The HepG2, Bel-7402, Huh7, PLC/PRF-5, SMMC7721 and LM3 liver cancer cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences Committee Type Culture Collection (Shanghai, China), and the normal L02 liver cell line was conserved at the Central Laboratory of Renmin Hospital of Wuhan University (Wuhan, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Zhejiang Tianhang Biotechnology Co., Ltd., Hangzhou, China) and 100 units penicillin/streptomycin. The cells were cultured at 37°C and 5% CO₂ in a humidified chamber. Rabbit polyclonal anti-MORC2 antibody was purchased from Abcam (Cambridge, UK). Mouse monoclonal anti-β-actin antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Promega Corporation (Madison, WI, USA).

The GSE14520 and GSE22058 mRNA expression profile were downloaded from the Gene Expression Omnibus (GEO) database (13–15). The Cancer Genome Atlas (TCGA) copy number-altered genome data for each patient was directly downloaded from cBioPortal for Cancer Genomics (16,17). All liver cancer samples and paired adjacent tissues were retrieved from patients receiving surgery between December 1 and December 31, 2014, from the Department of Pathology, Zhongnan Hospital of Wuhan University (Wuhan, China). All patients provided informed written consent prior to the investigation. The inclusion of human samples was approved by the Ethics Review Board of the Second People's Hospital of Guangdong Province (Guangdong, China; approval no. 2015-KYLL-023). The tissues were first stained with hematoxylin and eosin for histological examination. The deparaffinized sections were treated with 3% H₂O₂ and subjected to antigen retrieval by citric acid (pH 6.0). Following overnight incubation with primary antibody (anti-MORC2 antibody; 1:200) at 4°C, the sections were incubated for 30 min at room temperature with HRP-labeled polymer conjugated with secondary antibody (MaxVision™ kits) and incubated for 1 min with diaminobenzidine. The sections were then lightly counterstained with hematoxylin. Sections without primary antibody served as negative controls. The expression level of MORC2 was ascertained according to the average score of two pathologists' evaluations using a CKX41 microscope (Olympus Corporation, Tokyo, Japan). As MORC2 is mainly expressed in the nucleus, the positive nuclear staining of MORC2 was used to elucidate its expression level according to the following formula: Immunostaining score = percentage score × intensity score, where the percentage score represented the percentage of immunopositive cells, and was graded as 0 (<6%), 1 (6–33%), 2 (34–66%) and 3 (>66%). The intensity score represented the intensity of immunostaining, and was determined as 0 (absent), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). All cases were diagnosed by two certificated pathologists without discrepancy.

The siRNAs were designed and purchased commercially (Genepharma, Shanghai, China) as shown in Table I. The cells were transfected with 50 ng/µl targeting siRNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 48 h, following which the depletion efficiency was analyzed by western blot analysis. siRNA-homo-1760 was selected for the following experiments, as it exhibited the optimal efficiency. For the establishment of stable MORC2-overexpressing cells, the overexpression plasmid was constructed using the pLV-EGFP (2A) puro plasmid; the plasmid was packed using a lentivirus system (both from Inovogen Biotechnology Co., Ltd., Beijing, China). Following infection, the cells were selected for 4 weeks using DMEM with 10 µg/ml puromycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The expression of MORC2 was lowest in the PLC cell line, however, the overexpression efficiency was not satisfactory in this cell line, thus the SMMC7721 cell line was used to construct the stable MORC2-overexpressing cells in the present study.

In brief, cells were lysed in lysis buffer containing 50 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/l NaF, 1 mmol/l Na₃VO₄. Protein concentration was analyzed and adjusted using a bicinchoninic acid protein assay. Subsequently, equal quantities (50 µg) of samples were separated by 10% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20, the membranes were incubated with primary antibodies against MORC2 (1:400, bs-0354R; BIOSS, Beijing, China), cytochrome c (1:200, ab53056), caspase-3 (1:500, ab47131) (both from Abcam), caspase-9 (1:150, sc-56076; Santa Cruz Biotechnology, Inc.), poly (ADP-ribose) polymerase (PARP) (1:150, ab4830; Abcam), p53 (1:500, sc-6243), B-cell lymphoma-2 (Bcl-2) (1:500, sc-783), Bcl-2-associated X protein (Bax) (1:300, sc-493) (all from Santa Cruz Biotechnology, Inc.), p53 upregulated modulator of apoptosis (PUMA)α (1:5000, ab33906; Abcam), Yes-associated protein 1 (YAP1) (1:500, orb89757; Biorbyt Ltd., Cambridge, UK), phosphorylated (p-)S127-YAP1 (1:10,000, ab76252; Abcam), Transcriptional co-activator with PDZ-binding motif (TAZ) (1:500, #23306-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), p-S89-TAZ (1:500, sc-17610), and β-actin (1:1,000, sc-47778) (both from Santa Cruz Biotechnology, Inc.) at 4°C overnight. The primary antibodies coupled to sample proteins were then visualized by incubation for 1 h at 37°C with HRP-conjugated secondary antibodies (1:1,000, 074-1506 and 074-1806; KPL, Inc., Gaithersburg, MD, USA) using a chemiluminescence detection system (EMD Millipore) according to the manufacturer's protocol.

Total RNA from the tissues and cells was isolated using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 2 µg of total RNA from each sample was used for cDNA synthesis. RT-qPCR analysis was performed in triplicate with the SYBR^® Green PCR Master mix (Takara Bio, Inc., Otsu, Japan) and β-actin was used as an internal control according to the manufacturer's protocol. Each sample included: 4 µl cDNA, 2 µl primers, 12.5 µl 2X SYBR Green master mix and 6.5 µl ddH₂O. The PCR thermo-cycling conditions were as follows: 5 min at 94°C, followed by 40 cycles at 94°C for 20 sec, 60°C for 20 sec and at 72°C for 20 sec, and 5 min at 72°C. Relative expression levels of target genes were determined according to the 2^−ΔΔCq method (18), where Cq represents the quantification cycle for each transcript. The primer pairs for human MORC2 and β-actin were designed as shown in Table II.

In the cell proliferation assay, the cells (1×10⁵/well) were plated onto 96-well plates on the first day and allowed to attach overnight. The following day, 10 µl of CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubated at 37°C and 5% CO₂ for 1 h between days 1 and 5. The absorbance was then detected with the multifunctional microplate reader at 490 nm. For the chemoresistance assay, following seeding of the cells (1×10⁵/well) in the plates, the cells were treated with the chemotherapeutics, doxorubicin, 5-fluorouracil and cisplatin, at different concentrations (0, 1, 5, 10, 20, 40, 80 and 100 µM) for 72 h at 37°C, following which CCK-8 was added. Following incubation at 37°C and 5% CO₂ for 1 h, the absorbance was detected and the half maximal inhibitory concentration (IC₅₀) parameter was calculated.

The cells were digested with trypsin, centrifuged at 110 × g for 5 min at 37°C, resuspended in DMEM supplemented with 10% FBS and seeded in 6-well plates (1,000 cells/well), following which the cells in each group were cultured for 2 weeks. The cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and then stained with 0.1% crystal violet for 30 min. The dishes were then carefully washed with PBS until the background was clear. Finally, the number of colonies was counted under a microscope (CKX41; Olympus Corporation).

In brief, the cells were digested with trypsin, centrifuged at 110 × g for 5 min at 37°C, resuspended in PBS, washed twice with PBS and then fixed in 100% ice-cold methanol overnight at -20°C. The cells were then incubated with 50 mg/ml propidium iodide (PI) and 1 mg/ml RNAase in PBS for 20 min, following which the samples were analyzed with BD FACSAria (BD Biosciences, Franklin Lakes, NJ, USA).

In the scratch wound healing assay, cells were cultured in serum-free medium for 24 h and wounded with pipette tips. Subsequently, the medium was replaced with fresh medium. The wound healing procedure was observed after 48 h, and images of the cells were captured under a microscope (CKX41; Olympus Corporation). Cell migration and invasion assays were performed using Transwell chambers, as previously described (19). The cells were harvested and resuspended in serum-free medium, and then added to the upper chamber. Following incubation for 48 h, cells remaining on the upper side of membrane were removed with a cotton swab. The cells migrated to the lower membrane surface were fixed and stained with 0.1% crystal violet for 30 min, and the number of cells was counted under a microscope. To assess invasion ability, the membranes were pre-coated with diluted Matrigel, whereas the membranes in migration experiments were not pre-coated with Matrigel.

All animal experiments were approved by the Ethics Review Board of the Second People's Hospital of Guangdong Province (approval no. 2015-KYLL-063). For the experiments, 4-week-old male BALB/C nude mice (Hangzhou Hibio Technology Co., Ltd., Hangzhou, China) were used. The mice were maintained under the following pathogen-free conditions: 60% humidity; room temperature; 12-h light/dark cycle; ad libitum access to food and water. In a subcutaneous xenograft procedure, 1×10⁶ cells (SMMC7721^MORC2 or SMMC7721^Vector) were resuspended in PBS solution and then injected subcutaneously into the left and right side of each of the mice (n=6). The tumor size was measured every 3 days, and tumor volume was calculated using the following formula: 1/2 length × width². For the assessment of pulmonary metastasis, 10 mice were included in each group, and 1×10⁶ cells (SMMC7721^MORC2 or SMMC7721^Vector) were injected into the caudal vena. After 2 weeks, the mice were sacrificed and lung colonization was quantified by pathological examination.

Annexin V-FITC/PI staining was used to investigate whether MORC2 regulates the apoptosis of liver cancer cells. The cells were seeded into 6-well plates. When the cells in each group were at a log phase of growth, the cells were digested with trypsin, centrifuged at 110 × g for 5 min at 37°C and resuspended in PBS. The cells were then incubated with an ApoScreen Annexin V Apoptosis kit and PI. Every sample containing 10,000 cells was analyzed using BD FACSAria (BD Biosciences). The experiments were performed in triplicate.

Statistical significances between values of different experimental groups were analyzed using Student's t-test or one-way analysis of variance. A χ² test and Fisher's test were used in analyzing enumeration data and pathway analysis. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were conducted using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) or SPSS for Windows 17.0.1 software (SPSS, Inc., Chicago, IL, USA). Expression profile analysis was conducted on L02^MORC2 and L02^Vector data (Chen et al, unpublished data) using GCBI 1.0 software (Gminix Technology Co., Ltd., Shanghai, China) with the Kyoto Encyclopedia of Genes and Genomes database (http://www.genome.jp/kegg/) as reference.

By analyzing the expression profile of liver cancer samples from GEO datasets GSE14520 and GSE22058, it was found that the mRNA levels of MORC2 were significantly upregulated in the liver cancer tissues, compared with the matched non-tumorous tissues (Fig. 1). The expression levels of MORC2 in 73 liver cancer and corresponding adjacent liver tissues specimens were then examined using IHC staining. Consistent with previous reports, MORC2 was expressed mainly in the nucleus and was relatively weak in the cytoplasm (Fig. 2) (3,10). The results showed that MORC2 was overexpressed in the majority (72.6%, 53/73) of the liver cancer samples (Fig. 2). RT-qPCR analysis was also used to examine the levels of MORC2 in 10 pairs of liver cancer and pair-adjacent liver tissue specimens; accordingly, the expression of MORC2 was upregulated in almost all the cancerous tissues (Fig. 3A). The expression level of MORC2 was then examined in HepG2, Bel-7402, Huh7, PLC/PRF-5, SMMC7721, LM3 and L02 cells. Higher expression of MORC2 was observed in the majority of the liver cancer cells, compared with the L02 cells (Fig. 3B). From these results, it was concluded that MORC2 was overexpressed in liver cancer at the mRNA and protein level, demonstrating the important role of MORC2 in the pathogenesis of liver cancer.

Dysfunctional DNA damage repair results in genetic alterations, including somatic copy number alteration, and this characteristic is increasingly recognized as common feature of human liver cancer (20). It has been reported that MORC2 is associated with chromatin remodeling, promotes the induction of γ-H2AX and modulates DNA damage repair (6). Utilizing the data of TCGA database (16,17), it was detected that the fraction of copy number alterations was higher in the genome of patients with liver cancer and higher MORC2 (Fig. 4). This result indicated that the upregulation of MORC2 may result in liver cancer progression via accumulating extra copies of DNA.

Using the clinical information of GSE14520, the correlation between the expression of MORC2 and clinicopathological features was assessed to determine its clinical significance. The samples were divided into two groups according to the expression level and were analyzed with χ² test. As shown in Table III, a higher expression of MORC2 was associated with larger tumor volume (P=0.009) and higher American Joint Committee on Cancer T stage (P=0.007). These results indicated that MORC2 may be a potential prognostic biomarker in liver cancer.

Gene set enrichment analysis (GSEA) was performed in order to identify potential genes modulated by MORC2. The mRNA expression profiling data in GSE14520 were used to outline MORC2-correlated genes. In the GSEA analysis of Gene Ontology terms, it showed that gene sets involved in 'cell cycle process' and 'mitosis' were enriched in the MORC2-high expression samples (Fig. 5). Enhanced mitogenic signaling and aberrant process of cell cycle are essential for cell proliferation and cancer progression (20). The bioinformatics data provided a possible explanation of why a high expression of MORC2 was associated with unfavorable clinicopathological features, including tumor size and T stage (Table III). The results suggested that MORC2 has an important function in the proliferation of liver cancer cells.

To verify the biological role of MORC2 in the proliferation of liver cancer, MORC2 was knocked down using siRNAs in HepG2, Bel-7402 and LM3 cells, which had a higher expression of MORC2. Western blot analysis was used to determine the knockdown efficiency (Fig. 6A).

The effect of MORC2 on cell proliferation was then examined. As shown in Fig. 6B, compared with the control groups, following MORC2 knockdown, the HepG2, Bel-7402 and LM3 cells exhibited a significantly lower cell proliferation rate (Fig. 6B). Furthermore, cell proliferation was measured using a plate colony formation assay. Compared with the control cells, MORC2 knockdown in the HepG2, Bel-7402 and LM3 cells resulted in markedly decreased colony formation abilities (Fig. 6C).

Enhanced cell cycle progression and reduced apoptotic signaling are two important factors causing sustained proliferation. To examine the factors involved in MORC2 regulating the proliferation of liver cancer cells, the cell cycle was detected in liver cancer cells through flow cytometry. Following transfection with si-MORC2 or si-NC for 48 h, the inhibition of MORC2 led to a significant accumulation of cells at the G0/G1-phase and a marked decrease in cells at the S/G2/M-phase in HepG2, Bel-7402 and LM3 cells (Fig. 7A). Subsequently, the effects of MORC2 on apoptosis under normal condition or oxidative stress (treated with H₂O₂) were investigated. The knockdown of MORC2 markedly increased apoptosis of the HepG2 cells (Fig. 7B). These results indicated that the downregulation of MORC2 promoted cell cycle arrest and induced the apoptosis of liver cancer cells.

The clinicopathological analysis also revealed that a higher expression level of MORC2 was significantly associated with a gene expression signature of higher metastatic potential (P<0.001, Table III), however, the role of MORC2 in cancer cell metastasis has not been investigated previously. Cell migration and invasion are critical during the multistep process of cancer cell metastasis. The present study assessed whether MORC2 was a crucial molecule involved in cell migration and invasion using Transwell assays. MORC2-loss of function inhibited the migration and invasion rates of the HepG2, Bel-7402 and LM3 cells (Fig. 8A and B). To confirm this result, a scratch wound healing assay was also used to evaluate the effect of MORC2 on cell movement. Consistent with the previous observations, the inhibition of MORC2 attenuated the mobility of the liver cancer cells (Fig. 8C).

The SMMC7721 cell line, which had a relatively lower expression of MORC2, was used to construct cells overexpressing MORC2 using a lentivirus-mediated packed pLV-MORC2 vector. Western blot analysis was used to determine the knockdown and ectopic expression (Fig. 9A). Compared with the control group, SMMC7721 cells exhibited a significantly higher cell viability rate when MORC2 was overexpressed (Fig. 9B). Similarly, the MORC2-overexpressing SMMC7721 cells exhibited significantly increased colony formation (Fig. 9C). The overexpression of MORC2 increased the cell cycle progression and decreased the number of apoptotic cells (Fig. 9D and E). Furthermore, SMMC-7721 cells stably expressing MORC2 exhibited enhanced migration, invasion and movement, compared with the control cells (Fig. 10). The in vivo experiments showed that the overexpression of MORC2 significantly increased tumor volume in a subcutaneous xenograft model (Fig. 11A). Colonization at a distant site is the last key step in the metastatic cascade (21). The present study also used a tail vein injection model to imitate the pathophysiological process to determine whether MORC2 was involved in the distant colonization of liver cancer cells. At 2 weeks post-injection, the mice were sacrificed by cervical dislocation, and lung colonization was quantified by pathological examination. In line with the observation in vitro, all 10 mice exhibited severe lung metastasis in the MORC2-overexpression group, the incidence of which was significantly higher, compared that in the empty vector group (1/10; P<0.001) (Fig. 11B and C). These results revealed the promoting role of MORC2 in liver cancer metastasis.

The present study also examined whether MORC2 has the potential to be applied in clinical liver cancer treatment. Chemotherapy provides an optional strategy in the treatment of liver cancer, particularly for patients with advanced tumors. However, chemotherapy is unsatisfactory due to chemoresistance (22). The present study hypothesized that the negative effect of MORC2 on the apoptosis of liver cancer cells may also contribute to the drug resistance of liver cancer cells. To test this hypothesis, chemosensitivity to the three most common chemotherapeutic drugs, doxorubicin, cisplatin and 5-fluorouracil, was assayed in MORC2-knockdown groups and control groups. The results showed that the IC₅₀ values of all the chemotherapeutic drugs were significantly decreased by the knockdown of MORC2 (Table IV). Therefore, MORC2 contributed to enhancing the chemotherapeutic sensitivity of liver cancer cells.

To determine the mechanisms by which MORC2 affects apoptosis and chemoresistance, the expression levels of apoptosis-related proteins were examined following the knockdown of MORC2 in the cells. As shown in Fig. 12A, in the HepG2, Bel-7402 and LM3 cells, si-MORC2 transfection induced the cleavage of caspase-9, caspase-3 and PARP, and the release of mitochondrial cytochrome c into the cytosol. These results demonstrated that MORC2 affected apoptosis and chemoresistance by modulating the mitochondrial apoptotic pathway.

The p53 tumor-suppressor gene regulates apoptosis through the transcriptional activation of its target genes, and the mitochondrial apoptotic pathway is regulated by several p53-target genes, including Bax, Bcl-2, p53AIP1, Noxa and Puma (23–25). Therefore, the present study examined the possible association between p53 and MORC2 in liver cancer. By comparing the expression profile of L02^MORC2 and L02^Vector, we found that the overexpression of MORC2 significantly enriched genes involved in the p53 pathway (P<0.001) (Fig. 12B), which was consistent with our hypothesis. To confirm this result, the effect of MORC2-knockdown on the expression level of p53 and its target genes, including Bax, Bcl-2 and PUMAα, were examined. It was found that the knockdown of MORC2 increased the expression of p53, Bax and PUMAα, and reduced the expression of Bcl-2, compared with the control (Fig. 12C). The overexpression of MORC2 also notably enriched genes involved in the Hippo pathway (Fig. 12B). Further validating these results, the results of the western blot analysis showed that MORC2-knockdown increased phosphorylated Yes-associated protein (YAP) and TAZ (Fig. 12D). These results suggested that MORC2 may promote Hippo pathway activation in tumorigenesis. Collectively, the dysregulation of MORC2 in liver cancer disrupted the p53 and Hippo pathways during cancer progression.