Tumor samples from 59 cases of gastric carcinoma were obtained from the Minhang Central Hospital, (Shanghai, China) from 2012 to October 2014. The procedure was performed with patient informed consent and in accordance with the Helsinki Declaration and upon the approval of The Institutional Review Board of Minhang Hospital.

The human gastric carcinoma cell lines used in the present study were SGC-7901, AGS, NCI-N87, HS 746T, MKN-45, KATO III, NCI-SNU-1, SNU-5 and SNU-16 and were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, glutamine and ampicillin/streptomycin (all from Gibco Laboratories, Carlsbad, CA, USA) in a 5% CO₂ atmosphere. GAX023, GAXC031 and GAX066 are primary tumor cell lines established from PDX models (PDX-PDC; patient-derived xenograft, patient-derived cell line) at Shanghai ChemPartner as previously described (27–29).

The anti-UbcH10 (A-650) antibody was purchased from Boston Biochem (Boston, MA, USA). Anti-ERK1/2 (#4695), anti-phospho-ERK1/2 (T202/Y204, #4370), anti-p38 MAPK (D13E1, #8690), anti-phospho-p38 MAPK (Thr180/Tyr182, #9211), anti-Akt (#9272), anti-phospho-Akt (Ser473, #9271), anti-actin (13E5, #4970) and anti-GAPDH (14C10) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Annexin V-FITC Apoptosis Detection Kit I (cat. 556547) was obtained from BD Pharmingen (San Diego, CA, USA). Blocking buffer (PBS), goat anti-mouse (IRDye800CW 926-32210) and goat anti-rabbit (IRDye800CW 926-32211) were purchased from Odyssey. Hoechst 33258 (#861405) and MTT reagents (M2128) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The CDS sequences (121-660) of UBE2C transcript variant 1 mRNA (GeneBank NM_007019) were chemically synthesized and cloned into mammalian cell expression vector pLVX-UBE2C-IRES-eGFP.

Gastric carcinoma cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

siRNA directed against the UbcH10 transcript with the sequence: 5′-CCUGCAAGAAACCUACUCAdTdT3′ anti-sense, and a scrambled siRNA were obtained from GenePharma (Shanghai, China). In vitro transfections were performed using 0.8 μg (54 pM) siRNA in 24-well plates using Lipofectamine 2000 transfection reagent (1×10⁵ cells/well) following the manufacturer's protocol. Transfection of siRNA was performed at least 5 times for each cell line. Each experiment was repeated at least twice. Non-specific siRNAs (scrambled siRNAs) were used as negative controls.

Cells were lysed in T-PER tissue lysis buffer containing protease inhibitor (both from Thermo Fisher Scientific, Waltham, MA, USA), and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA). Twenty micrograms of protein/sample was loaded on 4–12% SDS-PAGE gels (Life Technologies, Carlsbad, CA, USA). Proteins were separated and transferred onto a nitrocellulose membrane. Membranes were incubated with blocking buffer (LI-COR, Inc., Lincoln, NE, USA) and incubated with primary antibodies overnight, followed by the Alexa Fluor 680-conjugated secondary antibody (Invitrogen). Blots were scanned on the Odyssey system (LI-COR, Inc.). Anti-p-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-Akt, anti-p-Akt (Ser473), anti-p38, anti-pp38 and anti-cyclin D1 antibodies were purchased from Cell Signaling Technology. Anti-actin (Cell Signaling Technology) or anti-GAPDH antibody (Epitomics, Burlingame, CA, USA) was used as a loading control.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to study the proliferation of the tumor cells. Cells were plated in 96-well plates at a density of 10⁴ cells/well to attach for 24 h and transfected with 50 nM different siRNAs. At 0, 24, 48 and 72 h after treatment, 10 μl of MTT reagent (5 mg/ml; Sigma) was added to each well. After a 4-h incubation at 37°C, the supernatant was gently aspirated and formazan crystals were dissolved in 100 μl of dimethyl sulfoxide (DMSO). The absorbance of each well was measured by an ELISA plate-reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 570 nm.

The cell cycle or apoptosis distribution was analyzed by flow cytometry. Cells were seeded into a 6-well plate, followed by transfection of siRNA or over-expression plasmids, and cultivation was maintained for 36 h. For cell cycle analyses, the harvested cells were washed with PBS and fixed with ice-cold 70% ethanol at 4°C overnight. The fixed cells were resuspended in PBS (containing 0.2 mg/ml RNAse and 10 mM PI) and incubated in the dark for 30 min at 4°C.

For cell apoptosis analyses, the cells were harvested and washed with PBS and stained with Annexin V and PI staining (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences, San Jose, CA, USA). A FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) was used for data acquisition and FlowJo (TreeStar, Palo Alto, CA, USA) was used for data analysis.

Protein cellular distribution of UbcH10 was assessed by immunohistochemical analysis. Formalin-fixed, paraffin-embedded, 6-mm tissue sections were deparaffined. Antigen retrieval was performed by water heating method at 100°C for 10 min with 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with methanol-hydrogen peroxide (3%) for 15 min. After blocking with unrelated antiserum, the slides were incubated with the UbcH10 primary monoclonal antibody (Boston Biochem, Cambridge, MA, USA) at 1:200 dilution and incubated overnight at 4°C. After washing with Tris-buffered saline and Tween-20 (TBST), the sections were incubated with secondary antibodies followed by peroxidase-labeled streptavidin (Dako, Carpinteria, CA, USA) for 20 min at room temperature, and visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin. Unrelated IgG was used instead of the primary antibody as a negative control.

To investigate the role of UbcH10 in gastric cancer, 12 gastric cancer cell lines (SGC-7901, AGS, NCI-N87, HS-746T, MKN-45, KATO III, NCI-SNU-1, SNU-5, SNU-16, GAXC023, GAXC031 and GAXC066) were examined to determine the UbcH10 expression levels by western blot analyses. Among these cell lines, GAXC023, GAXC03 and GAXC066 are primary tumor cell lines established recently in our laboratory from gastric cancer PDX models (30).

Diverse UbcH10 expression levels were observed in these 12 cell lines, with relative high expression levels observed in KATO III, SGC-7901, GAXC-023 and GAXC-031 cells, and much lower expression levels noted in NCI-N87, HS-746T and NCI-SNU-1 cells (Fig. 1A and B).

SGC-7901 and KATO III, both of which express UbcH10 at a high level, were selected for the RNA interference study. siRNA transfection led to 90 and 98% inhibition of UbcH10 expression in the SGC-7901 and KATO III cells, respectively (Fig. 2A).

Then, MTT cell proliferation assay was used to test the effect of UbcH10 knockdown. Cell growth was significantly slower in the UbcH10-knockdown SGC-7901 and KATO III cells when compared with those transfected with scramble RNAs (Fig. 2B).

Flow cytometric analysis with PI staining was then used to study cell cycle distribution. UbcH10-knockdown SGC-7901 cells displayed markedly reduced percentages of S and G2/M phase populations (Fig. 2C).

Cell proliferation can be induced by changes in signaling pathways. Cells with or without UbcH10 knockdown were analyzed using western blot analyses. The cells with siRNA transfection were seeded in DMEM without serum overnight. Serum (1%) was then used to stimulate cell signaling.

Phospho-ERK, phospho-Akt and phospho-p38 were attenuated with UbcH10 knockdown in the SGC-7901 cells (Fig. 3A and B). Similar changes were also observed in the same experiment using KATO III cells (data not shown).

The decrease in cell signaling and proliferation may be a sign of reduced cell fitness. To investigate this, the role of UbcH10 was then studied in cisplatin (10 μM)-induced gastric tumor cell apoptosis with flow cytometry using FITC-Annexin V and PI staining. Knockdown of UbcH10 in these 2 gastric cancer cell lines resulted in increases in apoptosis and eventual death following exposure to cisplatin treatment (Fig. 3C).

The data suggest that knockdown of UbcH10 in gastric cancer cells reduced tumor cell proliferation, serum-induced signal transduction and increased susceptibility to cisplatin-induced cell death.

To confirm these findings, we constructed the UbcH10 expression plasmid for the overexpression studies in the NCI-N87 and HS-746T cells, Two of the gastric cancer cell lines showed low UbcH10 expression (Fig. 1).

The western blot analysis showed the enhanced expression level of UbcH10 in the two cell lines after the transfection (Fig. 4A).

MTT assay was used to test the cell proliferation 24 h after transient transfection of UbcH10. In accordance with the previous gene knockdown data, the overexpression of UbcH10 promoted tumor cell growth (Fig. 4B).

The overexpression of UbcH10 also led to reduced cisplatin-induced cell apoptosis as detected by Hoechst staining and apoptotic nuclei quantification (Fig. 4C).

The role of UbcH10 in gastric cancer was supported both by the UbcH10 overexpression and the knockdown studies. Next, clinical gastric cancer patient samples were examined to evaluate the clinical relevance of the findings.

The expression of UbcH10 was examined in 59 cases of confirmed cases of gastric cancers by IHC analysis. Most of the cancer tissues showed different levels of UbcH10 expression at the tumor area; 19 samples displayed clear positive staining (score of +), 23 samples displayed strong staining (score of ++) and another 7 samples exhibited very strong staining (score of +++) (Fig. 5). No signals were detected in the neighboring mesenchymal or IgG-negative control. Overall, 49 out of 59 cases (83%) showed significant UbcH10 expression in the cancerous tissues when compared with that noted in the neigh-boring mesenchyma (Table I).