Human glioblastoma U251 and U87 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Primary human astrocytes were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO₂. All TMZ treatments were performed with 100 µM TMZ (T257; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h, at 37°C. The groups were divided as follow: Scramble (cells transfected with scramble siRNA), siRNA-1 (cells transfected with siRNA-1 targeting USP4) and siRNA-2 (cells transfected with siRNA-2 targeting USP4). TMZ+ indicates cells treated with 100 µM TMZ while PFT+ indicates cells treated with 10 µM PFT. PFT treatment were performed as previously described (33), with 10 µM PFT (Tocris Bioscience, Bristol, UK) for 24 h at 37°C. The cell lines were transfected with 50 nM scramble small interfering RNA (siRNA) or each of the two siRNAs designed for USP4 [siRNA-1: 5′-GGCUCUGGAACAAAUACAU-3′ and siRNA-2: 5′-GGUCGCAGAUGUGUAUAAU-3′]. All transfections were performed using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After 24 h of siRNA transfection, cells were treated with TMZ with or without 10 µM PFT for 24 h at 37°C prior to further analysis.

Human glioblastoma tissue samples and their adjacent controls (n=4) were obtained from patients who underwent surgical resection at the Department of Neurology at Xijing Hospital of the Fourth Military Medical University (Xi'an, China). These samples were confirmed as glioblastoma by pathological diagnosis (34). The present study was approved by the Ethics Committee of the Fourth Military Medical University. Written informed consent was obtained from all the patients.

USP4 siRNA was designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), according to the sequence information of the USP4 gene (GenBank accession no. NM_001251877). The two siRNA sequences used in the study were as follows: (siRNA-1), 5′-GGCUCUGGAACAAAUACAU-3′; and siRNA-2, 5′-GGUCGCAGAUGUGUAUAAU-3′. A siRNA with a scrambled sequence (5′-UUCUCCGAACGUGUCACGU-3′) was used as the negative control. U251 and U87 cells were seeded in 60-mm dishes at a density of 5×10⁵ cells/dish and incubated overnight at 37°C in a humidified atmosphere containing 5% CO₂. Subsequently, cells were transfected with siRNAs using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 24 h, cell lysates were collected for use in subsequent experiments.

Western blotting. U251 and U87 cells were washed with PBS twice and lysed in radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.). Proteins were quantified with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10 µg protein for each sample was separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked at room temperature with 5% milk in Tris-buffered saline/Tween 20 (TBST; containing 20 mM Tris-HCl, 150 mM NaCl and 0.1% Tween 20, pH 7.5) for at least 1 h and then incubated with each primary antibody overnight at 4°C. Primary antibodies against USP4 (ab38510; 1:500), p53 (ab131442; 1:1,000), poly(ADP-ribose) polymerase (PARP; ab32064; 1:1,000) were used, which were purchased from Abcam (Cambridge, MA, USA). Equal loading of samples was verified by immunoblotting for GAPDH (ab128915; 1:10,000; Abcam). Subsequently, the membrane was washed with TBST buffer for at least five times for 5 min each and then incubated with goat anti-rabbit secondary antibody (sc-2004, 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. The protein bands were visualized using a Pierce^™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from U251 and U87 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to manufacturer's protocol. For each sample, 500 ng total RNA was reverse-transcribed using a Promega Reverse Transcription System (Promega Corporation, Madison, WI, USA), and the resulting complementary DNA was diluted 40 times. qPCR was then performed using a QuantiTect SYBR^® Green PCR kit (204141; Qiagen China Co., Ltd., Shanghai, China) in 96-well optical reaction plates and the ABI StepOne Plus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR reaction cycle was 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 sec and an annealing/elongation step at 60°C for 30 sec. The following primer sequences were used: Human GAPDH, 5′-GAAGATGGTGATGGGATTTC-3′ (forward) and 5′-GAAGGTGAAGGTCGGAGTC-3′ (reverse); human USP4, 5′-CCTGGGCTCTGTGGACTTG-3′ (forward) and 5′-TGTTGATTTCGGCTTCATACTC-3′ (reverse). USP4 transcription level was normalized to GAPDH as reference gene. Relative expressions values for are presented as fold-increase in relation to control. The actual values were calculated using the 2^−ΔΔCq method (35).

U251/U87 cells transfected with control siRNA or USP4 siRNA were treated with 100 µM TMZ for 24 h at 37°C. TMZ-treated and untreated cells were plated at a density of 1×10⁴ cells/well in a 48-well plate. Viable cells were then stained with 0.25 mg/ml MTT [also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] for 1 h. The media were then removed, and the formazan crystals produced were dissolved by the addition of dimethyl sulfoxide. Cell viability was determined according to the absorbance at 540 nm.

Wild-type U251/U87 cells and U251/U87 cells transfected with control or USP4 siRNA were treated with 100 µM TMZ or 10 µM PFT for 24 h. The TMZ-treated and untreated cells were seeded into 6-well plates at a density of 1×10⁶ cells/well for 24 h. Subsequently, the cells were collected and stained with FITC Annexin V Apoptosis Detection Kit I (cat. no. 556547; BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol, and then analyzed by flow cytometry (FACSCalibur; BD Biosciences).

Tissues were fixed with 10% formalin for 2 h at room temperature, embedded in paraffin and then sectioned into 5-mm slides for USP4 expression analysis. The specimens were blocked for 1 h at room temperature with 10% sheep serum (Beyotime Institute of Biotechnology, Haimen, China). The primary antibody was anti-USP4 rabbit polyclonal antibody (ab3850; Abcam; 1:100 dilution) at 4°C overnight, and the secondary antibody was goat anti-rabbit horseradish peroxidase-conjugated antibody (ab6721; Abcam; 1:1,000) at room temperature for 1 h. The detailed IHC procedure was described previously (36).

Data are presented as the mean ± standard error of at least three separate experiments. To assess significant differences between two groups, Student's t-test was performed. Statistical calculations were performed using IBM SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). P-values of <0.05 were considered to denote differences that were statistically significant.

To investigate the role that USP4 plays in glioblastoma, the mRNA and protein expression levels of USP4 were first examined in the glioblastoma cell lines U251 and U87. The RT-qPCR results demonstrated that UPS4 mRNA expression was significantly increased in U251 and U87 cell lines by ~3 folds, as compared with primary human astrocytes as negative control cells (Fig. 1A). The protein level of USP4 was also evidently increased in the two cell lines (Fig. 1B). Next, the expression of USP4 in glioblastoma tissues was tested by immunohistochemical staining. The results revealed that USP4 was highly expressed in cancer tissues compared with control tissues (Fig. 1C).

In order to further explore the function of the elevated USP4 in glioblastoma, USP4 was knocked down by two different siRNA sequences in U251 and U87 glioblastoma cell lines. As shown in Fig. 2A, USP4 were efficiently knocked down by the two siRNAs in U251 and U87 cell lines. Subsequent to the UPS4 knockdown with these two siRNA sequences, glioblastoma cells were treated with TMZ. Notably, USP4 knockdown alone did not affect the cell viability, which was assessed by an MTT assay. However, when USP4 knockdown cells were treated with TMZ, the cell viability was significantly decreased compared with the scramble-transfected cells treated with TMZ, suggesting that USP4 attenuated the anti-cancer function of TMZ in the two glioblastoma cell lines (Fig. 2B and C). Furthermore, the level of cleaved PARP was also examined by western blotting in order to analyze the apoptotic status. The results demonstrated that cleaved PARP levels were elevated when cells were treated with TMZ. In addition, a marked increase in cleaved PARP was observed in cells transfected with USP4 siRNA, suggesting that USP4 inhibited the apoptosis induced by TMZ (Fig. 2D and E).

Since knockdown of USP4 promotes apoptosis, the expression of the apoptosis-associated protein P53 was then examined. The results revealed that USP4 knockdown by the two siRNA sequences led to a strong induction of P53 expression in U251 and U87 cells (Fig. 3A and B).

USP4 mediates chemoresistance via inhibiting P53 in glioblastoma. To further investigate the role of P53 in USP4-mediated chemoresistance, P53 activity was inhibited by a P53-specific inhibitor, pifithrin-α hydrobromide (PFT). As reported earlier, USP4 knockdown resulted in an increase in P53 expression, as well as apoptosis indicated by cleaved PARP. Following the administration of PFT, P53 protein level is slightly affected but the level of cleaved PARP reduced dramatically revealed by western blotting suggesting that USP4 facilitates chemoresistance by inhibiting P53 in both cell lines (Fig. 4A and B). Furthermore, the apoptosis mediated by USP4 knockdown was also analyzed by flow cytometry. In U251 cells, the results indicated that TMZ treatment increased the Annexin V and propidium iodide (PI) double-positive cells from 0.6 to 39%. When USP4 knockdown cells were treated with TMZ, the Annexin V and PI double-positive cells increased to 52.5% (siRNA-1) and 45.6% (siRNA-2), suggesting that an improved chemotherapy effect may be acquired when USP4 is absent in glioblastoma. Finally, when PFT was added in TMZ-treated USP4 knockdown cells, the percentage of apoptotic cells was decreased to 42.0% (siRNA-1) and 35.4% (siRNA-2), suggesting that USP4-mediated chemoresistance is dependent on P53 activity (Fig. 4C). Similarly, in U87 cells, USP4 knockdown facilitated the drug effect of TMZ, while P53 inhibitor reversed this effect (Fig. 4D).