Human HCC cell line SMMC-7721 and human 293T cells, purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin, and maintained in a humidified incubator with 5% CO₂ at 37°C.

The lentiviral vectors were prepared, and the lentiviruses encoding MOB2 (LV-MOB2) and control lentiviruses (LV-C) were generated and purified. Viral titers were determined by the Shanghai GeneChem Co., Ltd. (Shanghai, China). Following lentiviral infection, 1.0 µg/ml puromycin (cat. no. sc-205821; Santa Cruz Biotechnology, Inc., Dalla, TX, USA) was subsequently used to select stably transduced cell lines for two weeks. The cell lines that express a stable expression of control or MOB2 were established and screened by western blotting as previously described (8).

For clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated MOB2 gene knockout, the single-guide RNA (sgRNA) targeting MOB2 was generated using the online CRISPR Design Tool (http://crispr.mit.edu/), and the sgRNA-MOB2 sequence is 5′-AGAAGCCCGCTGCGGAGGAG-3′. The lentiCRISPRv2 vector (Addgene, Inc., Cambridge, MA, USA) harboring a puromycin resistance cassette was digested using BsmBI and ligated using annealing oligonucleotides (forward, 5′-CACCGAGAAGCCCGCTGCGGAGGAG-3′ and reverse, 5′-AAACCTCCTCCGCAGCGGGCTTCTC-3′). Once the sequence was verified by sequencing, the constructs were transfected into 293T cells, which were grown to 70–80% confluence in a 10 cm dish, using EndoFectin Lenti reagent (GeneCopoeia, Inc., Rockville, MD, USA) together with the lentiviral packaging vectors pSPAX2 and pCMV-VSV-G (all from Addgene, Inc.). After transfection for 48 h, the viral particles were harvested and purified, and 1.5×10⁶ SMMC-7721 cells were seeded in a 10 cm dish were infected with the indicated lentiviruses in the presence of polybrene (5 µg/ml; Shanghai GeneChem Co., Ltd.) for 14 h at 37°C. The infected SMMC-7721 cells were selected using puromycin 6 days following successful lentiviral transduction, followed by monoclonalization. The knockout of MOB2 expression was screened using western blotting. To construct the vector that express short hairpin RNA (shRNA) against human yes-associated protein (YAP) (shYAP), the primers: Forward, 5′-GATCCGCTGGTCAGAGATACTTCTTAATTCAAGAGATTAAGAAGTATCTCTGACCAGCTTTTTTA-3′ and reverse, 5′-CGCGTAAAAAAGCTGGTCAGAGATACTTCTTAATCTCTTGAATTAAGAAGTATCTCTGACCAGCG-3′ were synthesized and annealed, and followed by cloning into the BamH I and MluI sites of the pLent-U6-GFP-Puro vector (ViGene Biosciences Inc., Rockville, MD, USA), and the non-silencing control shRNA vector (shNC) was also generated. The MOB2 knockout SMMC-7721 cells were transfected with shYAP or with shNC for 72 h using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The knockdown of YAP expression was screened by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting.

In total, 5.0×10⁵ of the SMMC-7721 cells that overexpressed MOB2 (LV-MOB2), MOB2-knocked out cells (LV-sgMOB2) and the corresponding vector controls (LV-C and LV-sgC) were seeded onto 6-well culture plates and serum-starved overnight at 37°C. The cell monolayers were wounded by scratching with a sterile 200 µl plastic pipette tip and gently washed three times with phosphate-buffered saline (PBS) and captured under a phase-contrast microscope at ×100 magnification and marked as 0 h. The cells were further cultured with DMEM supplemented with 1% FBS at 37°C for 48 h, and wound closure was observed and captured under an inverted microscope at a magnification of ×100. The relative migration of cells was calculated. All experiments were performed in triplicate and repeated three times.

Transwell migration and invasion assays were performed using Boyden chambers (diameter, 6.5 mm; pore size, 8.0 µm; Corning Incorporated, Corning, NY, USA) as described previously (8). The migrated or invaded cells on the lower surface of the inserts were fixed with methanol for 15 min at room temperature, stained with 0.1% crystal violet for 20 min at room temperature and counted from six random fields using a phase-contrast microscope at a magnification of ×100 per insert from triplicate wells. A total of three separate experiments were performed.

Total RNA was isolated and purified from the SMMC-7721 cells that overexpressed MOB2 (LV-MOB2), MOB2-knocked out cells (LV-sgMOB2) and the corresponding vector controls (LV-C and LV-sgC) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was obtained using the HiScript First Strand cDNA Synthesis kit (Vazyme, Piscataway, NJ, USA) according to the manufacturer's protocol. qPCR was performed using the SYBR Green qPCR system (Takara Biotechnology, Co., Ltd., Dalian, China) according to the manufacturer's protocol, and the qPCR thermocycling conditions were 95°C for 5 min followed by 40 cycles of 95°C for 10 sec and 60°C for 34 sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. The primer sequences were as follows: Connective tissue growth factor (CTGF) forward, 5′-AGGAGTGGGTGTGTGACGA-3′ and reverse, 5′-CCAGGCAGTTGGCTCTAATC-3′; cysteine-rich angiogenic inducer 61 (CYR61) forward, 5′-AGCCTCGCATCCTATACAACC-3′ and reverse, 5′-TTCTTTCACAAGGCGGCACTC-3′; YAP forward, 5′-CTCGAACCCCAGATGACTTC-3′ and reverse, 5′-CCAGGAATGGCTTCAAGGTA-3′; and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The relative expression of the indicated mRNAs (normalized to GAPDH expression) was calculated using the 2^−ΔΔCq method as described (9). All experiments were performed in triplicate and repeated three times.

Protein extraction and subsequent western blotting were performed following standard methods as described previously (8). In brief, the cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with protease inhibitors for 30 min at 4°C, and proteins were quantified using the Bradford method, and approximately 40 µg cellular proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat dried milk in tris-buffered saline with Tween-20 for 2 h at room temperature, and incubated with primary antibodies at 4°C overnight followed by incubation for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. The bands were detected using the Pierce ECL Plus western blotting substrate (Thermo Fisher Scientific, Inc.). GAPDH served as the loading control, and quantification of the western blotting results was performed using ImageJ 2× software (National Institutes of Health, Bethesda, MD, USA). A total of three independent experiments were performed. The antibodies used were as follows: Rabbit polyclonal anti-MOB2 (cat. no. SAB1301138; 1:500; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); rabbit polyclonal anti-NDR1/2 (cat. no. sc-271703; 1:200; Santa Cruz Biotechnology, Inc., Dalla, TX, USA); rabbit monoclonal anti-YAP (cat. no. 14074; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-pS127YAP (cat. no. 13008; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-pS397YAP (cat. no. 13619; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-LATS1 (cat. no. 3477; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-pY1079LATS1 (cat. no. 8654; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-pS909 LATS1 (cat. no. 9157; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-MOB1 (cat. no. 13730; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-pY35 MOB1 (cat. no. 8699; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), HRP-conjugated anti-mouse IgG and HRP-linked anti-rabbit IgG (cat. no. 7076 and 7074, respectively; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA); mouse monoclonal anti-GAPDH (cat. no. KC-5G4; 1:1,000; KangChen Bio-tech Co., Ltd., Shanghai, China). The rabbit polyclonal antibody against NDR1/2 (pThr444/442) was generated as described previously (10,11). The peptides (KDWVFINYT(PO4)YKRFEG) were synthesized and conjugated to keyhole limpet hemocyanin in the laboratory. The rabbit injections and bleed collection were performed by DGpeptidesCo., Ltd (Hangzhou, China), and the antisera were purified and extensively characterized in the laboratory.

Immunoprecipitation was performed using the Pierce Classic IP kit (cat. no. 26146; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, the cells were washed with PBS three times and lysed at 4°C for 10 min in IP lysis/wash buffer (pH 7.4) containing 0.025M Tris, 0.15M NaCl, 0.001M EDTA, 1% NP-40 and 5% glycerol, and incubated for 20 min at 4°C. The samples were mixed periodically. Subsequent to centrifugation at 13,000 × g and 4°C for 15 min to pellet the cell debris, the protein concentration of the supernatant was determined using the Bradford method, and equivalent quantities of protein (800 µg) were pre-cleared using control agarose resin (component of the Pierce Classic IP kit). The pre-cleared cell lysates were incubated with the rabbit polyclonal anti-NDR1/2 (3 µg per sample) or rabbit monoclonal anti-LATS1 (dilution, 1:100) antibodies overnight at 4°C to form the immune complex followed by incubation at 4°C with 20 µl Protein A/G Plus Agarose beads (component of the Pierce Classic IP kit) for 1 h. Following four washes with the IP lysis/wash buffer, the beads were washed once with conditioning buffer and heated at 100°C for 10 min in Lane Marker Sample buffer (both buffers are components of the Pierce Classic IP kit) supplemented with dithiotheritol to a final concentration of 20 mM. The samples were then subjected to SDS-PAGE and western blotting as aforementioned.

All data were expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). The analyses were performed using unpaired Student's t-test or a one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The inhibitory effects of MOB2 on the motility of the HCC cell lines SMMC-7721 and HepG2 were previously reported, where migration and invasion were markedly suppressed in MOB2-overexpressing cells and cell motility was decreased in MOB2-knocked down cells (8). In the present study, the aim was to further confirm this role of MOB2 in the regulation of migration and invasion of HCC cells. MOB2-knockout SMMC-7721 cells were generated by infecting the cells with lentiCRISPRv2 viruses that encode the Cas9 nuclease and single-guide RNA (sgRNA) that targets MOB2. Gene knockout was confirmed by western blotting. As presented in Fig. 1A, the expression of MOB2 was undetectable in cells that stably express MOB2-targeted sgRNA (LV-sgMOB2) when compared with the blank vector-transduced cells (LV-sgC) and the normal control cells (NC). In addition, the efficiency of MOB2 overexpression was also confirmed in LV-MOB2-transduced SMMC-772 cells by western blotting (Fig. 1B). Therefore, SMMC-7721 cells that stably overexpress MOB2, SMMC-7721 MOB2 knockout cells and their corresponding blank vector-transduced cells (LV-sgC and LV-C) were successfully established in order to perform subsequent experiments.

Subsequently, wound-healing and Transwell assays were performed in order to evaluate the effect of the knockout or overexpression of MOB2 on the migration and invasion of SMMC-7721 cells. The results of the wound-healing assay revealed that MOB2 knockout cells were able to repair the wound areas significantly faster compared with LV-sgC-transduced cells while MOB2-overexpressing SMMC-7721 cells exhibited a decreased wound-healing capacity compared with LV-C-transduced cells (P<0.05; Fig. 2). The effect of MOB2 on the migratory and invasive capacities of SMMC-7721 cells was also assessed using Transwell assays. Compared with LV-sgC, the knockout of MOB2 significantly increased the number of cells that migrated and invaded, whereas opposite results were observed in MOB2-overexpressing cells when compared with LV-C (Fig. 2). Therefore, these results clearly demonstrate that silencing MOB2 significantly promoted cell migration and invasion. By contrast, the overexpression of MOB2 significantly decreased the motility of SMMC-7721 cells, which is consistent with the results of a previous study (8), thus providing further confirmation that MOB2 regulates the migratory and invasive abilities of HCC cells.

Accumulating evidence has suggested that MOB2 may perform its functions by competing with MOB1 for interaction with NDR1/2, where the binding of MOB2 to NDR1/2 blocks the activity of NDR kinase, and the binding of MOB1 to NDR1/2 leads to increased activation of NDR1/2 kinases (1–4). NDR1/2 and LATS1/2 function as YAP kinases and may be considered as members of the Hippo core cassette (4,12,13). The phosphorylation of YAP leads to decreased nuclear YAP transcriptional activity, which serves critical roles in cell motility (14–17).

Therefore, in the present study, the expression of NDR1/2 and YAP in SMMC-7721 cells that stably overexpress MOB2, MOB2 knockout-SMMC-7721 cells and their corresponding vector control cells were examined. It was revealed that there was a significant decrease in the level of Thr444/442 phosphorylation (pT444/442) at the hydrophobic motif of NDR1/2 in MOB2-overexpressing cells compared with their corresponding vector control cells (P<0.05; Fig. 3A and B). There was also a significant increase in the level of YAP phosphorylation at S127 (pS127YAP) and S397 (pS397YAP) sites in MOB2-overexpressing cells compared with their corresponding control (P<0.05; Fig. 3C). By contrast, the knockout of MOB2 resulted in the significant upregulation of pT444/442 proteins and the downregulation of pS127YAP and pS397YAP compared with the corresponding vector controls (P<0.05; Fig. 3A-C). There were no significant differences observed between the empty vector-infected cells and the NC cells (data not shown). It is widely accepted that the phosphorylation of YAP results in its cytoplasmic retention and reduced nuclear YAP and transcriptional activity of TEA domain family (14–16). Therefore, RT-qPCR was performed in order to analyze the expression of CTGF and CYR61, two well-characterized YAP target genes (18,19), in SMMC-7721 cells MOB2 overexpressing-cells, MOB2 knockout cells and their corresponding vector control cells. It was revealed that the overexpression of MOB2 significantly decreased the expression of CTGF and CYR61, while the knockout of MOB2 significantly promoted the transcription of CTGF and CYR61 compared with the levels in their corresponding vector control cells (P<0.05; Fig. 3D and E).

To evaluate the requirement of YAP for MOB2-regulated cell motility, YAP was knocked down in MOB2 knockout cells as verified by RT-qPCR and western blotting (P<0.05; Fig. 4A and B) followed by Transwell cell migration and invasion assays. Compared with the MOB2-silenced cells that were transfected with non-targeting shRNA vector (shNC), the silencing of YAP in MOB2 knockout cells significantly decreased cell migration and invasion (P<0.05; Fig. 4C-F), suggesting that YAP is involved in the modulation of the motility of SMMC-7721 cells via MOB2. Although NDR1/2 kinases have been reported to function as the upstream kinases of YAP and directly phosphorylate YAP (12), the results of the present study suggest that MOB2-mediated regulation of YAP phosphorylation appears to be independent of NDR1/2 activation.

A key function of the Hippo tumor suppressor pathway is to inhibit the activity of YAP transcriptional co-activators by MOB1-LATS signaling through LATS-mediated direct phosphorylation of YAP (20,21). Therefore, the present study aimed to investigate the effect of MOB2 on the expression of MOB1 and LATS1 in SMMC-7721 cells. It was revealed that the overexpression of MOB2 significantly upregulated the levels of MOB1 phosphorylation (pMOB1) and LATS1 phosphorylation at Y1079 (pY1079LATS1) and S909 (pS909LATS1) sites compared with their corresponding controls (Fig. 5A-C). By contrast, the knockout of MOB2 significantly decreased the levels of pMOB1 and pLATS1 (pY1079LATS1 and pS909LATS1) compared with the corresponding vector control (P<0.05; Fig. 5A-C). Together, these results indicated that MOB2-induced YAP activation may be mediated through activating MOB1-LATS signaling.

Mammalian NDR kinases are the only reported binding partners of MOB2, whereas MOB1 may associate with NDR and LATS kinases (1,5). To further dissect the molecular mechanism of MOB2 in LATS/YAP activation, immunoprecipitation assay was performed followed by western blotting. As hypothesized, it was confirmed that MOB2 may interact with NDR but not LATS, while MOB1 may bind to NDR1/2 and LATS1 (Fig. 6). It was revealed that MOB1 was enriched in LATS1 precipitates but reduced in NDR1/2 precipitates in MOB2-overexpressing cells. Furthermore, the knockout of MOB2 resulted in accumulation of MOB1 in NDR1/2 precipitates and reduction of MOB1 in LATS1 precipitates (Fig. 6). These observations demonstrate a role of MOB2 in regulating the alternative interaction of MOB1 with LATS1 and NDR1/2.