7/CDDP (Fig. 4B) cells. In addition, VP treatment significantly downregulated the expression levels of IL-6 and TGF-β in both HepG2/CDDP (Fig. 4C) and Huh-7/CDDP (Fig. 4D) cells. On the other hand, the expression levels of IL-6 and TGF-β in both HepG2/CDDP (Fig. 4E) and Huh-7/CDDP (Fig. 4F) cells were significantly upregulated compared with in their corresponding control cells. The current results suggested that YAP may regulate the expression levels of IL-6 and TGF-β in LC/CDDP cells.

The current study further analyzed whether IL-6 and TGF-β were involved in the YAP-mediated chemoresistance of LC cells. The data demonstrated that neutralization antibodies anti-IL-6 (Fig. 5A) and anti-TGF-β (Fig. 5B) significantly increased the CDDP sensitivity of HepG2/CDDP cells. In addition, rIL-6 (Fig. 5C) and rTGF-β (Fig. 5D) significantly attenuated the VP-induced CDDP sensitivity of HepG2/CDDP cells. All these data indicated that IL-6 and TGF-β may be involved in the YAP-mediated chemoresistance of LC cells.

The potential mechanisms responsible for the upregulation of YAP expression in LC/CDDP cells were subsequently investigated. The protein stability of YAP in HepG2 and HepG2/CDDP cells following CHX treatment was similar to each other (Fig. 6A). Additionally, the expression levels of the precursor mRNA of YAP, analyzed by RT-qPCR, were not significantly different between HepG2 and HepG2/CDDP cells or between Huh-7 and Huh-7/CDDP cells (Fig. 6B). In addition, the nuclear turnover rate of YAP was not significantly different between HepG2 and HepG2/CDDP cells, as analyzed by RT-qPCR (Fig. 6C). However, the data revealed that the mRNA stability of YAP in HepG2/CDDP cells following Act-D treatment was markedly increased compared with in HepG2 cells (Fig. 6D). Consistently, the mRNA stability of YAP in Huh-7/CDDP cells was also increased compared with in Huh-7 cells (Fig. 6E). These results indicated that increased mRNA stability may be responsible for the upregulation of YAP expression in LC/CDDP cells.

miRNAs can decrease mRNA stability via binding to the 3′-untranslated regions of mRNA (22). It has been revealed that miR-375 (23), miR-506 (24), miR-132 (25) and miR-129 (26) directly target YAP mRNA to downregulate its expression. Thus, the expression levels of these miRNAs in both LC/CDDP and LC cells were subsequently analyzed. The data revealed that, among all miRNAs, only the expression levels of miR-375 were significantly downregulated in both HepG2/CDDP (Fig. 7A) and Huh-7/CDDP (Fig. 7B) cells. Furthermore, the overexpression of miR-375 (Fig. 7C) using miR-375 mimics significantly downregulated the mRNA expression levels of YAP in both HepG2/CDDP and Huh-7/CDDP cells (Fig. 7D). This was due to the fact that miR-375 decreased the mRNA stability of YAP (Fig. 7E).