Five human HCC cell lines including Hep3B, HepG2, MHCC97L, MHCC97H and HCCLM3 (from the Liver Surgery Department at Zhongshan Hospital, Fudan University.) with consecutively increased metastasis potentials were routinely cultured at 37℃ in 5% CO2 in high-glucose MEM or DMEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, UT, USA). Briefly, the cells were grown to 90% confluency and harvested by treating with 0.25% trypsin and 0.02% EDTA. After rinsed three times with PBS, cells were centrifuged for further RNA isolation and protein extraction.
Lentivirus-induced SLC35A2 Deficiency (Lv-shSLC35A2)
Three different sequences targeted to 3 different sites of SLC35A2 mRNA (NM_0005660) were designed without off-target effects and cloned into expression vector pGCsi-SLC35A2shRNA. After validation of inhibited efficiency in HCC cells, a most efficient target sequence (5’-CAGUAUGUUGCCAUCUCUA-3’) was identified and then modified their structure to form short hairpin RNA (shRNA-2): 5’-CCGGCAGTATGTTGCCATCTCT ATTCAAGAGATAGAGATGGCAACATACTGTTTTTG-3’. Viral vector generation was obtained by co-transfection of 293T cells by the calcium phosphate precipitation (CPP) method on 10-cm plates with 20 μg of PGC-LV, 15μg of pHelper1.0EGFP, and 10 μg of pHelper2.0. Infectious lentiviruses were harvested, centrifuged to eliminate cell debris, and then filtered through 0.22-μm cellulose acetate filters. Infectious titer was determined by fluorescence-activated cell sorting analysis of EGFP positive in 293T cells. A multiplicity of infection (MOI) of 20 for HCC cells in serum-free growth medium was used. Transduced cells were selected by 1μg/mL puromycin and used in subsequent assays.
Construction of pcDNA3.1-based wild-type and mutant vectors
The coding sequences of SLC35A2 and B4GalT1 were amplified by RT-PCR and cloned respectively into pcDNA3.1(+)-flag and pcDNA3.1(+)-HA eukaryotic expression vectors. Moreover, de novo mutant in SLC35A2 gene (c.797G > T, p.G266V) was constructed by using site-directed mutagenesis system. These cloned sequences were confirmed by carrying out a sequencing analysis. The transfection of these vectors into HCC cells was Lipofactamin 3000-mediated, according to the manufacturer’s protocol. Co-location of SLC35A2 with B4GalT1 in HCC cells through co-transfecting pcDNA3.1-HA-SLC35A2 and pcDNA3.1-Flag-B4GalT1 was observed by fluorescence microscope (Leica, DM500).
Equivalent protein amounts were separated on a denaturing SDS polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membrane. After blocked with 5% nonfat dry milk in PBS containing 0.05% Tween-20, membranes were incubated with primary antibodies. Visualization used horseradish peroxidase linked anti-rabbit secondary antibodies (Cell Signaling) and ECL-Plus blotting substrate detection kit (Pierce). Quantification of Western blots was analyzed densitometrically by using Quantity One software (Bio-Rad Laboratories).
Polymerase Chain Response
RT-PCR assay was carried out according to manufacturer’s instruction. Briefly, 1-2μg RNA was incubated at 70℃ for 5 min and then placed on ice. After addition of oligo-(dT)18 primer, RT reaction was performed at 42℃ for 60 min followed by 97℃ for 2 min. For PCR amplification, 1μL cDNA from RT was used in a final volume of 25μL, PCR program for SLC35A2 was performed as 95℃ for 5 min, followed by 25 cycles of 95℃ for 30s, 60℃ for 50s and 72℃ for 1 min and a final extension at 72℃ for 10min. The products were separated by electrophoresis in a 1.2% agarose gel and visualized after staining with ethidium bromide.
For quantitative RT-PCR (qRT-PCR), Platinum® SYBR® Green qPCR Super Mix kit (Invitrogen, Carlsbad, CA, USA) was used. Reactions were carried out using three independent technical replicates for each sample which was quantified in IQ5 real-time PCR system (Bio-rad). The copy number was adjusted by the geometric mean of two house-keeping genes (GAPDH and ACTB).
Invasion and migration assay
In vitro invasion assay was performed using 24-well Transwell unit with polycarbonate filters transwell cell culture plates (8-μm pore size; Costar, Acton, MA, USA). Briefly, 1x105 cells were seeded onto Matrigel-coated (0.8 μg/μL) upper chambers of the membrane and the lower chambers were filled with 600 μL NIH-3T3 conditioned media as a resource of chemoattractants. After 36 hours, cells were fixed with 0.5% glutaraldehyde and stained with Gimsa. Cotton swabs were used to remove the cells from the upper surface of the membrane, leaving the cells on the underside. For the migration assay, experimental procedures are the same as the in vitro invasion assay described above except that the filter was not coated with Matrigel. The membrane was removed and mounted onto glass slides and scanned.
Cell adhesion assay
Cell adhesion assay was performed by using CytoSelectTM cell adhesion assay (Cell Biolabs, Inc.). In brief, under sterile conditions, allow the Fibronectin, Collagen Ⅳ and Laminin Adhesion Plate or flat-bottom culture plates were coated with E-selectin to warm up at room temperature for 10 minutes. Prepare a cell suspension containing 0.5×106 cells/ml in serum free media. Add 150 μL of the cell suspension to the inside of each well (BSA-coated wells are provided as a negative control). Incubate for 60 min in a cell culture incubator. Carefully aspirate the media from each well. Gently wash each well 4-5 times with 250 μL PBS. Aspirate the PBS from each well and add 200 μL of Cell Stain Solution. Incubate for 10 minutes at room temperature. Discard the Cell Stain Solution from the wells. Gently wash each well 4-5 times with 500 μL deionized water. Discard the final wash and let the wells air dry. Add 200 μL of Extraction Solution per well, and then incubate 10 minutes on an orbital shaker. Transfer 150 μL from each extracted sample to a 96-well microtiter plate and measure the OD 560nm in a plate reader.
For cell-cell adhesion assay, human endothelial cells (HEC) were seeded in 96-well plates and cultured for 48-72 hr until 100% confluent. Then pretreated or untreated HCC cells with sustained expression of EGFP protein were added to each well as 2.0×104/well and incubated at 37℃ for 60 min. After washed three times, plates were subjected to fluorescence microscopy and remaining HCC cells were counted.
In vivo metastasis assay
An orthotopic liver xenograft model was established for tumor growth and metastasis analysis as previously described. In brief, 1×107 stably transfected MHCC-97H cells were resuspended in PBS buffer and injected subcutaneously into the capsule of the left hepatic lobe of male BALB/c nude mice. Tumour volume was monitored and calculated as volume (mm3) = [width2 (mm2) × length(mm)]/2. After 7 weeks, mice were sacrificed and the lungs were removed and fixed in formalin, embedded in paraffin. Lung colonization was carefully counted by histological examination under microscope after consecutive tissue sections (5 μm/each). The Animal Experimentation Ethics Committees of our institutes approved the animal study.
Immunoprecipitation of E-cadherin
Equal amounts of total protein of each canine cell line (750 μg) were precleared with 50 μl of protein G-sepharose beads for 1.2 h. After centrifugation, the supernatant was incubated overnight with 5 μg of monoclonal antibody (mAb) against E-cadherin. After that, incubation with protein G-sepharose for 2 h was performed. Next, the beads were washed three times with an immunoprecipitation buffer. The immune complexes were released by boiling 5 min at 95℃ in Laemmli sampling buffer and the immunoprecipitants were subjected to 7.5% SDS–PAGE and the separated proteins were transferred to a nitrocellulose membrane. The mobility shift was evaluated.
Cell surface glycosylation pattern analysis
13 kinds of tumor-associated lectins (Vector laboratory, Burlingame, CA, USA) were dissolved with chip-spotting buffer (CapitalBio, Beijing, China) at a concentration of 1 mg/mL respectively, and spotted on gel-substrate chip using a microarray printing robot Smart Arrayer-48 (CapitalBio, Beijing, China). The diameter of each point is 150µm, and distance between two points is 400µm. Each lectin point has 4 repeats. Then the chip was incubated in a vacuum chamber with humidity greater than 80% at 25℃ overnight to immobilize the lectins.
Cells were cultured and reached greater than 80% confluence. Then cells were harvested by cell-scraping and collected by centrifugation at 1200 rpm for 5 min. The resulting pellet was washed 4-5 times with warmed PBS (PH 7.4) and cells were resuspended in 0.5-1 ml cy3 in NaHCO3-NaH2CO3 (PH 9.3) at 106 cells/ml. After incubation at room temperature for 30-60 min, stained cells were washed three times in PBS and resuspended in 500 ml PBS. The lectin arrays were blocked by 20% BSA-PBS at room temperature for 1 hr, followed by washing with 0.1% Tween 20-PBS for 5 min, three times. Stained cells were added into sub-array and incubated in the dark for 15-30 min in humidified incubator at 37℃. The binding fluorescence signals of the glycoproteins with lectins were obtained with fluorescence scanner LuxScan 3.0(CapitalBio, Beijing, China). The net intensity value for each spot was calculated by subtracting background value. Median rectification was used to calculate the dye-bias-corrected ratios. Median corrected data were used to calculate q value by T-statistical analysis with Significance Analysis of Microarrays (SAM, version 2.1, Stanford University, CA, USA) software.
In vitro co-immunoprecipitation assay
The cell culture dishes were placed on ice and the cells were washed with ice cold PBS. After the PBS was drained, then the cells were treated with buffer (170 mM NaCl, 50 mM Tris-HCl, pH 8.0, 50 mM NaF, 0.5% NP-40) containing protease inhibitors. After clearing with protein-G-sepharose, supernatants were incubated with protein-G-sepharose conjugated with anti-GP73 monoclonal antibody at 4 °C overnight. Subsequently, protein–beads complexes were washed with PBS for three times and then re-suspended in 20 μL loading buffer. Lastly, the proteins were detected by western blotting.
The expression data of patients with live hepatocellular carcinoma (LIHC, n=371) was downloaded from TCGA database (https://portal.gdc.cancer.gov) by using the data transfer tool. The differential expression analysis of SLC35A2 among a variety of groups in LIHC was performed through GEPIA2 web server (http://gepia2.cancer-pku.cn). Overall survival probability and progress-free survival probability was plotted by using the Kaplan-Meier analysis. The comparison was performed at the 0.05 level of significance. The gene list positively correlated with SLC35A2 in human hepatocellular carcinoma was downloaded from the public cancer UALCAN databases (http://ualcan.path.uab.edu). Then Metascape database (http://www.metascape.org, v3.5.20220101) was used for pathway enrichment analysis, in which P value less than 0.05 was considered as significant.
Data were expressed as mean ± standard error (SE) and analyzed using analysis of variance (ANOVA). Student’s t-test was used in two-group comparisons. The association between the various factors was determined using the Pearson correlation. P<0.05 was considered to be statistically significant.