A total of three human lung cancer cell lines (H460, H1299 and A549) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. 11965-092) supplemented with 10% fetal bovine serum (FBS) (cat. no. 10270-106) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained at 37°C in an incubator containing 5% CO₂. Dimethyl sulfoxide, Trichostatin A (TSA) and nicotinamide (NAM) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), and 7.5 mM NAM or 0.5 nM TSA was applied to A549, H1299 or H460 cells for 24 h at 37°C in an incubator containing 5% CO₂.

The full-length cDNA of human HDAC7 and plakoglobin were cloned into the pSin-puro vector (Focus Bioscience Co., Ltd., Nanchang, China), and the restriction sites were EcoRI and NheI. The full-length cDNA of human HDAC (1-11) was constructed by inserting a Flag tag into the HDAC N-terminus in a pcDNA3.1(+) vector (cat. no. V790-20, Invitrogen; Thermo Fisher Scientific, Inc.) and the plasmids were transfected into H460 cells using Lipofectamine 2000 (no. 11668019; Thermo Fisher Scientific, Inc.). All recombinant plasmids were verified by DNA sequencing (Ruiboxingke Biotech Co., Ltd., Beijing, China).

Human anti-plakoglobin (cat. no. 75550), anti-GAPDH (cat. no. 5174), anti-HDAC7 (cat. no. 33418), anti-Flag (cat. no. 14793) and the Acetyl-Histone Antibody Sampler kits containing anti-H3K9, -H3K18, -H3K27 and -histone 3 (cat. nos. 9927), and anti-H4K5, H4K12, histone 4 (cat. no. 8346) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-β-actin (cat. no. 66009-1-1g) was procured from ProteinTech Group, Inc. (Chicago, IL, USA).

H1299 cells stably expressing scrambled (Scr) or HDAC7 short hairpin RNA (shRNA) were established using the Sigma-Aldrich shRNA system (Merck KGaA), according to the manufacturer's protocols. Scr shRNA (with no known targets in the human genome) with the following sequence: 5′-GGGCGAGGAGCTGTTCACCG-3′, and the oligonucleotides for human HDAC7 shRNA#1 and #2 were 5′-GATCCGGGTGCAAGTAAATA-3′ and 5′-CCACTATTCCTGGCTCTGCAA-3′, respectively. A total of 3 µg pSin-puro delivering HDAC7 or plakoglobin, 3 µg pSin-puro-empty vector, 3 µg shRNA-NC, 3 µg shRNA-HDAC7#1 or #2 was co-transfected with 3 µg pMD2.G and 3 µg psPAX2 into 293 cells (ATCC) for 48 h. using Lipofectamine 2000. The recombinant viruses were subsequently collected and applied to H1299, A549 or H460 cells cultured with 8 µg/ml Polybrene for 24 h. The stable lines were selected with 1 µg/ml puromycin for 2 weeks.

RT-qPCR was performed as described (27). Briefly, total RNA obtained from cell lines was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. First-strand cDNA was synthesized at 42°C for 60 min using the Revert Aid™ First Strand cDNA Synthesis kit (cat. no. 6110A; Takara Bio, Inc., Otsu, Japan). Subsequently, the qPCR reaction was performed in a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR^®-Green mix (Tiangen Biotech Co., Ltd., Beijing, China). Thermal cycling of the qPCR reaction was initiated with a denaturation step at 95°C for 15 min, and consisted of 40 cycles (denaturation at 95°C 15 sec, annealing at 60°C for 30 sec and elongation at 72°C for 30 sec). The amplified products were examined using the 2^−ΔΔCq method (28), and each sample was calibrated to the expression levels of the housekeeping gene GAPDH. The primers used for amplifying HDAC7, plakoglobin and GAPDH were as follows: HDAC7, forward, 5′-GGCGGCCCTAGAAAGAACAG-3′ and reverse, 5′-CTTGGGCTTATAGCGCAGCTT-3′; plakoglobin, forward, 5′-TCTCCAACCTGACATGCAACA-3′ and reverse, 5′-CATAGTTGAGACGCACAGAGTTC-3′; and GAPDH, forward, 5′-ACAGTCAGCCGCATCTTCTT-3′ and reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′.

In vitro cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. For cell proliferation, cells were seeded in 96-well plates at a density of 1,000 cells/well and incubated for 1, 2, 3, 4 or 5 days; 10 µl of CCK-8 reagent (Beyotime Institute of Biotechnology, Haimen, China) was then added to each well, followed by incubation for 1.5 h at 37°C. The absorbance value (optical density) of each well was measured at 450 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc.).

The ChIP assay was performed using the ChIP kit (cat. no. 53008, Active Motif, Carlsbad, CA, USA) as described previously (29). Briefly, to fix the cells, Complete Cell Fixative Solution (included in kit) was added to the existing culture medium for the cells at 80% confluence at room temperature, and the fixation reaction was stopped by adding Stop Solution (included in kit) to the existing culture medium. The cells were collected by centrifugation at 1,000 × g for 5 min at 4°C. Subsequently, the nuclear pellet was resuspended in ChIP Buffer (included in kit). The cell lysate was subjected to shearing using a sonication instrument (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) to a fragment length of 200-500 bp. Total genomic DNA (input) was quantified and 20 µg of chromatin from each sample was immunoprecipitated overnight at 4°C with 5 µg anti-HDAC7 (cat. no. 33418; Cell Signaling Technology, Inc.), anti-acetyl-H3K9 (cat. no. 39917) or anti-acetyl-H4K5 (cat. no. 39699) (both from Active Motif), or normal IgG as a negative control. Then, nucleosome complexes were isolated with the protein G agarose beads for 3 h at 4°C. Bound DNA-protein complexes were eluted and cross-links were reversed after a series of washes using the washing reagent contain in the ChIP kit. Purified DNA was resuspended in TE buffer. Subsequently, the PCR reaction was performed using PrimeSTAR^® Max DNA Polymerase (cat. no. R045A; Takara Bio, Inc.). Thermal cycling of the qPCR reaction was initiated with a denaturation step at 94°C for 2 min, and consisted of 35 cycles (denaturation at 98°C 10 sec, annealing at 60°C for 15 sec and elongation at 72°C for 30 sec). The primers for the plakoglobin were as follows: Plakoglobin-ChIP, forward, 5′-GGACAGTCAGGCGAGATAGC-3′ and reverse, 5′-CGAACAAAAGGCGAAGAGAC-3′

For the Transwell migration assay, 4.0×10⁴ (A549) or 1.5×10⁵ (H1299) cells in 200 µl serum-free DMEM were added to cell culture inserts with an 8-µm microporous filter, without an extracellular matrix coating (BD Biosciences, Franklin Lakes, NJ, USA). DMEM medium containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.; cat. no. 10270-106) was then added to the bottom chamber. After 24 h of incubation at 37°C in an incubator containing 5% CO₂, the cells on the lower surface of the filter were fixed, stained and examined under a light microscope. The number of migrated cells in three random optical fields (magnification, ×10) from triplicate filters was averaged. For the Transwell invasion assay, 8.0×10⁴ (A549) or 3.0×10⁵ (H1299) cells resuspended in 200 µl serum-free DMEM were added to cell culture inserts, which contained 8-µm microporous filters and were coated with Matrigel (BD Biosciences; cat. no. 354480). DMEM containing 10% FBS was then added to the bottom chamber. After 24 h of incubation at 37°C in an incubator containing 5% CO₂, the cells on the lower surface of the filter were fixed with 10% formalin for 15 min at room temperature, stained with 0.1% crystal violet for 60 min at room temperature and examined under a light microscope. The number of migrated cells in three random optical fields (magnification, ×100) from triplicate filters was averaged.

Western blotting procedures were performed as described previously (3), including the experimental conditions of the gels. Briefly, cultured cells from all cell lines were lysed in ice-cold radioimmunoprecipitation assay lysis buffer (cat. no. P0013C; Beyotime Institute of Biotechnology) at 4°C for 30 min. Following centrifugation (12,000 × g) at 4°C for 20 min, the lysates were obtained and protein concentration was determined with the BCA method. Equal amounts of protein (30 µg/lane) were separated by 10% SDS-PAGE. The conditions were as follows: Voltage of 100 V for 2.5 h at room temperature. Proteins were then transferred to polyvinylidene fluoride membranes with an electrical current of 250 mA at 4°C for 2 h. To block the non-specific binding sites, the membranes were incubated with 5% non-fat milk (in Tris-buffered saline with 0.1% Tween-20) at room temperature for 60 min, and then membranes were then incubated with the following primary antibodies: Human anti-plakoglobin (cat. no. 75550, anti-GAPDH (cat. no. 5174), anti-HDAC7 (cat. no. 33418), anti-Flag (cat. no. 14793) and the Acetyl-Histone Antibody Sampler kit (cat. nos. 9927 and 8346) (all from Cell signaling Technology, Inc.), anti-β-actin (cat. no. 66009-1-1g; ProteinTech Group, Inc.) at a dilution of 1:1,000 overnight at 4°C. Subsequently, the membranes were incubated with anti-rabbit IgG Secondary antibody peroxidase-conjugated (cat. no. W401B; Promega Corporation, Madison, WI, USA) and anti-Mouse IgG Secondary Antibody Peroxidase Conjugated (cat. no. W402B; Promega Corporation) with the dilution 1:10,000 at room temperature for 2 h. Specific protein bands were visualized using an enhanced chemiluminescence detection system (cat. no. P0018F; Beyotime Institute of Biotechnology) and exposed to radiographic film (Carestream Health, Rochester, NY, USA; cat. no. 6535876).

All animal studies were performed in accordance with protocols approved by the Research Animal Resource Center of Sun Yat-Sen University (Guangzhou, China). The mice were maintained in specific pathogen free conditions at a temperature of 20-25°C, a 50-70% humidity, under a light/dark cycle of 12 h, with free access to water and food. A total of 18 male athymic nude mice at 4 weeks of age were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). For subcutaneous injection, 2×10⁶ H1299 cells with stably expressing Scr or shHDAC7-1 were mixed with 0.2 ml PBS (pH 7.4) and 30% (v/v) Matrigel matrix (BD Biosciences). Suspensions were injected subcutaneously into the flanks of 4-week-old male athymic nude mice, which were monitored over 5 weeks. For tail vein injection, 3×10⁶ H1299 cells with stably expressing Scr or shHDAC7-1 were resuspended in 300 µl PBS (Biological Industries, Beit Haemek, Israel) and injected into the lateral tail vein of mice that did not receive a subcutaneous injection of cells (3×10⁶ cells/animal). Mice were sacrificed 7 weeks following injection, and nodules were counted and compared between the H1299-scr and H1299-shHDAC7-1 groups.

Samples were fixed in 10% formalin for 10 h at room temperature and embedded in paraffin. Sections (3-µm thick) were prepared and mounted onto positively-charged glass slides. Sections for HDAC7 staining were incubated in 10 mM citrate buffer (pH 6.0) and boiled in a microwave oven for 15 min. After incubation with Protein Block Serum-Free (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), sections were incubated overnight with anti-HDAC7 (1:250) at 4°C in a humidified container. Following washing with PBS three times, the tissue slides were treated with a non-biotin horseradish peroxidase detection system according to the manufacturer's protocols (Dako; Agilent Technologies, Inc.). IHC staining was evaluated by two independent pathologists who were experts in diagnosing lung cancer. HDAC7 detection was quantified as described (3). Briefly, the HDAC7 signal was detected in the cytoplasm and the nucleus, and the intensity in tissues was categorized into four categories: 0, absent; 1, weak; 2, moderate; or 3, strong. The percentage of stained cells was categorized as: 0, no staining; 1, 1-10%; 2, 11-50%; 3, 51-80%; and 4, 81-100%. The staining score for each tissue was calculated by multiplying the intensity value by the percentage value, and the average of the scores from the two pathologists was used as the final score.

The association between HDAC7, and the clinical characteristics or survival of lung cancer patients were analyzed using the online KMplot database (30). The median value of expression was selected as the cutoff in the online database; Cox's proportional hazards model was used to estimate the hazard ratio.

A total of 35 pairs of specimens were collected from patients with lung cancer (median age, 45 years; age range, 33-78 years; male/female ratio, 3:4) with resection between March 2014 and September 2016, and inclusion criteria were as follow: Initial diagnosis of lung cancer. Exclusion criteria included: Previous radiotherapy, chemotherapy or surgery, and previous malignancy or other concomitant malignant disease. Normal lung tissues were collected at >5 cm from the edge of the tumor. The use of human lung cancer tissues was reviewed and approved by the Ethics Committee of the Sun Yat-Sen University Cancer Center. Informed consent was obtained from patients.

The raw data for the correlation between plakoglobin and HDAC7 expression were obtained from the MethHC 1.0.3. software (http://methhc.mbc.nctu.edu.tw/php/index.php).

All statistical analyses were performed using SPSS for Windows, version 16.0 (SPSS, Inc., Chicago, IL, USA). All values from the in vitro assays were expressed as the mean ± standard deviation or standard error of the mean of at least three independent experiments or replicates. A paired Student's t-test was used for the comparison of two groups, multiple group comparisons were performed by using one-way analysis of variance followed by a Tukey-Kramer post-hoc test. The correlation between plakoglobin and HDAC7 expression was analyzed by a Pearson's correlation analysis. The Mann-Whitney test was used to analyze the difference expression of HADC7 in lung cancer tissues and paired normal tissues. P<0.05 was considered to indicate a statistically significant difference.

H460, A549 and H1299 cells were treated with HDAC inhibitors TSA and NAM for 24 h; these reagents are inhibitors of class I and II HDACs and class III HDACs (31,32), respectively. It was identified that the addition of TSA, but not NAM, significantly increased the mRNA expression levels of plakoglobin compared with the control (Fig. 1A-C); notable increases in the protein expression levels of plakoglobin were observed following TSA treatment. The results indicated that plakoglobin may be regulated by class I and II HDACs in lung cancer cells.

In order to determine which class I and II HDAC was responsible for the suppression of plakoglobin expression, class I and II HDACs were ectopically expressed in lung cancer cells. As presented in Fig. 2A and B, only ectopic HDAC7 significantly suppressed the mRNA levels of plakoglobin in H460 cells; the protein expression levels of HDA7 were notably downregulated. Furthermore, the expression levels of HDAC in three cancer cell lines was determined (Fig. 2C). It was evaluated whether overexpression of HDAC7 could decrease plakoglobin expression in other lung cancer cells according to the endogenous levels of HDAC7 in the H460, H1299 and A549 cell lines. RT-qPCR indicated that stable expression of HDAC7 significantly suppressed the mRNA expression levels of plakoglobin in A549 cells compared with the control; the protein expression levels of plakoglobin following HDAC7 overexpression were notably reduced (Fig. 2D). Thus, plakoglobin expression was downregulated by overexpression of HDAC7. The opposite effect was observed with HDAC7 knockdown in H1299 cells. As indicated in Fig. 2E, the two shRNA sequences effectively decreased the expression of endogenous HDAC7 levels compared with the control, whereas the mRNA and protein expression levels of plakoglobin increased. Furthermore, the protein expression levels of plakoglobin decreased following stable overexpression of HDAC7 in H1299 cells (Fig. 2F).

To investigate the mechanisms by which HDAC7 participates in silencing plakoglobin expression, a ChIP assay was performed to analyze whether HDAC7 directly binds to the plakoglobin promoter. The results indicated that HDAC7 binds to the plakoglobin promoter in H1299 cells (Fig. 3A). Acetylation of histones 3 and 4 was assessed by western blot analysis using antibodies targeting lysine residues that are known to be acetylated. It was identified that the levels of acetylation at H3K9 and H4K5 markedly increased following knockdown of HDAC7 with shDAC7 #2 in H1299 cells compared with the control (Fig. 3B); however, overexpression of HDAC7 decreased the levels of the H3K9 and H4K5 acetylation in A549 and H1299 cells (Fig. 3B). To investigate whether H3K9 and H4K5 acetylation was decreased in the promoter of plakoglobin, a ChIP assay was performed to analyze H3K9 and H4K5 acetylation within the plakoglobin promoter regions. The results indicated that the degree of histone acetylation at this promoter was decreased in HDAC7-overexpressing A549 and H1299 cells (Fig. 3C and D). By contrast, the level of histone acetylation at this promoter increased in HDAC7-knocked down H1299 cells (Fig. 3E and F). Acetylation of H3K9 and H4K5 contributes to chromatin decondensation (33), and the enrichment of acetylated H3K9 and H4K5 near a transcription initiation site is associated with the transcriptional activity of the gene (33,34). Thus, HDAC7 may directly inhibit the transcription of plakoglobin via binding to the promoter region of plakoglobin and deacetylating histones H3K9 and H4K5 in lung cancer.

As plakoglobin is downregulated in lung cancer tissues (9), the expression of HDAC7 in lung cancer tissues was evaluated using IHC. The results indicated that HDAC7 expression significantly increased in lung cancer tissues compared with paired normal tissues (Fig. 4A); representative images were presented (Fig. 4B). Using an online database (35), an inverse correlation was identified between HDAC7 and plakoglobin in tissues (Fig. 4C). In addition, whether the mRNA expression levels of HDAC7 is clinically relevant in lung cancer was determined in the present study. Based on the HDAC7 mRNA levels, lung cancer samples were subdivided into two groups, and the associated OS was analyzed. Individuals with high HDAC7 levels were observed to exhibit shorter OS compared with those with low levels (Fig. 4D) using a large public clinical microarray database of lung tumors from 2,170 patients (30). Therefore, HDAC7 expression levels may be a clinical predictor in lung cancer.

Subsequently, it was investigated whether HDAC7 regulates the biological behaviors of lung cancer cells. Stable cell lines with either ectopic expression of HDAC7 in A549 and H1299 cells, or silenced HDAC7 in H1299 cells, were constructed (Fig. 2C-E). Cell proliferation assays indicated that the ectopic expression of HDAC7 significantly promoted cell proliferation of A549 cells compared with the control (Fig. 5A). The migration and invasion of lung cancer cells was analyzed. The ectopic expression of HDAC7 significantly enhanced cell migration and invasion abilities of A549 cells compared with the control (Fig. 5B and C). The proliferative ability was notably increased following HDAC7 overexpression in H1299 cells (Fig. 5D); however, significant increases in migration and invasion were observed compared with the control (Fig. 5E and F). On the contrary, knockdown of HDAC7 markedly reduced cell proliferation (Fig. 5G), and significantly inhibited H1299 cell migration and invasion compared with the control (Fig. 5H and I). In summary, these findings indicate that HDAC7 promotes lung cancer cell proliferation, migration and invasion.

As HDAC7 was observed to promote lung cancer cell proliferation, migration and invasion in vitro, whether HDAC7 promotes lung cancer cell tumorigenesis and metastasis was investigated in vivo. To confirm the promoting effects of HDAC7, an in vivo tumorigenesis study was performed by inoculating shHDAC7 H1299 cells into nude mice. Mice in the shHDAC7#1-H1299 and Scr groups were sacrificed 5 weeks after inoculation, with average tumor weights of 0.04 and 1.313 g, respectively (P<0.01; Fig. 5J-L). These results suggested a significant inhibitory effect of decreased HDAC7 on in vivo tumorigenesis.

To further evaluate the role of HDAC7 in lung cancer cell migration and invasion in vivo, lung cancer cells with or without HDAC7 knockdown were injected into nude mice via the tail vein. Cells with endogenous HDAC7 formed colonies in the lungs within 7 weeks of injection, whereas the HDAC7 knockdown cells resulted in significantly reduced pulmonary metastatic colonization ability (Fig. 5M and N). These results suggest that HDAC7 promotes lung tumor growth and metastasis in vivo.

As the aforementioned experiments demonstrated that plakoglobin was a direct target of HDAC7, whether loss of plakoglobin contributes to the oncogenic role of HDAC7 in lung cancer was determined. Plakoglobin was stably induced into HDAC7-overexpressing lung cancer cells to observe whether restoration of plakoglobin expression could rescue the promoted cell proliferation, migration and invasion of HDAC7-overexpressing cells in vitro (Fig. 6A). Notably, overexpression of plakoglobin significantly reduced the effects of HDAC7 overexpression on cell proliferation, migration and invasion compared with the control (Fig. 6B-D). These results indicated that suppression of plakoglobin by HDAC7 promoted cell proliferation, migration and invasion in lung cancer.