Decreased expression of ARID1A associates with poor prognosis and promotes metastases of hepatocellular carcinoma

Sixty-four patient-derived paired HCC and adjacent nontumorous tissue samples were collected at the Zhongshan Hospital of Xiamen University. Written informed consent was obtained from all patients, and the study was approved by the Clinical Research Ethics Committee of Zhongshan Hospital of Xiamen University.

Total RNA was isolated using TRIzol® Reagent (#15596-018, Life Technologies, New York, USA) according to manufacturers’ instructions. Subsequently, cDNA was generated using the PrimeScript™ RT reagent Kit with gDNA eraser (Takara Bio Inc., Dalian, China), and quantitative real-time reverse transcription polymerase chain reaction (qPCR) was performed using the Real-Time PCR detection system (#7500, Applied Biosystems, Shanghai, China) with 2× SYBR Green II/ROX qPCR Master Mix (Takara Bio Inc.). Relative mRNA expression was calculated using the delta threshold cycle (ΔΔCT) method and normalized to β-actin (ACTB) expression. The PCR primers are listed in Additional file 1: Table S1.

Surgically excised tumor specimens were fixed in 10 % neutral formalin and embedded in paraffin, and 4-μm thick sections were prepared using a microtome (#HM315, Thermo Scientific, Waltham, USA). Free-floating section immunostaining was performed using the avidin–biotin–peroxidase complex method (UltraSensitive™, Maixin, Fuzhou, China). Sections were deparaffinized in xylene, and rehydrated in a graded ethanol series. They were then placed in EDTA antigen retrieval buffer (pH 9.0, MVS-0099 Maixin, Fuzhou, China) and incubated at 121 °C for 3 min in an autoclave. Endogenous peroxidase activity was blocked by placing the specimens in 3 % hydrogen peroxide solution for 10 min. Sections were incubated overnight at 4 °C in an anti-human ARID1A antibody (rabbit polyclonal, 1:500, HPA005456, Sigma, USA). Sections were stained in parallel with non-immune immunoglobulin G as a negative control. Antibody binding was detected using an Elivision plus kit (Elivision™ super KIT9922, Maixin, Fuzhou, China), which uses 3, 3’-diaminobenzidine for visualization. Sections were counterstained with hematoxylin, then dehydrated, and coverslips were mounted onto slide-fixed specimens for microscopy. Slides were examined by 2 investigators in an independent and random manner. Five views per slide with 100 cells/view were evaluated at 400× magnification using a light microscope (#Axio Scope A1 pol, Carl Zeiss, Germany). Nuclear staining was considered as positive. Immunohistochemical grading was performed using the following scoring system: 0, 0–10 %; 1, 11–29 %; 2, 30–59 %; and 3, > 60 %. Samples had a higher score in HCC than in notumor tissue were defined as ARID1A high. Otherwise, they were be defined as ARID1A low. Hematoxylin-eosin (HE) stain was performed as previous described [23].

A panel of human HCC cell lines including MHCC-97H, LM3, SK-Hep1, SMMC-7721, HepG2, and Huh7 were purchased from the Cell Bank of Shanghai, Institutes for Biological Sciences, China. HEK293T and GP2-293 cells were generously gifted by the Medical College of Xiamen University. All clonal cells except SMMC-7721 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10 % fetal bovine serum in a humidified incubator at 37 °C and 5 % CO₂. The short hairpin RNA (shRNA) retroviral plasmid (RNAi-Ready pSIREN-RetroQ), which contains a puromycin resistance gene, was purchased from Clontech. The ARID1A shRNA sequences cloned into this vector are shown in Additional file 1 Table S2. The full coding sequence of ARID1A was cloned into the lentiviral pLV-CS2.0 vector that contains an EF1α promoter to drive expression. All transfections were performed using Turbofect Transfection Reagent (#R0531, Thermo Scientific, Waltham, USA). Puromycin was used to generate cells with stable knockdown of ARID1A.

Total protein was extracted from cells in RIPA lysis buffer (#P0013B, Beyotime, Shanghai, China) and quantified using a Bradford assay. In total, 30 μg of protein was separated using 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked in a 5 % powdered milk solution and incubated in primary antibody overnight at 4 °C. After washing, the membrane was incubated with a horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, USA.) at 37 °C for 1 h. Protein bands were visualized using Western Bright ECL (#K-12049-D50, Advansta, CA, USA) and detected using ImageQuant LAS4000mini (General Electric, USA). Relative protein levels were calculated based on a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. Antibodies used included E-cadherin (#3915S, Cell Signaling technology, Boston, USA), Vimentin (#5741S, Cell Signaling technology), Fibronectin (#610077, BD Bioscience, Bedford, USA), GAPDH (#AB-M-M 001, Hangzhou Xianzhi Biotechnology, Hangzhou, China), and ARID1A (#04-080, Millipore, Shanghai, China).

Migration of HCC cells was assessed using the 24-well polycarbonate membrane cell migration assay kit (#3422, Corning Incorporated Costar, Tewksbury, USA) according to the manufacturer’s instructions. Briefly, HCC cell lines were transfected with control and ARID1A shRNAs and were incubated in serum-free medium for 24 h. The cells were then transferred to the upper chamber of a Transwell plate by seeding 2 × 10⁵ cells per well in 200 μL serum-free medium. Next, 0.5 mL of 10 % fetal bovine serum-containing medium was added to the lower chamber as a chemoattractant. Cells were incubated for 24–48 h (depending upon migration capability) at 37 °C. Non-migrating cells on the upper membrane surface were scraped off using cotton swabs. Cells that migrated to the bottom of the membrane were stained with 0.1 % crystal violet for 30 min, followed by washing with water for 30 s to remove residual dye. Four views were examined per transwell and cells/view were counted at 200× magnification. Each experiment was performed in triplicate. The invasion assay was performed in a similar fashion using BD BioCoat™ Matrigel™ Invasion Chambers (#354480, BD Biosciences), except that the upper chambers were precoated with ECMatrix™ gel.

Cell growth was determined using a Cell Counting Kit-8 (CCK-8) cell viability assay (#YB-K001, Yiyuan Biotechnologies, Guangzhou, China) according to the manufacturer’s instructions as described previously [24].

Cell apoptosis detection was performed with Annexin V/PI (propidiumiodide) double staining (#KGA108KeyGEN Biotech, Nanjing, China). Briefly, 48 h after transfection and 12 h Cisplatin (20 μM) treatment, cells were harvested by 0.25 % trypsin (without EDTA), washed twice with chilled PBS, followed by resuspension in 200 μL of binding buffer. Staining solution containing Annexin V/FITC and PI was added to the cell suspension. After incubation in the dark for 30 min, the cells were analyzed by FACS Gallios flow cytometer (Beckman Coulter, USA).

All animal protocols were approved by the Animal Care and Use Committee of Xiamen University. We purchased male BALB/c nude mice (4–5weeks old) from Shanghai Experimental Animal Center of Chinese Academic of Sciences (Shanghai, China). Animals were kept under standard pathogen-free conditions and allowed to acclimate for 1 week before use. MHCC-97H cells (5 × 10⁶/0.2 mL of PBS) stably transfected with either control or ARID1A shRNA expression vectors were subcutaneously injected into right flank of each mouse (n =8 mice/group). Tumor growth was monitored once a week using a caliper, and the tumor volume was calculated using the following formula: volume = π/6 × length × width². We monitored tumor growth over an 8-week period.

The statistical package SPSS 19.0 (SPSS, Chicago, IL, USA) was used for all analyses. All values are expressed as mean ± SEM. Correlations of ARID1A expression with clinicopathological characteristics were evaluated with a χ ² test using R language. Survival analyses were conducted using the Kaplan-Meier method with the log-rank test. Other results were analyzed using a Student’s t test. All p-values were two-sided, and p <0.05 indicated statistical significance