Anti-Cx32, Cx43 and Cx26 antibodies for immunohistochemistry were purchased from BOSTER (Wuhan, China). PV-9000 DAB detection Kit was obtained from ZSGB-Bio (Beijing, China). The DEME (high glucose) and RPMI-1640 media, Lipofectamine™ 3000, anti-Cx26 antibody were purchased from Invitrogen (Carlsbad, CA, USA). 2-aminoethoxydiphenyl-borate (2-APB), streptonigrin (SN), polybrene, anti-Cx43, anti-β-tubulin antibodies, and HRP-conjugated secondary antibodies were acquired form Sigma-Aldrich (St. Louis, MO, USA). Polyethylenimine (PEI) was purchased from Polysciences, Inc. (Warrington, PA, USA). Annexin V-FITC / PI apoptosis detection kit was from Biotool (Houston, TX, USA). TRIzol was obtained from Life Technologies (California, USA). cDNA Synthesis SuperMix kit and qPCR SuperMix kit were purchased from Transgen Biotech (Beijing, China). BCA protein assay kit was from Bio-Rad (Hercules, CA, USA). Chemiluminescent HRP Substrate Kit was from Millipore Corporation (Billerica, MA, USA). Anti-Cx32 antibody was purchased from Santa Cruz (Dallas, TX, USA). Anti-GAPDH antibody was obtained from Ray Antibody Biotech (Beijing, China). EGFR, p-EGFR (Tyr845), PARP, cleaved-Caspase3, Erk 1/2, p-Erk 1/2 (Thr202/Tyr204), STAT3, p-STAT3 (Tyr705), Bcl-2, Bak, Bax, and Src primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Nonspecific mouse or rabbit IgG was purchased from Beytime (Shanghai, China). Protein G Plus/Protein A Agarose Suspension was from Merck Millipore (Billerica, MA, USA). HRP-conjugated secondary antibodies (light chain specific or heavy chain specific) were obtained from Abbikine (Wuhan, China).
Tissue specimens and clinical data
From May 2011 to November 2013, 96 HCC patients underwent hepatectomy at the Affiliated Cancer Hospital of Xinjiang Medical University, Xinjiang, China. All the flesh HCC specimens, corresponding peritumoral tissue (< 3 cm distance from the tumor tissue), and remote normal liver tissues (5 cm away from the tumor tissue) were collected within 10 min after hepatectomy and then stored in liquid nitrogen for protein extraction and paraffin embedding. The cohort of 13 female and 83 male patients had a median age of 53.53 ± 10.88 years (range 24 ~ 78 years), with a median follow-up time of 27.5 months (range 2~ 44 months). The mean tumor size was 7.50 ± 3.53 cm (range: 1.5~20 cm). All patients and their corresponding tissue samples had been confirmed by pathology. None of the patients had received any chemoradiotherapeutic agents in the preoperation. Clinical variables including age, sex, pathological differentiation, TNM stages, serum alpha-fetoprotein (AFP), presence of hepatitis B surface antigen (HBsAg), and tumor size were recorded. Histological grading, according to the Edmondson-Steiner (ES) criteria, showed that ES grade I (well-differentiated), II (moderately differentiated), III (poorly differentiated) and IV (undifferentiated) tumors were found in 21 (21.8%), 36 (37.5%), 24 (25.0%), and 15 (15.6%) cases. Cancer clinical staging was performed according to the AJCC/UICC tumor–node–metastasis (TNM) stage (2010), which showed that TNM stage I, II, and III tumors were found in 15 (15.6%), 18 (18.7%), and 63 (65.6%) cases.
IHC was performed as described previously . Sections were incubated overnight at 4 °C with primary antibody (1:100) and were then visualized using a PV-9000 DAB detection kit according to the manufacturer’s protocol. Sections were counterstained with hematoxylin and observed under a BX-51 microscope (Olympus, Tokyo, Japan). The degree of immunostaining in the sections was reviewed and scored independently by 2 observers based on both the percentage of positive-stained tumor cells and the staining intensity. The number of positive cells was divided into five grades (percentage scores): ≤10% (0), 11–25% (1), 26–50% (2), 51–75% (3), and > 75% (4). The staining intensity was graded in four categories on a scale from 0 to 3 (intensity scores): no staining (0), light-brown staining (1), brown staining (2) and dark-brown staining (3). Protein staining was evaluated using the following formula: overall staining score = intensity score × percentage score. A final score of ≤4 was defined as negative staining, and a final score of > 5 was defined as positive staining.
Cell culture and high−/low-density cultures
HCC cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells and SMMC-7721 cells were cultured in DMEM (high-glucose) and RPMI-1640 medium, respectively, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in an atmosphere containing 5% CO2. For low-density cultures, 1 × 105 cells were seeded in a 150 mm dish to physically inhibit GJ formation (the cells were not in direct contact with each other). For high-density cultures, 1 × 105 cells seeded in each well of a 6-well plate were allowed to form GJs [22, 23]. In high-density cultures, 2 h of pretreatment with 50 μm 2-APB was used to inhibit the function of GJs (Additional file 1: Figure S2) [22,23,24].
Apoptosis induction and analysis
Apoptosis was induced in cells by stimulation with 1 μM streptonigrin (SN) for 7 h [22, 23]. Overall apoptosis was measured by staining cells using an Annexin V-FITC / PI apoptosis detection kit according to the manufacturer’s protocol, followed by analysis using a FACScan (Beckman Instruments, Fullerton, CA, USA). Early apoptotic cells were considered to be Annexin V-positive but PI-negative. Cell apoptosis was analyzed using FlowJo 7.6 software.
Cells were seeded and grown to 30–50% confluence, and complexes of sequence-nonspecific siRNAs (NC) or targeted siRNAs (50 nM) (RiboBio, Guangzhou, China) and 5 μl of Lipofectamine™ 3000 were added to cells in each well according to the manufacturer’s instructions. Cells were incubated for an additional 48 h for the experiments. The sequences of the synthesized Cx32, EGFR and Src siRNA were as follows, and siCx32_2, siEGFR_1, siSrc_1 were chosen for subsequent experiments.
siCx32_1: 5′-CCGGCATTCTACTGCCATT-3′, siCx32_2: 5′-GGCTCACCAGCAACACATA-3′, siCx32_3: 5′-GCAACAGCGTTTGCTATGA-3′. siEGFR_1: 5′-GGCTGGTTATGTCCTCATT-3′, siEGFR_2: 5′-CCTTAGCAGTCTTATCTAA-3′, siEGFR_3: 5′-GGAACTGGATATTCTGAAA-3′. siSrc_1: 5′-CAAGAGCAAGCCCAAGGAT-3′, siSrc_2: 5′-CAGGCTGAGGAGTGGTATT-3′, siSrc_3: 5′-GCAGTTGTATGCTGTGGTT-3′.
Plasmid transfection and establishment of stable cell lines
For transient transfection, cells were seeded and grown to 80% confluence. Following the manufacturer’s instructions, 2.5 μg of each DNA plasmid was mixed with 5 μl of Lipofectamine™ 3000 and 5 μl of P3000™. Then, the mixture was added to the cell culture medium. Cells were incubated for an additional 48 h for the experiments. Plasmid vectors overexpressing Cx32 (EX-A0514-M02–5), EGFR (EX-A0275-M98–5), or Src (EX-B0107-M09) along with the corresponding control vectors, were constructed by GeneCopoeia (Rockville, MD, USA).
To generate stably transfected cells, SMMC-7721 cells were transfected with lentiviral plasmids containing Cx32 (EX-A0514-Lv105) or with empty vector (pEZ-Lv105), which were constructed by GeneCopoeia, and HepG2 cells were transfected with lentiviral plasmids containing the shCx32 sequence or with the negative control vector (pLVX-shRNA-tdTomato-Puro), which were constructed by Landbiology (Guangzhou, China). Lentiviral particles were produced by transfecting 293 T cells with 6 μg of lentiviral plasmid, 4.5 μg of psPAX2 and 1.5 μg of pMD2.G using 30 μl of PEI (100 μM) in a 10 cm culture plate. Then, HCC cells were incubated in medium containing the virus and polybrene (6 μg/ml) for 48 h. After infection, cells were selected by culture with puromycin (2 μg/ml) for 2 weeks.
The sequences for the short-hairpin RNA targeting Cx32 (shCx32) was as follows: 5′-GCTGCAACAGCGTTTGCTACTCGAGTAGCAAACGCTGTTGCAGCTTTTTTT-3′.
Quantitative real-time polymerase chain reaction (qPCR)
After total RNA extraction using TRIzol, RNA concentrations were measuring using a Nanodrop 2000 (Thermo Fisher, Waltham, MA, USA). Reverse transcription to cDNA was performed using a cDNA Synthesis SuperMix kit on a C1000 Thermal Cycler (Bio-Rad, CA, USA). Next, cDNA samples (2 μl) were used for qPCR with the qPCR SuperMix kit, and amplification was conducted for 40 cycles on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cycle time (Ct) values of the selected genes were first normalized to the CT value for GAPDH for the same sample, and fold changes were calculated by relative quantification (2-ΔΔCt). The following formula was used: 2-ΔΔCt = 2control group (Ct value of target gene - Ct value of GAPDH) - experiment group (Ct value of target gene - Ct value of GAPDH). All experiments were repeated 3 times. The primer sequences synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) were as follows:
EGFR: 5′-CCCACTCATGCTCTACAACCC-3′ (Forward), 5′-TCGCACTTCTTACACTTGCGG-3′ (Reverse); Src: 5′-TGGCAAGATCACCAGACGG-3′ (Forward), 5′-GGCACCTTTCGTGGTCTCAC-3′ (Reverse); Cx32: 5′-ACACCTTGCTCAGTGGCGTGA-3′ (Forward), 5′-AGGGACCACAGCCGCACATGG-3′ (Reverse); GAPDH: 5′-TGTGGGCATCAATGGATTTGG-3′ (Forward), 5′-ACACCATGTATTCCGGGTCAAT-3′ (Reverse).
Western blotting (WB)
Cells were washed with cold PBS and then harvested in cell lysis buffer. Whole cell lysates were sonicated and were then centrifuged at 12,000 rpm for 30 min at 4 °C. The protein concentration was determined using a BCA protein assay kit. Fifteen micrograms of protein from each sample was separated by SDS-PAGE followed by transfer to a nitrocellulose membrane. Membranes were blocked with 5% (w/v) skim milk and were then incubated with primary antibodies (1:1000) overnight at 4 °C. Immunoreactive bands were visualized using a Chemiluminescent HRP Substrate Kit and scanned with an ImageQuant LAS 4000™ (GE Healthcare, Piscataway, NJ, USA). The expression values for each target protein were normalized to the corresponding GAPDH or β-tubulin expression values.
Total protein was extracted in cell lysis buffer. The supernatant was incubated with 2 μg of anti-Cx32, anti-p-EGFR or anti-Src antibody on a spinning wheel at 4 °C for 4 h, respectively, with parallel samples containing 2 μg of nonspecific mouse or rabbit IgG as the negative controls (IgG group). Additionally, a certain proportion of supernatant was incubated without any antibody as the positive control (input group). Next, 30 μl of Protein G Plus/Protein A Agarose Suspension was added dropwise to bind to the antibodies overnight at 4 °C. The beads were washed 5 times with lysis buffer and resuspended in sample buffer, followed by boiling for 5 min. The samples were separated by SDS-PAGE and reacted with the corresponding primary antibodies (1:1000), followed by incubation with HRP-conjugated (light chain specific or heavy chain specific) secondary antibodies (1:5000).
Xenograft tumor model
BALB/c-nu mice (male, 4 weeks of age, 18–20 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. The nude mice were first randomly divided into 2 groups (n = 12). These two groups were subcutaneously inoculated with stable cell lines, including SMMC-Vector cells and SMMC-Cx32 cells (5 × 106 in 200 μl of PBS), near the right scapula. When the tumors had grown to an appropriate size (200 mm3), each group was further randomized into 2 groups (n = 6) according to the tumor volume and body weight. The control group was orally administered vehicle (PBS), and the treatment group was orally administered 0.5 mg/kg SN (dissolved in PBS) once every two days for a total of 7 treatment . Tumors sizes were as measured daily using a caliper, and body weights were recorded. Tumor volumes were calculated using the following formula: V = (A × B2)/2, where V is the volume (mm3), A is the long diameter, and B is the short diameter (both in mm). Mice were sacrificed 24 h after the final intragastric administration.
Immunofluorescence, Cytomembrane protein extraction and Parachute dye-coupling assay
The details are shown in Additional file 3 (Supplemental Methods).
All experiments were repeated at least three times. Parametric data were analyzed by one-way ANOVA or Student’s t-test, and nonparametric data were analyzed by Fisher’s exact test or the Chi-square test. The association between Cx32 expression and clinicopathological characteristics was statistically determined using the Pearson χ2 test. The Kaplan-Meier method and log-rank test were applied to generate and compare survival curves. A Cox proportional hazards model was used to determine independent factors for survival based on the variables selected in the univariate and multivariate analyses. A two-tailed value of P < 0.05 was considered statistically significant. Quantitative data are presented as the means ± SDs (standard deviations). The data were statistically analyzed by using the SPSS software package (version 16.0).