USP39 expression was evaluated by means of immunohistochemical analysis with a rabbit monoclonal anti-USP39 antibody. Briefly, tissue sections from breast cancer specimens were incubated with a rabbit monoclonal anti-USP39 antibody (ab131244; Abcam, Cambridge, MA, USA) at 4°C overnight. After being washed, the expression of USP39 in tissue sections was detected using a polymer detection system with polymer helper and polyperoxidase-anti-mouse/rabbit IgG (PV-9000; Zhongshan Golden Bridge Biotechnology Co., Beijing, China), followed by counterstaining with Mayer’s hematoxylin. The percentage of positive cells in a total of 10 fields from each slide was examined and graded as 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%) by 2 pathologists. The intensity of the immunohistochemical staining was graded as follows: 0 (no staining), 1 (bright yellow), 2 (orange), or 3 (brown). The staining was quantified according to the sum of the positive cells and the intensity of the immunohistochemical staining as 0, 1–2, 3–4, or 5–7, which represent negative (−), weakly positive (+), positive (++) or hadro-positive (+++), respectively.

Small interfering RNAs (siRNA) targeting the USP39 sequence (AAGGTTAAGGTGAGCTCA TCG) and the non-silencing sequence (AATTCTCCGAACG TGTCACGT) were transformed into short hairpin RNA (shRNA) (stem-loop-stem structure) and were cloned into the pGCSIL-GFP lentiviral vector (GeneChem Co. Ltd., Shanghai, China) with AgeI/EcoRI sites. Then, the recombined pGCSIL-GFP vector and two-helper vector system (GeneChem) were transfected into the human embryonic kidney cell line 293T via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) to generate the lentivirus. After 3 days of incubation, the lentivirus from the culture medium was collected and concentrated with Centricon Plus-20 (Millipore, Billerica, MA, USA).

The human breast cancer MCF-7 cell line and the human embryonic kidney cell line 293T were purchased from the American Type Culture Collection. The MCF-7 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) containing 4 mM L-glutamine, 0.1 mM MEM non-essential amino acid solution (Sigma-Aldrich) and 10% fetal bovine serum (FBS; Gibco-BRL) at 37°C in 5% CO₂. The 293T cell line was cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sigma-Aldrich) at 37°C in 5% CO₂. For lentivirus infection, MCF-7 cells were cultured in 6-well plates, and the USP39 shRNA-expressing lentivirus (USP39-shRNA) or nontargeting shRNA-expressing lentivirus (control) was then added, with a multiplicity of infection (MOI) of 10. After 5 days of infection, cells were observed under fluorescence microscopy (DMI4000B; Leica Microsystems, Germany).

Total RNAs were prepared from cells using TRIzol reagent (Invitrogen). The reverse transcription reactions were carried out following the manufacturer’s protocol (Promega), and each reverse transcription reaction mixture contained 2 μg total RNA. Then, 25 μl of qRT-PCR mixtures containing 0.1 μM primers, 10 μl 2X SYBR Premix Ex Taq and 20–100 ng cDNA sample was assayed on TP800 (both from Takara Bio, Inc.). The primers used were as follows: USP39, 5′-TTTCCTCAACCTCCACA-3′ and 5′-ATTCAGTCCCACAATACCC-3′; GAPDH, 5′-TGACT TCAACAGCGACACCCA-3′ and 5′-CACCCTGTTGCTGTA GCCAAA-3′. The relative expression of USP39 mRNA was calculated with the 2^−ΔΔCt method, using GAPDH mRNA expression level for normalization. All experiments were repeated at least 3 times.

Total protein was isolated from whole cells using ice-cold protein lysis buffer (1% Triton X-100; 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.1% SDS; 1 mM PMSF; 1 mM EDTA), followed by 30 min of incubation on ice and centrifugation at 10,000 × g for 10 min at 4°C. Protein concentration was determined by the BCA protein assay (Pierce Biotechnology Inc., Rockford, IL, USA). Protein extracts were separated on a SDS-polyacrylamide gel, blotted onto a polyvinylidene fluoride membrane and incubated with mouse anti-FLAG antibody (Sigma-Aldrich) or mouse anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Western blotting was developed using horseradish peroxidase-conjugated goat anti-mouse IgG and was detected with enhanced chemiluminescence reagent (both from Santa Cruz Biotechnology, Inc.).

After 5 days of infection, cells were trypsinized, resuspended and seeded into 6-well plates at a concentration of 200 cells/well and cultured at 37°C for 14 days. The media were replaced every 3–4 days. At the end of incubation, the cells were washed with phosphate-buffered saline (PBS) twice and fixed with paraformaldehyde. Then, the cells were washed with PBS twice, stained with Giemsa (Sigma-Aldrich) for 10 min, and washed with ddH₂O 3 times, sequentially. The plates were photographed with a digital camera.

The monolayer culture growth rate was determined using a Cellomics ArrayScan (Thermo Scientific). Briefly, after being infected with virus for 5 days, cells at the same density were seeded into flat-bottom 96-well plates and grown under normal conditions. Cultured cells were observed at 1, 2, 3, 4 and 5 days using a Cellomics ArrayScan to evaluate the cell growth with GFP signal. Subsequently, the growth curves of the cells were measured after each experiment. Each experiment was performed in triplicate.

Eighty-five percent confluent lentivirus-infected cells were harvested by centrifugation at 1,200 rpm for 5 min. The pellets were washed twice with cold PBS, fixed with cold 70% ethanol, centrifuged at 1,500 rpm for 5 min to discard ethanol, and resuspended with PBS, sequentially. The suspensions were filtrated through a 400-mesh membrane and centrifuged at 1,200 rpm for 5 min. The cells were stained with propidium iodide (PI) or Annexin V-APC (eBioscience Inc.) and then were analyzed using a BD FACSCalibur Flow Cytometer (BD Biosciences, San Diego, CA, USA). Each experiment was performed in triplicate.

The data shown are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical significance was determined with the Student’s t-test. A p-value <0.05 was considered to indicate a statistically significant difference.

Since there are no reports on the USP39 expression level in breast cancers, the levels of USP39 were detected by immunohistochemical analysis to determine the USP39 expression in human breast cancer tissues. The overall distribution of the USP39 protein in the different histological tissue types is summarized in Table I. Stronger staining of USP39 was observed in the breast tumor tissues than that in the normal breast tissues (fewer normal samples were used due to the difficulty in clinical collection) (Fig. 1). No significant correlation between the metastatic lymph node and non-metastatic lymph node subgroups was noted in regards to USP39 expression levels (Table II). RT-PCR analysis revealed that the expression of USP39 was positive in the SK-BR-3, MCF-7, T-47D, HCC1937 and MDA-MB-231 breast cell lines (Fig. 2A). These data clearly indicate that high levels of USP39 expressed selectively in human breast tumor tissues may contribute to the development of high risk breast cancer.

To investigate the role of USP39 in breast cancer, shRNA targeting USP39 or non-siliencing sequences were cloned into the pGCSIL-GFP lentiviral vector, respectively. Then, the USP39-shRNA lentivirus or the non-silencing shRNA lentivirus expressing GFP were generated and infected into MCF-7 cells. The infection efficiency of lentivirus was >90% after 5 days of infection (Fig. 2B). The qRT-PCR assay revealed that the USP39 mRNA level was reduced by ~88.6% (Fig. 2C). We also determined the level of USP39 protein in USP39-shRNA construct-transfected cells via western blot analysis. In 293T cells co-transfected with the USP39 plasmid and USP39-shRNA construct, the protein level of overexpressed USP39 was significantly reduced by >60% (Fig. 2D). These results indicate that this USP39-shRNA was specific and efficient in inhibiting the expression of USP39.

To examine the effect of USP39-shRNA-mediated downregulation of USP39 on the growth of breast cancer cells, MCF-7 cell proliferation was assayed. Five days after infection, USP39-shRNA and non-silencing shRNA-infected MCF-7 cells were seeded into 96-well plates at the same density, and cell numbers were detected through GFP signal at indicated time points using Cellomics ArrayScan. Compared to the control, USP39-shRNA-infected MCF-7 cells displayed significant decrease in cell proliferation with no obvious growth observed during the entire assay period (5 days) (Fig. 3). This finding indicates that the knockdown of USP39 markedly diminished the cell proliferative ability in breast cancer cells.

We then studied the colony formation capacity of MCF-7 cells treated with the USP39-shRNA lentivirus. The control group and the USP39-shRNA-infected MCF-7 cells were allowed to grow for 14 days to form colonies. USP39 knockdown resulted in a nearly 5-fold decrease in the number of MCF-7 colonies, as compared to the control (Fig. 4).

Based on the finding that inhibition of USP39 in MCF-7 cells markedly inhibits cell proliferation and represses cell colony formation, we further employed cell cycle analysis to uncover the mechanism governing the inhibitory effect of USP39-shRNA on cell proliferation and colony formation. An obvious increase in the G0/G1-phase cell population was observed in the USP39-shRNA-infected MCF-7 cells accompanied by a decrease in the S and G2/M-phase cell population, when compared to these percentages in the control (Fig. 5A and B). Our results revealed that USP39-shRNA exerted an inhibitory effect on breast cancer cell proliferation via G0/G1 cell cycle arrest. In addition, we found that knockdown of USP39 led to an increase in MCF-7 cell apoptosis (Fig. 5C and D).