Access to public datasets
Fifteen datasets including GSE6764(19), GSE10143(20), GSE14323(21), GSE14520(22, 23), GSE22058(24, 25), GSE25097(26–28), GSE36376(29), GSE46444(30), GSE54236(31), GSE63898(32), GSE76427(33), GSE112790(34), GSE64041(35), the Liver Hepatocellular Carcinoma Project of The Cancer Genome Atlas (TCGA-LIHC)*, and the Liver Cancer-RIKEN, JP Project from the International Cancer Genome Consortium (ICJC-LIRI-JP)(36) were retrieved from the Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB)(37), and analyzed for DLG2 mRNA expression in HCCs and adjacent hepatic normal tissues (NTs). These datasets included 1431NTs and 2,321 HCCs. Four of these datasets compared gene transcription between HCC and adjacent cirrhotic tissues (GSE10143, GSE14323, GSE25097, and GSE6764), two of them included liver tissues from healthy volunteers (GSE64041 and GSE25097), and one of them also included tissues from different disease stages (GSE6764). The TCGA-LIHC dataset and ICJC-LIRI-JP dataset were used to validate survival analyses from a tissue microarray (TMA) described below. To explore potential mechanisms of DLG2 in HCC progression, a GSEA was employed using the TCGA-LIHC dataset*.
Tissue microarray and immunohistochemistry (IHC)
Microarray sections of HCCs and neighboring NTs containing 90 paired HCCs and NTs from patients [the TMA cohort] were prepared by Shanghai Tufei Biotech Co., Ltd (Cat. No. TFHCC-01; Shanghai, China). The clinicopathological characteristics of these patients are shown in Table 1.
Table 1
Clinical and pathologic features of HCC patients* (n = 90).
Variable | No. of patients (%) |
Sex | |
Male | 70(77.8) |
Female | 20(22.2) |
Age | |
≤ 60 | 64(71.1) |
> 60 | 26(28.9) |
Differentiation | |
G1/2 | 54(60) |
G3 | 36(40) |
Tumor size (cm) | |
≤ 5 | 47(52.2) |
> 5 | 43(47.8) |
HBV infection | |
Negative | 19(21.1) |
Positive | 71(78.9) |
AFP(µg/L) | |
≤ 200 | 45(50) |
> 200 | 45(50) |
Tumor stage | |
T1 | 58(64.5) |
T2 | 17(18.9) |
T3 | 3(3.3) |
T4 | 12(13.3) |
Nodal stage | |
N0 | 85(94.4) |
N1 | 5(5.6) |
M stage | |
M0 | 88(97.8) |
M1 | 2(2.2) |
TNM stage | |
I | 56(62.2) |
II | 17(18.9) |
III | 16(17.8) |
NA | 1(1.1) |
Vessel invasion | |
No | 50(55.6) |
Yes | 25(27.8) |
NA | 15(16.6) |
*Data shown here may be duplicated with those from other published resources that are based on the same cohorts. Abbreviations: NA, information not available. |
As described previously(38), after deparaffination, rehydration in graded ethanol, antigen retrieval with citrate buffer pH 6.0 (1:300 dilution; cat. no. Zli-9065; oriGene Technologies, inc.) and blocking with goat serum (1:20 dilution; cat. no. C0265; Beyotime Institute of Biotechnology) at room temperature for 1 h, slides were stained with a rabbit polyclonal antibody against human DLG2 (1:50 dilution; cat. no. abs141074; Absin Bioscience Inc, Shanghai, China) at 4˚C overnight. Normal rat immunoglobulin G (1:50 dilution; cat. no. D110504; Sangon Biotech Co., Ltd.) instead of the primary antibody was used as a control.
Subsequently, after washing with PBS, a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000; goat anti‑rabbit, cat. no. A0208; Beyotime Institute of Biotechnology, Haimen, China) was added and incubated at room temperature for 1 h. Then, these sections were stained using 3,3'-diaminobenzidine (DAB) (cat. no. GK500705; Shanghai GeneTech Co.,Ltd., Shanghai, China) at room temperature for 5 min and counterstained with 100% hematoxylin (cat. no. C0107; Beyotime Institute of Biotechnology) at room temperature for 2 min. A modifed H score system was used to semiquantitate DLG2 expression, as previously described(39). Briefly, the maximal intensity of staining (0, negative; 1, weak; 2, moderate; and 3, strong) was multiplied by the percentage of positive tumor cells (0100%) to generate the modifed H score (range, 0300). DLG2 expression was categorized as high or low based on the median H score.
Cell lines and culture conditions
A hepatic cell line (L02) and four HCC cell lines (HCCLM3, MHCC97-H, SMMC-7721, Huh7) were kindly provided by Dr. Lijie Ma at Zhongshan Hospital, Fudan university (Shanghai, China). HCC cell lines SK-HEP-1 and Hep3B2.1-7 were kind gifts from Prof. Yongzhong Liu at the Cancer Research Institute, Shanghai Jiao Tong University (Shanghai, China); HCC cell line HepG2 was kindly provided by Prof. Xiuping Liu at the Shanghai Cancer Hospital, Fudan university (Shanghai, China). All cell lines used in this study were regularly authenticated by morphological observation and tested for the absence of mycoplasma contamination (MycoAlert; Lonza Rockland, Rockland, ME, USA).
Cells were cultured in Dulbecco’s Modified Essential Medium (DMEM; BBI Life Sciences, Shanghai, China) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin, and 100 mg/mL streptomycin at 37 °C with 5% CO2 in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA).
Construction of DLG2-expressing plasmids and DLG2targeting shRNAs and packaging of lentiviruses
GFP-expressing lentivirus particles were prepared by Genechem (Shanghai, China). The vectors used were GV358 (for DLG2 and the vector control) and GV248 (for shDLG2 and the scrambled control). The target sequences for shDLG2 were GCCAGTACAATGACAATTT (shDLG2-1), GATCAATGACGACTTGATA (shDLG2-2), GTGAACAAACTATGTGATA (shDLG2-3) and TTGTGGAAATCAAACTGTT (shDLG24). Lentiviral stocks were prepared and purifed as previously described(40).
Infection of HCC cells with lentiviruses
Cells were seeded in 6-well plates at a density of 2 × 105/ml and cultivated for 24 h. Then, 20 µl of lentivirus solution and 1 ml fresh medium containing 10 µg/ml polybrene (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were added to each well. The medium was changed after 24 h and an effcient lentiviral transduction was confirmed by a fluorescence microscope at 48 h and 72 h after infection.
RNA extraction and quantitative polymerase chain reaction (qPCR)
Total RNA was isolated from cell cultures using RNAiso Plus (Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions, and reverse-transcribed and subjected to real-time reverse transcription-PCR using the 2-ΔΔCT method(41). Each sample was determined in duplicate. All PCR products were confirmed by 2.0% agarose gel electrophoresis. The sequences for RT-PCR primers were the following: DLG2 forward primer, 5′- ATGTTCGGCACCTGTCTGTG-3′; and DLG2 reverse primer, 5′- CCTCCAAAACAAAACCCTTTGG-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. Experiments were repeated three times, in duplicate.
Western blotting
Total protein was extracted and quantitated following the established protocol described previously (42). Western blotting was performed using a rabbit anti-DLG2 polyclonal antibody (1:1,000 dilution; cat. no. abs141074; Absin Bioscience Inc, Shanghai, China). GAPDH (1:2,000 dilution, rabbit anti-human; Beyotime Biotechnology) was detected as a loading control(43).
Cell proliferation assay
Briefly, stably transfected SMMC-7721 or HCCLM3 cells (2 × 103 cells/well) were seeded in 96-well plates and cultivated for 24, 48, 72, or 96 h. Then, 10 µL cholecystokinin octapeptide-8 reagent (CCK-8) [10% (v/v) in serum-free DMEM; Beyotime Biotechnology] was added to each well and incubated at 37 °C for 1 h. The absorbance at 450 nm was measured using a microplate reader (BioTek Synergy 2; BioTek, Winooski, VT, USA). The detailed protocol of cell proliferation assays could be retrieved from the previous article(42).
Cell colony formation assay
As previously described(42), stably transfected SMMC-7721 or HCCLM3 cells (2 × 103 cells/well) were seeded in 12-well plates and cultivated in DMEM complete medium at 37˚C for 14 days. The cell colonies were washed with phosphate-buffered saline (PBS) twice, fixed with methanol for 20 min, and stained with crystal violet solution (C0121; Beyotime Institute of Biotechnology) for 15 min. The plates were scanned and colonies containing > 50 cells were counted manually, and the experiments were performed in triplicate.
Scratch wound healing assay
Following the previously described protocol(42), stably transfected SMMC-7721 or HCCLM3 cells (4 × 105 cells/well) were seeded in 12-well plates and grown to nearly 100% confluence. After 24 h of serum starvation, a scratch wound was generated with a 200 µL pipette tip. Wound closure was photographed at 0 and 24 h under a microscope using the ImageScope software (Leica Biosystems Nussloch GmbH) with a magnification of x40.
Gene set enrichment analysis
A Gene Set Enrichment Analysis (GSEA) was performed as previously described(42). Briefly, the TCGA LIHC RNA-seq data were loaded into GSEA and run with the latest version of Hallmarks gene sets. Patient data were divided into DLG2 high and low groups by the median of DLG2 expression. Then GSEA was performed according to the default weighted enrichment statistics and genes were ranked by the Pearson method. Gene sets were considered significantly enriched if P < 0.05 and FDR < 0.25. Those gene sets that were significantly enriched in the DLG2 high group were designated as DLG2-positively related gene sets, while those that were significantly enriched in the DLG2 low group were designated as DLG2-negatively correlated gene sets.
Statistical analysis.
Statistical analyses were performed using GraphPad Prism7 (GraphPad Software Inc., San Diego, CA, USA), SPSS statistical software for Windows, version 22 (IBM Corp., Armonk, NY, USA), and Microsoft Excel 2010 (Microsoft, Redmond, WA, USA). Student's t-tests were performed for continuous variables between two groups and Wilcoxon signed-rank tests were used if the differences between pairs of data did not follow a Gaussian distribution. One-way analysis of variance (ANOVA) was performed for statistical comparisons among multiple groups along with Bonferroni's post hoc test. Pearson's χ2 test and Fisher's exact test were used for categorical comparisons. Pearson's R correlation test was used to describe correlations between continuous variables. Survival analyses were conducted using the Kaplan-Meier method and differences in survival were examined using the log-rank test. Univariate and multivariate survival analyses were conducted using the Cox proportional hazards regression model. Statistical significance was defined as a value of P < 0.05. All statistical tests were two-sided.