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Medical School of Nantong University, Nantong, 226007, ChinaDepartment of Oncology, Affiliated Hospital of Nantong University, Nantong, 226001, ChinaDepartment of Radiotherapy, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Radiotherapy, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Pneumology, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of General Medicine, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Radiotherapy, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
USP15 was highly expressed in NSCLC cancerous tissues and cells.
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USP15 knockdown reduced NSCLC progression in vitro and in vivo.
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USP15 knockdown inhibited the expression of MMP3 and MMP9 in NSCLC cells.
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High USP15 and MMP3 expression associated with poor prognosis.
Abstract
Aberrant ubiquitin modifications caused by an imbalance in the activities of ubiquitinases and de-ubiquitinases are emerging as important mechanisms underlying non-small cell lung cancer (NSCLC) progression. The deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) has been identified as an important factor in oncogenesis and a potential therapeutic target. However, the expression profile and function of USP15 in NSCLC remain elusive. In the present study, we investigated the expression pattern and the potential biological functions of USP15 in NSCLC both in cells and animal models. Our data revealed that USP15 was highly expressed in NSCLC tissues and cells compared with normal counterpart. We subsequently knocked down USP15 expression in two NSCLC cell lines, which significantly suppressed cell proliferation. In addition, knocking down USP15 expression reduced NSCLC cell migration and invasion according to the results from Matrigel-Transwell analysis. NSCLC animal model results showed that USP15 knockdown also reduced NSCLC size. Biochemical analysis revealed that USP15 knockdown inhibited matrix metalloproteinase (MMP)3 and MMP9 expression. Furthermore, high levels of USP15 and MMP3 expression were associated with poor prognosis in NSCLC. In conclusion, the results from the present study suggest that the high expression of USP15 promotes NSCLC tumorigenesis. Therefore, it is proposed that USP15 and MMPs may represent novel biomarkers for NSCLC progression and prognosis.
]. Although 30-40% of the patients exhibit good responses to cytotoxic therapy initially, most, if not all, of the patients will eventually relapse during or after treatment [
]. However, the downstream function mediated by the majority of DUBs remains poorly understood. DUBs belong to a family of proteases that are ubiquitin-specific [
]. First discovered in 1999, ubiquitin-specific peptidase 15 (USP15) is a ubiquitously-expressed DUB that contains a zinc finger domain critical for its de-ubiquitinase activity [
]. In another study, USP15 was shown to specifically deubiquitinate kelch like ECH associated protein 1 and increase nuclear factor-erythroid factor 2-related factor 2 expression, which enhanced chemoresistance in breast cancer [
]. Recent studies have also revealed a de novo stabilizing function of USP15 on newly synthesized proteins, which sheds new light on the potentially multifaceted roles of DUBs [
Matrix metalloproteinases (MMPs) have the ability to degrade extracellular matrix (ECM) proteins and expose binding sites within the matrix molecules to facilitate tumor progression and metastasis [
In the present study, we found that compared with that in the normal control tissues and cell lines, USP15 was highly expressed in NSCLC tumor tissues and cells. USP15 knockdown significantly reduced the proliferation and invasion of NSCLC cells by downregulating the expression of MMP3 and MMP9. In addition, results from the NSCLC animal models showed that USP15 knockdown inhibited NSCLC growth. High levels of USP15 and MMP3 expression were also associated with poor prognosis. Therefore, this study suggests that upregulation of USP15 is important for NSCLC pathogenesis by regulating the expression of matrix metalloproteinases, rendering USP15 to be a novel target for NSCLC treatment.
Materials and methods
Tissues
NSCLC and healthy control tissues (30 cases, 14 male and 16 female) were collected from patients at The First Affiliated Hospital of Soochow University (Soochow, China) between Jan 2017 to Mar 2018. This project obtained the subjects’ written informed consent and institutional review board approval of The First Affiliated Hospital of Soochow University.
Cells and plasmids
The lung epithelial cell line BEAS-2B and lung cancer cell A549, were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and maintained in DMEM (Hyclone, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., USA) and penicillin and streptomycin (100 μg/ml). Lung cancer cell H1299 were also purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and maintained in RPMI-1640 (Hyclone, USA) medium supplemented with 10 % FBS and penicillin and streptomycin (100 μg/ml). All cells were cultured at 37˚C in a humidified atmosphere of 5% CO2 and were confirmed to be free of mycoplasma contamination.
The USP15 knockdown lentivirus was purchased from Hanyin Biotech, Co. Ltd. (Shanghai, china). To obtain stable cell lines, lentivirus supernatant was added to the NSCLC cells (MOI:10), followed by puromycin (1 μg/ml) selection after 2 weeks at 37˚C in a humidified atmosphere of 5% CO2.
Immunofluorescence
Immunofluorescence experiments were performed. The slides were fixed with 4 % paraformaldehyde and blocked with PBS containing 0.3 % TritonX-100 and 5 % bovine serum albumin (BSA) for 30 min. Antibodies were incubated at 4°C overnight. The slides were washed 6 times with PBST (PBS containing 0.3 % TritonX-100) for a total of an hour and incubated with secondary antibodies for 1 hour at room temperature. The samples were observed with a microscope (Nikon).
Cell Counting Kit-8 (CCK-8) assay
A CCK-8 assay was conducted according to the manufacturer's protocols (Dojindo, Kumamoto, Japan). Briefly, NSCLC cells with or without USP15 knockdown were seeded into 96-well plates (2,000 cells/ well) and detected for 5 days at 37˚C in a humidified atmosphere of 5% CO2. Following the incubation, 10 µl CCK-8 reagent (5 mg/ml) diluted in the culture medium was added to the cells and cultured for 2 h at 37˚C. The absorbance in each well was detected at 490 nm using a microplate reader (Biotek ELx800; BioTek Instruments, Inc.).
Matrigel-Transwell assay
Cell invasion after USP15 knockdown was examined. Briefly, NSCLC cells were suspended in serum-free DMEM or 1640 medium and plated into the upper chamber of Matrigel (BD)-coated Transwell inserts (8-μm). At the same time, medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h at 37˚C, cells on the upper surface of the insert were gently removed with a cotton swab. Cells that had invaded onto the lower surface of the insert were fixed with 4% paraformaldehyde for 20min and stained with 1% crystal violet for 30 mins at room temperature. Stained cells were visualized in five randomly selected microscopic fields of view.
Western blot analysis
The cells were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China) containing protease inhibitors (Roche, Complete Mini). The total protein was quantitated by bicinchoninic acid assay kit (Beyotime, Haimen, China). Proteins (30-50 μg/lane) were isolated and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk at room temperature for 60 min and incubated with primary antibodies (1:1,000 dilution) overnight at 4˚C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1h at room temperature (Jackson lab, 1:10,000 dilution) and detected by chemiluminescence (ECL, millipore). The following antibodies were used: Rabbit anti-human USP15 (cat. no. GTX104644; GeneTex, Inc.), mouse anti-β-actin (cat. no. 3700P; Cell Signaling Technology, Inc.), rabbit anti-human MMP2 (cat. no. ab192082; Abcam), rabbit anti-human MMP3 (cat. no. ab53015; Abcam) and rabbit anti-human MMP9 (cat. no. 3852; Cell Signaling Technology, Inc.).
Animal models
16 male-nude mice (6 to 8 weeks-old) purchased from Lingchang Biotech (Shanghai, China) were maintained and treated in accordance to the Institutional Animal Care and Use Committee of the Soochow University. H1299 cells (1 × 106 suspended in 1640) with or without USP15 knockdown were injected into the tail veins of 6–8-week-old nude mice (6-8 mice/group). After 6 weeks, mice were euthanized using CO2 before the lungs were removed and fixed in Bouin's solution overnight at room temperature. The number of nodes that were >0.5 mm in diameter in every lobe of lung were counted.
Hematoxylin-Eosin (H&E) Staining
For H&E staining, paraffin embedded sections are dyed with hematoxylin for 5 minutes after they are deparaffinized and hydrated. Then washed with soft running water, treated with 1 % hydrochloric acid alcohol for seconds and quickly turn blue with 0.6 % ammonia. After that the slices are dyed in the eosin dye solution for 3min. After dehydration, use neutral resin to seal, and the images are captured by a microscope (Nikon).
Statistical analysis
Data were presented as mean±SD. One-way ANOVA followed by the Student-Newman-Keuls tests was performed to determine any differences among the treatment groups using SPSS 13.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference, Repeat each experiment three times.
Results
USP15 expression is upregulated in NSCLC tissues and cell lines
The expression of the USP15 gene has been previously found to be increased in glioblastoma, breast and ovarian cancer compared with normal counterpart [
]. We therefore measured USP15 expression in NSCLC and adjacent normal tissues. We found that the USP15 expression level was significantly higher in NSCLC tissues compared with that in adjacent normal tissues (Fig. 1A and B). We next measured USP15 expression in two different NSCLC cell lines, A549 and H1299, and in the lung normal epithelial cell line, BEAS-2B. Western blot analysis revealed that USP15 expression was higher in the NSCLC cells compared with that in BEAS-2B cells (Fig. 1C).
Fig. 1Expression of USP15 in NSCLC tissues and cells. A, Immunofluorescence analysis of USP15 expression in NSCLC and adjacent control normal tissues. B, Western blot analysis of USP15 expression in NSCLC and adjacent normal tissues (n=5). C, Western blot analysis of USP15 expression in the normal lung epithelial cell line BEAS-2B and in NSCLC cell lines. USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer. Repeat each experiment three times.
To explore the possible function of USP15 during NSCLC progression, we constructed lentivirus particles encoding short hairpin RNA (shRNA) targeting USP15 to knock down USP15 expression in A549 and H1299 cells. USP15 protein expression was significantly decreased in cells transfected with the lentivirus expressing USP15-shRNA compared to negative control (Fig. 1D and 1E).
USP15 inhibition reduces the proliferation and invasion of NSCLC cell lines
To explore the possible physiological function of USP15 in NSCLC, we performed CCK-8 assays to assess the viability of the NSCLC cell lines, A549 (Fig. 2A) and H1299 (Fig. 2B). The CCK-8 assay results showed that the viability of the NSCLC cells was reduced following USP15 knockdown (Fig. 2C).
Fig. 2Knockdown of USP15 reduces NSCLC cell proliferation and invasion. A, Western blotting data following USP15 knockdown in A549 cells. B, Western blotting data following USP15 knockdown in H1299 cells. C, CCK-8 analysis of cell proliferation in negative control and USP15 knockdown A549 cells. D, CCK-8 analysis of cell proliferation in negative control and USP15 knockdown H1299 cells. E and F, Matrigel-Transwell assay in A549 and H1299 cells after USP15 knockdown. G and H, Semi-quantification of the Transwell assay results in E and F. CCK-8, Cell Counting Kit-8; USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer. Repeat each experiment three times.
The effect of USP15 knockdown on cell migration and invasion was subsequently evaluated using Matrigel-Transwell assays. Knocking down USP15 expression significantly decreased cell migration and invasion (Fig. 2C-2F). These results suggest that USP15 serves an active role in NSCLC cell proliferation and invasion.
Inhibition of USP15 reduces the progression of NSCLC in vivo
To study the functional role of USP15 in NSCLC progression further, we injected H1299 cells into the tail vein of nude mice to induce pulmonary tumor nodules (Fig. 3). Compared with that in mice injected with control cells, knockdown of USP15 resulted in the significantly reduced formation of tumor nodules (Fig. 3C). Consistently, the tumor weight was also found to be significantly lower in the USP15-knockdown group compared with that in the control group (Fig. 3D).
Fig. 3Knockdown of USP15 reduces the progression of NSCLC in vivo.
], we determined whether MMPs can regulate USP15-mediated NSCLC cell proliferation and invasion. Western blotting results showed that the knockdown of USP15 significantly decreased the expression of MMP3 and MMP9 in NSCLC cells compared to negative control (Fig. 4A and 4B).
Fig. 4Knockdown of USP15 inhibits the expression of MMPs in NSCLC.
Higher levels USP15 and MMP3 expression are associated with poor prognosis
We then investigated the effects of USP15 and the associated MMPs on the prognosis of patients with NSCLC using the public Gene Expression Profiling Interactive Analysis (GEPIA) database (https://gepia.cancer-pku.cn/detail.php?gene). The results showed that high USP15 and MMP3 expression levels were associated with a poor disease-free survival (P=0.041; n=481; Fig. 5B). However, USP15 and MMP2 or MMP9 were not significantly associated with disease-free survival (Fig. 5A and 5C).
Fig. 5High USP15 and MMP3 expression is associated with poor prognosis in terms of NSCLC.
A, Kaplan-Meier analysis of the effects of USP15 and MMP2 expression on the prognosis of patients with NSCLC using the public GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene).
B, Kaplan-Meier analysis of the effects of USP15 and MMP3 expression on the prognosis of patients with NSCLC using the public GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene).
C, Kaplan-Meier analysis of the effects of USP15 and MMP9 expression on the prognosis of patients with NSCLC using the public GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene).
D, The amount of USP15 protein in shUSP15 after overexpression of MMP9 in rescue experiment*** P < 0.001, * * P < 0.01, Repeat each experiment three times.
Altogether, the results from our study suggest that USP15 promotes the growth and invasion of NSCLC by regulating MMP expression, which may provide a novel target for NSCLC treatment.
Discussion
USP15 is a deubiquitinating enzyme that was first discovered in 1999 and has been reported to serve active roles in various types of malignancies, including brain tumors, colorectal cancer and several other types of cancer [
]. In the present study, USP15 was revealed to be highly expressed in NSCLC, suggesting that USP15 may be used as a beneficial biomarker for NSCLC.
In a previous study, USP15 gene expression was found to be significantly reduced in docetaxel-resistant gastric cancer cells compared with that in their corresponding parental cells using microarray analysis [
]. These observations were subsequently validated using reverse transcription-quantitative PCR[36]. Further analysis was conducted in 11 cancer cell lines of the digestive system, which demonstrated that USP15 expression levels were significantly correlated with docetaxel sensitivity [
]. However, the function and underlying mechanism of USP15 in NSCLC remain to be elucidated. The results of the present study showed that USP15 knockdown significantly reduced the tumorigenic ability of NSCLC cells. In addition, USP15 regulated the migration and invasion of NSCLC cells, which were mediated by modulating MMP3 and MMP9 expression. MMPs are proteolytic enzymes that have been documented to contribute to most stages of tumor progression, including the later stages of invasion and metastasis [
]. The expression of MMP3 in tumor cells has been previously reported to be a prognostic predictor of poor survival in pancreatic, pulmonary and mammary carcinoma [
]. Our in vivo results found that USP15 regulates NSCLC tumor growth and invasion. However, although USP6 expression was previously found to induce MMPs by activating NF-κB [
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