Cell lines, antibodies, and siRNAs
Human immortalized hepatocytes LO2 and HCC cells Hep3B, HepG2, Huh7, and SNU449 were purchased from the Cellcook Biotech (Guangzhou, Guangdong, China) or the Cell Bank at the Chinese Academy of Sciences (Shanghai, China). The short tandem repeat (STR) typing data of these cell lines were provided with purchase. BRIT1-overexpressed cells Hep3B.BRIT1 were constructed by using pMSCVpuro-BRIT1 vector (Addgene #16205) (pMSCVpuro as empty control) as described previously . BRIT1-specific siRNA transiently knockdown cells siSNU449 were used as a BRIT1-low control compared to parental SNU449 cells. LO2, Hep3B, Hep3B.BRIT1, and HepG2 cells were cultured in DMEM, while SNU449 and siSNU449 cells were in RPMI 1640 (Hyclone, GE Healthcare Life Sciences, Utah, USA), both supplemented with 10% FBS (PAN-Biotech GmbH, Germany) and penicillin-streptomycin (Solarbio, Beijing, China), and maintained at 37˚C in an atmosphere of 5% CO2. Primary antibodies used in this study were anti-BRIT1 (#4120, Cell Signaling Technology, UAS), anti-SERPINB2 (ab47742, Abcam, USA), anti-SERPINA5 (ab172060, Abcam, USA), anti-b-Actin (ab8226, Abcam, USA), and anti-GAPDH (E12-052, EnoGene, China). The confirmatory BRIT1-specific siRNAs were described previously  and listed below: AGGAAGUUGGAAGGAUCCAdTdT (sense), UGGAUCCUUCCAACUUCCUTT (antisense). The negative control siRNAs (Ctrl siRNA) were as follows: UUCUCCGAACGUGUCACGUTT (sense), ACGUGACACGUUCGGAGAATT (antisense). All siRNAs were synthesized by GenePharma (Shanghai, China), and each siRNA was resuspended in nuclease-free water and stock solutions were stored at -20°C until use.
siRNA transfection assay
BRIT1-proficient SNU449 cells were transiently transfected with either BRIT1 siRNA or Ctrl siRNA at final concentration of 30 pM by using Lipofectamine RNAi MAX (HB-TRLF-1000, Hanbio, China) according to the manufacturer’s instructions. siRNA transfection was first performed in 24-well plates (Corning Costar, Cambridge, MA, USA). As instructed, 1.5 μl of siRNA duplex (30 pM) and 1 μl transfection reagent were individually mixed in 50 μl Opti-MEM medium, the siRNA-transfection reagent combined complex (100 μl) was added into 24-well plates and incubated with 500 μl of diluted cells in opti-MEM with 10% FBS (1x105 cells/ well) for 24 h at 37°C under 5% CO2. For measuring siRNA knockdown efficiency, the siRNAs-treated cells were cultured for up to 72 h and harvested at the indicated time intervals. For examining cell proliferation, the siRNAs-treated cells after 24 h transfection were resuspended and seeded into 96-well plates, and then assessed by using Cell Counting Kit-8 (CCK8) at the indicated time points. For monitoring cell migration and invasion, the siRNA-transfected cells from 24-well plates were planted into Transwell cambers, and the other steps were done as described below.
Quantitative reverse transcription PCR (qRT-PCR)
qRT-PCR was performed as described previously . Briefly, total RNAs were prepared and reverse transcribed to cDNA by using First Strand cDNA Synthesis kits (Roche, USA). PCR reactions were performed using a Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The baseline cycles and threshold (CT) were autocalculated in PCR baseline-subtracted mode using the system's software. Gene expression levels were normalized against that of GAPDH and determined relative to controls using the 2− ΔΔCT method. The qRT-PCR primer sequences for the selected SERPIN genes and GAPDH were provided upon request.
Cell proliferation assay
Cell proliferation assay was performed by using a CCK8 kit (Transgene, Beijing, China). Briefly, cells (3×103 cells/well) were seeded into 96-well plates and incubated for 1, 2, 3, 4, and 5 days. At the indicated time point, 100 µl fresh medium plus10 µl CCK8 solution was added to each well pre-emptied. The plate was then incubated for additional 2 hours before measuring the absorbance at 450 nm wavelength using a microplate reader (M5e, MD Co. USA). The assays for each time point were performed in triplicate and the experiments were biologically repeated at least three times.
HCC tissue microarrays (TMA) were purchased from Alenobio (cat# LV2084) (Xi’an, Shaanxi, China). This TMA contained 83 HCCs, 5 cholangiocellular carcinomas, 2 adenosquamous carcinomas, 1 each of mixed carcinoma, carcinoma sarcomatodes, squamous cell carcinoma, and papillary carcinoma, and 10 normal liver tissues. The TMA was generated in duplicate so as to compare the staining in different areas of the same sample. Tumor characteristics of these TMA samples were available including age, gender, tumor type, histological grade, tumor stage, and metastasis status (Supplementary Table S1).
Immunohistochemical analysis of BRIT1 and SERPINA5 was performed on the TMA slides with rabbit anti-BRIT1 antibody (E2A4831, EnoGene, China, 1:80) and rabbit anti-SERPINA5 antibody (ab172060, Abcam, USA, 1:500), respectively. Briefly, the TMA slides of 4 µm were deparaffinized in graded alcohols. Antigen retrieval was carried out by pressure cooking slides for 5 min at full pressure in 0.01 M citrate buffer (pH6.0). Endogenous peroxidase activity was inhibited by immersing the sections in 3% H2O2. The slides were then incubated with primary antibodies at 4ºC for overnight. Bound antibodies were detected with EliVisionTMplus (Bioz, Palo Alto, CA, USA) and visualized with diaminobenzine (Sigma). The slides were counterstained with Mayer’s haematoxylin. Negative controls, without primary antibody and positive controls of normal tissue, were included in each batch of immunohistochemistry. The TMA sections were scanned by using high resolution digital images (Olympus BX51, Japan) at 20 x magnifications. The cytoplasmic and/or nuclear staining were quantitated as a H-score, i.e. a percentage of the positive cells in relation to the total number of tumor cells (0-100%) weighted with the staining intensity (0-3) .
Wound healing assay
Wound healing assay was used to determine the migratory capacity as described previously . Cells (5×105 cells/well) were planted into the wells of 6-well plates and cultured 24 h until the culture was subconfluent. After that, a wound track was scored in each well, and the complete medium was replaced with 1% FBS-containing medium to reduce the influence of cell proliferation on cell migration to the least degree. Replicated wells were terminated and cell movements were examined under inverted microscopy at 0, 12, 24, 36, 48, 60, and 72 h after wounding. The experiments were duplicated for statistical analysis.
Transwell migration and invasion assays
Cell migration and invasion were also assayed by using Transwell chambers (8 μm pore, 353097, Corning, New York, USA) as described previously . For migration assay, about 5×104 cells in 1% FBS-containing medium were seeded into the upper chambers. The lower chambers contained the medium with 10% FBS to stimulate cell migration. For invasion assay, the upper chambers were pre-coated with Matrigel (354480, Corning Costar, Cambridge, MA, USA) to mimic the basement membrane, and the other steps were the same as migration assay. At 12, 24, 36, and 48 h after incubation, the cells located on the lower surface of the chambers were fixed with cool methanol for 10 min, stained with 0.1% DAPI (Solarbio, Beijing, China) for 20 min, and counted using a fluorescent microscope (Olympus, Tokyo, Japan). The number of migratory or invasive cells was showed as the average of 5 random fields under the microscope at ×200 magnification. The experiments were repeated at least three times.
Western blotting analysis
Cultured cells were collected and lysed with a RIPA Buffer (Boster, Wuhan, China) with 0.1% Protease Inhibitors Cocktail (B14001, Biotool, USA). Protein concentrations were measured by using conventional BCA assay kit (PC0020, Solarbio, China). Around 30 μg of total lysates were subjected to electrophoresis on 8%-12% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and then wet-transferred onto a Polyvinyl difluoride (PVDF) membrane (Immobilon-P, 0.45 μm, Merck Millipore, USA) at 100 V for 1.5 h. The membranes were blocked with 5% non-fat dry milk in TBS-T for 1 h at room temperature, and then incubated with the indicated primary antibodies (anti-BRIT1, #4120, Cell Signaling Technology, UAS, SERPINA5, ab172060, Abcam, USA, anti-b-Actin, ab8226, Abcam, USA, GAPDH, E12-052, EnoGene, China) at 4°C for overnight. After that, the membranes were extensively washed with TBS-T, and incubated with secondary antibodies conjugated with goat anti-mouse IgG H&L (ab6789, Abcam, USA) or goat anti-rabbit IgG H&L (ab6721, Abcam, USA) for 1 h at room temperature. Following an additional wash with TBS-T, chemiluminescent reaction was recorded using ECL prime western blot detection kit (WBKLS0100, Millipore, USA), as per manufacturer’s guidelines. The signals were captured, and the intensity of the bands was quantified using Image LabTM Software (Bio-Rad).
Promoter activity analysis
SERPINA5 promoter region spanning -1445 nt to +49 nt (+1 representing the first nucleotide of AUG in coding sequencing, the position 94581369 in the human chromosome 14 reference GRch38.p12 primary assembly (NC_000014.9)) was constructed as original template (promoter region P1), and the subcloned promoter regions including P2 (-1102nt to -31nt), P3 (-822 nt to +49nt), P4 (-563 nt to +49 nt), P5 (-340 nt to +49 nt), and P6 (-365 nt to -31 nt) subsequently constructed. E2F1 wild-type (E2F1 wt) and its N-terminus (E2F1 mutant) expression plasmids were constructed according to the literature . For promoter activity analysis, Hep3B cells were transfected with P5 promoter-Luc (firefly luciferase) and pRL (Renilla luciferase)-TK (a constitutively active plasmid for controlling transfection efficiency containing only a portion of the herpes simplex virus TK promoter and lacking E2F1 binding sites) with or without BRIT1 plasmid, E2F1 wt/mutant plasmids. Firefly and Renilla luciferase activity were assayed by using dual-luciferase reporter assay system, and the firefly luciferase activity representing the SERPINA5 promoter activity were normalized with Renilla activity.
HCC datasets (including BRIT1’s or SERPINA5’s copy number variation and mRNA expression level, and the association of BRIT1 or SERPINA5 with recurrence, vascular invasion, or metastasis) from several other cohorts were retrieved from Oncomine (https://www.oncomine.org/) or the cBioPortal for Cancer Genomics(www.cbioportal.org). The promoter region of the SERPINA5 gene was retrieved from UCSC Genome Browser on human Dec 2013 (GRCh38/hg38) assembly (https://genome.ucsc.edu). The E2F1 binding sites in the SERPINA5 promoter region was predicted by using PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3), a virtual laboratory for the study of transcription factor binding sites in DNA sequences .
Each experiment was performed in triplicate for all data. Data were expressed as mean ± standard error of the mean (SEM). Statistical comparisons were performed using the Student’s t test and ANOVA. P values less than 0.05 were considered to be statistically significant.