Introduction
Glioma is one of the deadliest tumours, which essentially occurs in the central nervous system [31], and poor outcome is commonly observed in glioma patients due to the high invasiveness and resistance [22,30]. Vemurafenib is a selective small molecule in-hibitor of mutant BRAFV600E, and it has an 50% re-sponse in patients with the BRAFV600E mutant [1].A previous clinical trial showed that usage of this inhibitor induced apoptosis of cells, and then caused resistance and progression [27]. However, effective therapeutic for colorectal cancer (CRC) was still lacking. Deubiquitinases (DUBs) comprise a group of enzymes that can remove ubiquitin from proteins, leading to the changes in stability and activity of target proteins [4,19]. Members of DUBs could be divided into 5 groups, including ubiquitin-specific proteases (USPs), ovarian tumour proteases (OTUs), ubiquitin C-terminal hydrolases (UCHs), Machado-
Joseph disease protein domain proteases (MJDs), and JAMM motif proteases [3,15]. Among them, a recent study found that depletion of USP38 in mice presents the genome aberration in comparison with wild type mice; besides, a recent study noted that ubiquitin-specific protease 38 (USP38) plays an important role in the maintenance of the integrity of genome in cells and mice, and thus reduction of USP38 expression might lead to tumourigenesis [7,11,13]. Thus, we thought that overexpression of USP38 might suppress the development of colon cancer. In this study, we found that overexpression of USP38 combined with vemurafenib treatment decreased the expression of MMPs and LAMPs, with the increased expression of pro-apoptotic proteins, resulting in the reduction in cellular proliferation rate and tumour metastasis ability. We further noticed that these effects might be mediated by inhibition of JAK2/STAT3 signalling pathway. Thus, we thought that overexpression of USP38 might be a therapeutic target in the treatment of glioma.
Material and methods
Reagents
SNX10 (SAB2107086) was purchased from Sigma. USP38 (ab72244), p53 (ab26), BAX (ab32503), JAK2 (ab108596), JAK2 (phospho Y1007 + Y1008) (ab32101), STAT3 (ab119352), STAT3 (phospho Y705) (ab76315), LAMP2 (ab13524), LAMP1 (ab25630), MMP-9 (ab38898), MMP-2 (ab97779), E-cadherin (ab76055), MMP-2 (ab254516), MMP-9 (ab253227), ICAM-1 (ab252355), and VCAM-1 (ab201278) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Abcam.
Cell culture and model construction
U-87MG (HTB-14) and U-138MG (HTB-16) cell lines were purchased from ATCC. 293T (GNHu17) cells were purchased from the cell bank of the typical culture preservation committee of the Chinese Academy of Sciences. Full length of USP38 DNA was obtained using PCR method with the following primer: Forward: 5˘-CGCTGCCGGTCTACGATAAA-3˘, Reverse: 5˘-TCGATGGCACGGTTGTACTT-3˘. DNA of USP38 and pcDNA3.1-blank vector were digested with BsmBI (R0580S, NEB) and BamHI (R3136S, NEB), then the USP38 overexpression vector was constructed after being linked with DNA ligase (M0202S, NEB). USP38 overexpression vector was transfected into HCT 116 cells using Lipofectamine 2000 Transfection Reagent (11668500, Thermo). Stable expressed cells were screened using 800 µg/ml. USP38 knockdown vector was constructed according to previous description [39]. USP38 oligos were obtained using the following primer: Forward: 5˘-CACCGAGGCTTGCCAATGCTACAGA-3˘, Reverse: 5˘-AACTCCGAACGGTTACGATGTCTC-3˘. Oligos and CRISRP vector were digested with BsmBI (37°C for 30 min). Then, digested vector and oligos were liked with Quick Ligase and transfected into 293T cells to construct a lentiviral vector. U-87MG and U-138MG cells were transfected with lentiviral vector, and stable expressed cells were screened using 2 µg/ml puro. Cells were cultured in H-DMEM medium with 10% fetal bovine serum (FBS) at a 37°C atmosphere supplied with 5% CO2. Then, cells were divided into 4 groups: normal group (NC in U-87MG cell model, MC in U-138MG cell model), vemurafenib-treated group (VT in U-87MG cell model, MT in U-138MG cell model), vemurafenib-treated combined with USP38 overexpression group (VO in U-87MG cell model, MO in U-138MG cell model), and vemurafenib-treated combined with USP38 inhibition group (VI in U-87MG cell model, MI in U-138MG cell model). In the vemurafenib treated group, cells were treated with 10 µM vemurafenib for 24 h [23].
CCK-8 assay
Cell Counting Kit-8 (CCK-8) assay was performed according to the protocol of CCK-8 kit (CA1210, Solarbio). Briefly, cells were firstly seeded into each well of a 96-well plate at a concentration of 1 × 105, and then they were divided into 4 groups and treated as previously described. Then, cells were incubated with CCK-8 reagent for 4 h, and the OD value at 450 nm was measured using a microplate reader (UV-3200S, MAPADA). The viability rate was measured using the following formula: viability rate = (ODExperimental – ODBlank)/(ODControl – ODBlank). Each experiment was repeated 3 times independently.
Wound healing assay
Cells were seeded into a 6-well plate at a concentration of 1 × 105, and then they were treated as previously described. Then a cell straight line was made using 10 µl tips. After washing with sterile PBS the cells were cultured and the image was acquired after being cultured for 24 h.
RNA extraction
RNA extraction was performed according to the protocol of total RNA extraction kits (R1200, Solarbio). Cells and tumour tissues were firstly lysed with lysis buffer, and then incubated with chloroform for 5 min. Then, samples were transferred into an absorption tube and centrifuged at 12,000 rpm for 2 min. After washing with buffer, RNA samples were eluted with elution buffer. The concentration of RNA was measured using an ultramicro-DNA analyser (NanoGenius, MAPADA). RNA samples were stored at –80°C until the following experiments were performed.
Reverse transcription and real-time quantitative polymerase chain reaction
Reverse transcription and quantitative polymerase chain reaction (qPCR) were performed according to the protocol of TaqMan One Step RT-qPCR Kit (T2210, Solarbio). Briefly, reaction mixture was made up as recommended; the primers used in this experiment are as follows: MMP-2: Forward: 5˘-AACCCTTGGAGCCAATGGAGACT-3˘, Reverse: 5˘-GGTTCTAAGGCAGCCAGCAGTG-3˘; MMP-9: Forward: 5˘-ATCCCTCAACATCGCAACTGT-3˘, Reverse: 5˘-CAGCCTC TGGTAGATTATCAAGC-3˘; ICAM-1: Forward: 5˘-CTAGCTAG CATGGCTCCCAGCAGCCCC-3˘, Reverse: 5˘-CGGATATCGGGAGGCGTGGCTTGTGTG-3˘; VCAM-1: Forward: 5˘-GTGA ATATGACATGCTTGAGCCAGG-3˘, Reverse: 5˘-CATCCTTCA ACTGGGCCTTTCG-3˘. The reaction was performed with the following steps: Reverse transcription at 50°C for 20 min, degeneration at 95°C for 3 min, and the following steps repeated for 40 cycles: degeneration at 95°C for 15 s, annealing at 58°C for 30 s, and extending at 72°C for 60 s. Each experiment was repeated 3 times independently. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Expression of each target gene was calculated using the 2–DDCq method [17].
Western blotting analysis
Cells were grouped and treated as previously described, then cells and tumour tissues were lysed with lysis buffer, and protein samples were collected after being centrifuged at 12,000 rpm for 10 min. Concentrations of protein samples were measured using BCA assay, and 60 µg protein samples were used to perform the Western blotting analysis. Briefly, proteins were separated using 10% SDS-PAGE electrophoresis, and proteins were transferred onto a PVDF membrane using a semi-dry transfer blotter. Membranes were blocked with 5% skimmed milk and then incubated with primary antibody at 4°C overnight and incubated with secondary antibody for 1 h at room temperature. Grey value of proteins were detected using chemiluminescent immunoassay. GAPDH was used as an internal control, and each experiment was repeated 3 times independently.
ELISA
ELISA was performed according to the protocol of ELISA kits. Briefly, cultured medium was collected and added into each well of a 96-well plate. Then, samples were incubated with antibody for 1 h at room temperature, and after washed with washing buffer, samples were incubated with TMB solution for 10 min at room temperature. After incubation with stop solution for 10 min at room temperature, the OD value at 450 nm was detected using a microplate reader (UV-3200S, MAPADA).
Statistical analysis
The data are presented as the mean ± S.E.M. Each experiment was repeated 3 times independently. One-way ANOVA was used to analyse the differences between groups using SPSS 22.0 software. P-value < 0.05 was set as a statistically significant difference.
Results
Detection of cellular viability rate of cells
The viability rate in the NC, VT, VO, and VI groups of U-87MG cells was 100.0 ±5.3, 82.3 ±4.3, 60.1 ±3.2, and 94.6 ±5.0, respectively, and the viability rate of NC, VT, VO, and VI groups of U-138MG cells was 100.0 ±4.8, 86.5 ±4.1, 64.2 ±2.8, and 94.7 ±4.6, respectively (Fig. 1A). The viability rate was significantly decreased in the VT and VO groups compared with the NC group (p < 0.05), and the viability rate was significantly decreased in the VO group and significantly increased in VI group compared with the VT group (p < 0.05, Fig. 1A). The expression of USP38 in the normal, overexpression, and inhibition groups of the U-87MG and U-138MG cell models are shown in Figure 1B, and significantly increased expression of USP38 was detected in the overexpression group while significant reduction of USP38 expression was detected in the inhibition group, indicating the successful establishment of the cell model. And as shown in Figure 1C, the distance of scratch in the NC, VT, VO, and VI groups of U-87MG cells was 1.3 ±0.1, 1.1 ±0.1, 1.9 ±0.2, and 0.7 ±0.1 mm and 0.9 ±0.1, 1.3 ±0.1, 1.7 ±0.1, and 0.8 ±0.1 mm, respectively. These results indicate that overexpression of USP38 enhances the effect of vemurafenib on inhibition of the proliferation and metastasis ability of cancer cells.
Detection of metastasis-related actors in cell models
As shown in Figures 2 and 3, the expression of MMP-2 in cultured medium of the NC, VT, VO, and VI groups of U-87MG cells was 1.7 ±0.1, 1.3 ±0.1, 0.6 ±0.1, and 1.6 ±0.1, respectively, and the expression was 2.5 ±0.2, 2.1 ±0.2, 1.4 ±0.1, and 2.6 ±0.2, respectively, in culture medium of U-138MG cells in the MC, MT, MO, and MI groups. The expression of MMP-9 in each group of U-87MG cells was 1.9 ±0.1, 1.6 ±0.1, 1.1 ±0.1, and 1.8 ±0.1, and the expression was 2.8 ±0.2, 2.2 ±0.2, 1.5 ±0.1, and 2.7 ±0.2, respectively, in the culture medium of U-138MG cells.
The expression of ICAM-1 in each group of the U-87MG cell model was 2.3 ±0.2, 1.9 ±0.1, 1.4 ±0.1, and 2.2 ±0.2, respectively, and the expression was 2.9 ±0.2, 2.5 ±0.2, 1.8 ±0.1, and 2.7 ±0.2, respectively, in the culture medium of U-138MG cells. The expression of VCAM-1 in each group of the U-87MG cell model was 2.6 ±0.2, 2.3 ±0.2, 1.5 ±0.1, and 2.7 ±0.2, respectively, and the expression was 3.2 ±0.2, 2.7 ±0.2, 2.1 ±0.2, and 3.1 ±0.2, respectively, in the culture medium of U-138MG cells.
Detection of metastasis-related actors in cultured medium and serum sample
As shown in Figures 4 and 5, the concentration of MMP-2 in cultured medium of the NC, VT, VO, and VI groups of U-87MG cells was 423.2 ±24.9, 394.1 ±23.2, 286.3 ±16.8, and 445.7 ±26.2 pg/ml, respectively, and the concentrations of MMP-2 were 652.1 ±38.4, 588.4 ±34.6, 496.2 ±29.2, and 646.5 ±38.0 pg/ml in the MC, MT, MO, and MI groups of the U-138MG cell model, respectively. The concentration of MMP-9 in each group of the U-87MG cell model was 472.3 ±27.8, 415.2 ±24.4, 326.1 ±19.2, and 498.4 ±29.3 pg/ml, respectively, and the concentration in each group was 772.1 ±45.4, 695.8 ±40.9, 604.2 ±35.5, and 732.6 ±43.1 pg/ml, respectively, in each group of the U-138MG cell model. The concentration of ICAM-1 in each group of the U-87MG cell model was 132.1 ±7.8, 112.6 ±6.6, 85.4 ±5.0, and 129.6 ±7.6 pg/ml, respectively, and the concentration was 362.1 ±21.3, 300.8 ±17.7, 231.6 ±13.6, and 348.7 ±20.5 pg/ml, respectively, in each group of the U-138MG cell model. The concentration of VCAM-1 in each group of the U-87MG cell model was 230.6 ±13.6, 204.2 ±12.0, 145.3 ±8.5, and 229.1 ±13.5 pg/ml, respectively, and the concentration was 485.1 ±28.5, 432 ±25.4, 348.5 ±20.5, and 475.3 ±28.0 pg/ml, respectively, in each group of the U-138MG cell model.
Expression of metastasis-related proteins in cell models
As shown in Figures 6 and 7, the expression of E-cadherin in the NC, VT, VO, and VI groups of the U-87MG cell model was 0.79 ±0.05, 1.25 ±0.07, 1.47 ±0.09, and 1.03 ±0.06, respectively, and the expression of E-cadherin in the MC, MT, MO, and MI groups of the U-138MG cell model was 0.54 ±0.03, 0.62 ±0.04, 0.74 ±0.04, and 0.06 ±0.01, respectively. The expression of MMP-2 in each group of the U-87MG cell model was 0.97 ±0.06, 0.96 ±0.06, 0.82 ±0.05, and 1.35 ±0.08, respectively, and the expression was 1.02 ±0.06, 0.76 ±0.04, 0.42 ±0.02, and 1.00 ±0.06, respectively, in each group of the U-138MG cell model. The expression of MMP-9 in each group of the U-87MG cell model was 1.13 ±0.07, 0.74 ±0.04, 0.21 ±0.01, and 0.97 ±0.06, respectively, and the expression was 1.12 ±0.07, 1.05 ±0.06, 0.55 ±0.03, and 0.89 ±0.05, respectively, in each group of the U-138MG cell model. The expression of LAMP-1 in each group of the U-87MG cell model was 1.10 ±0.06, 1.10 ±0.06, 0.72 ±0.04, and 1.66 ±0.10, respectively,
and the expression was 0.82 ±0.05, 0.70 ±0.04, 0.32 ±0.02, and 0.82 ±0.05, respectively, in each group of the U-138MG cell model. The expression of LAMP-2 in each group of the U-87MG cell model was 1.21 ±0.07, 1.08 ±0.06, 0.80 ±0.05, and 1.64 ±0.10, respectively, and it was 1.61 ±0.09, 1.53 ±0.09, 1.14 ±0.07, and 1.89 ±0.11, respectively, in each group of U-138MG cells.
Activation of JAK2/STAT3 signalling pathway in cell models
As shown in Figures 8 and 9, the ratio of p-STAT3/STAT3 in NC, VT, VO, and VI groups of the U-87MG cell model was 0.26 ±0.02, 0.19 ±0.01, 0.17 ±0.01, and 0.23 ±0.01, respectively, and the ratio was 0.30 ±0.02, 0.31 ±0.02, 0.12 ±0.01, and 0.24 ±0.01, respectively, in the MC, MT, MO, and MI groups of the U-138MG model. The ratio of p-JAK2/JAK2 in each group of the U-87MG cell model was 0.21 ±0.01, 0.17 ±0.01, 0.12 ±0.01, and 0.20 ±0.01, respectively, and the ratio was 0.23 ±0.01, 0.22 ±0.01, 0.10 ±0.01, and 0.19 ±0.01, respectively, in each group of the U-138MG cell model. The expression of SNX10 in each group of the U-87MG cell model was 0.31 ±0.02, 0.60 ±0.04, 0.96 ±0.06, and 0.40 ±0.02, respectively, and the expression was 0.11 ±0.01, 0.38 ±0.02, 0.53 ±0.03, and 0.03 ±0.01, respectively, in each group of the U-138MG cell model. The expression of BAX in each group of the U-87MG cell model was 0.45 ±0.03, 0.80 ±0.05, 0.96 ±0.06, and 0.55 ±0.03, respectively, and the expression was 0.52 ±0.03, 1.15 ±0.07, 1.32 ±0.08, and 0.81 ±0.05, respectively, in each group of the U-138MG cell model. The expression of p-53 in each group of the U-87MG cell model was 0.36 ±0.02, 0.76 ±0.04, 0.90 ±0.05, and 0.48 ±0.03, respectively, and the expression was 0.56 ±0.03, 1.10 ±0.06, 1.28 ±0.08, and 0.85 ±0.05, respectively, in each group of the U-138MG cell model. The expression of SOX11
in each group of the U-87MG cell model was 0.15 ±0.01, 0.59 ±0.03, 0.92 ±0.05, and 0.47 ±0.03, respectively, and the expression was 0.30 ±0.02, 0.61 ±0.04, 0.74 ±0.04, and 0.18 ±0.01, respectively, in each group of the U-138MG cell model.
Discussion
Glioma is one of the most frequent tumours in adults. The proportion of glioma in intracranial tumours is 40.49% according to a previous study [20], and the average survival period of glioma patients after diagnosis is less than 5 years [2]; thus, glioma is a malignant disease with high mortality and disability. A small molecule inhibitor of BRAFV600E, vemurafenib has been successfully used in the treatment of metastatic melanoma patients [16]; however, few studies focus on the effect of vemurafenib on the treatment of glioma.
Metastasis is the final step of tumour formation and leads to the morbidity and mortality of cancer patients, in which the spread of cancer cells from the primary tumour to nearby tissues plays an important role [34]. The underlying mechanism of cancer metastasis is called epithelial-mesenchymal transition (EMT), which includes destruction of intercellular relationships, extracellular matrix (ECM) breakdown, and regulation of cleavage of basement membrane by matrix metalloproteinase (MMP) activity [24]. A previous study found that MMPs are critical factors in the promotion of angiogenesis and metastasis processes via regulation of EMT [12]. MMPs contains a family of metalloproteinases with a zinc-binding domain, and present a critical function in degradation of ECM components. They were proven to be involved in the regulation of cancer metastasis over 50 years ago [8], and recently a study indicated that MMPs also play a critical role in the regulation of angiogenesis [12], resulting in migration of tumour cells into nearby tissues. MMP-2 and MMP-9 are the subtypes of the MMPs family, which can degrade type IV collagen and are highly expressed in human cancer. Using an in vivo experiment, researchers found that MMP-2 and MMP-9 presents a cooperative effect in promoting the invasiveness of tumour keratinocytes [18]. Another study also found that MMP-2 and MMP-9 lead to the angiogenesis of cancer via activation of transforming growth factor b (TGF-b) via a CD44-dependent manner, resulting in the promotion of tumour growth and invasion [37]. LAMP-1 and LAMP-2 are major constituents of lysosomal membrane and are classified as type I transmembrane proteins [33]. A previous study showed that knockout of LAMP-1 and LAMP-2 in mice developed into embryonic lethality, indicating that these 2 proteins play an important role in maintaining cellular homeostasis [9]. More and more research has shown that LAMP-1 and LAMP-2 play a pro-invasion and pro-metastasis role in progression of cancer, and elevated expression of LAMP-1 has been found in breast cancer cells associated with the migration of cancer cells [28]. Besides, LAMP-1 and LAMP-2 expression also binds with E-selectin and galectin-3 through poly-N-acetyl-lactosamine (polyLacNAc), and further activates ERK and p38 signalling pathways, leading to the expression of MMPs and degradation of ECM [6]. Here, we found that the expression of pro-metastasis and pro-invasion was decreased in vemurafenib combined with USP38 overexpression in the treatment group, indicating that overexpression of USP38 presented an anti-tumour effect, while the detailed mechanism was not clear.
Sex-determining region Y-related HMG-box 11 (SOX11) is a type of structurally related transcription factor and belongs to group C of the SOX family. Expression of transcription factors is strictly regulated by binding with DNA domains; under this mechanism, SOXs regulate differentiation, organogenesis, and other cellular process. A recent study discovered that overexpression of SOX11 would suppress the migration and invasion of prostate cancer cells [26], and activation of SOX11 promoter was significantly higher in cancer cells compared with normal group [32]. A previous study noted that STAT3 is one of the downstream targets of SOX11 via activation of JAK signalling pathway [21]. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway play a critical role in regulation of tumour development and survival. Among family members of the JAK and STAT family, JAK2/STAT3 is the most studied signalling pathway in the pathogenesis of cancer via induction of EMT [36]. It was previously shown that activation of JAK2/STAT3 increases the metastasis ability of cancer cells via up-regulation of EMT-inducing transcription factors [14]. JAK2/STAT3 also activates in a gastrin-dependent manner and further inhibits the cellular adhesion process via inhibition of MMP secretion and the expression of E-cadherin [10]. In the present study, we found that overexpression of USP38 enhanced the effect of vemurafenib on inhibition of JAK/STAT3 signalling pathway activation, and that might be the mechanism of inhibition of pro-metastasis and pro-invasion molecule expression, leading to the anti-tumour effect.
P53 could induce the expression of many genes involved in multiple signalling pathways related to cellular apoptosis [25]. BAX is a member of the BH3 domain-only pro-apoptotic proteins; activation of p-53 further activates the BH3-only proteins, leading to the permeabilization of the mitochondrial outer membrane [29]. Sorting nexin 10 (SNX10) is a member of the sorting nexins (SNXs) family, which contains the phox homology domain, targeting SNXs to endosome membrane via binding with phosphoinositide to regulate endosomal transport [5]. Recently a study found that SNX10 plays a critical role in alcohol-induced liver injury and steatosis via regulation the degradation of LAMP2 [35], and thus has been regarded as a tumour suppressor protein in CRC [38]. In this study, we found that overexpression of USP38 enhanced the inhibition effect of vemurafenib on expression of SNX10, presenting an anti-tumour effect.
Here, we noticed that vemurafenib treatment elevated the expression of BAX and p53, inducing the reduction of cellular proliferation rate. Also, vemurafenib treatment reduced the expression of MMPs and LAMPs, resulting in a reduction of the degradation of ECM and metastasis ability of cancer cells. And we also noticed that these effects were increased by overexpression of USP38, and these effects might be mediated by reduction of activation of JAK2/STAT3 signalling pathway. Thus, USP38 might be a therapeutic target in the treatment of glioma.
Disclosure
The authors report no conflict of interest.
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