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The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth
GENE-40647; No. of pages: 8; 4C: Gene xxx (2015) xxx–xxx
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth Ying-Li Liu a, Jie Zheng b, Li-Juan Tang a, Wei Han a, Jian-Min Wang a, Dian-Wu Liu a, Qing-Bao Tian a,⁎ a b
Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, China Department of Functional Neurosurgery, Hebei General Hospital, Shijiazhuang, China
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 10 June 2015 Accepted 28 June 2015 Available online xxxx Keywords: USP22 Deubiquitinating enzyme activity HeLa cell Small interfering RNA Cell cycle Apoptosis
a b s t r a c t Ubiquitin-specific protease 22 (USP22) can regulate the cell cycle and apoptosis in many cancer cell types, while it is still unclear whether the deubiquitinating enzyme activity of USP22 is necessary for these processes. As little is known about the impact of USP22 on the growth of HeLa cell, we observed whether USP22 can effectively regulate HeLa cell growth as well as the necessity of deubiquitinating enzyme activity for these processes in HeLa cell. In this study, we demonstrate that USP22 can regulate cell cycle but not apoptosis in HeLa cell. The deubiquitinating enzyme activity of USP22 is necessary for this process as confirmed by an activity-deleted mutant (C185S) and an activity-decreased mutant (Y513C). In addition, the deubiquitinating enzyme activity of USP22 is related to the levels of BMI-1, c-Myc, cyclin D2 and p53. Our findings indicate that the deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, and it promotes cell proliferation via the c-Myc/cyclin D2, BMI-1 and p53 pathways in HeLa cell. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ubiquitin-specific protease (USP), a subfamily of deubiquitinating enzymes (DUBs) (D'Andrea and Pellman, 1998; Reyes-Turcu and Wilkinson, 2009), is responsible for the removal of ubiquitin or polyubiquitin from target proteins, the processing of ubiquitin precursors, and the disassembly of unanchored polyubiquitin by catalyzing the hydrolysis of isopeptide bonds in ubiquitin-protein conjugates (Takayama and Toda, 2010). The growth of many cancers is regulated by USPs: USP8 is related to non-small cell lung cancer (Baykara et al., 2013); USP28 is a potential prognostic marker for bladder cancer (Guo et al., 2014); and USP9X expression correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma (Peng et al., 2013). Therefore, USPs are important factors for cancer. Recent observations have identified an 11-gene poly-comb/cancer stem cell signature that could predict the likelihood of treatment failure in cancer patients (Glinsky, 2005). Ubiquitin-specific protease 22 (USP22) is a new putative cancer stem cell marker involved in the 11-gene polycomb/cancer stem cell signature (Zhang et al., 2008a,b). It belongs to a large family of proteins with ubiquitin hydrolase activity. USP22 is a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA)
Abbreviations: USP, ubiquitin-specific protease; DUBs, deubiquitinating enzymes; USP22, ubiquitin-specific protease 22; qRT-PCR, quantitative real-time polymerase chain reaction. ⁎ Corresponding author. E-mail address: [email protected] (Q.-B. Tian).
transcriptional cofactor complex. Within hSAGA, USP22 regulates the transcription of downstream genes related to epigenetic alteration and cancer progression by removing ubiquitin from histone H2A and H2B (Zhang et al., 2008b). Moreover, USP22 can regulate tumor recurrence, distant metastasis, therapeutic failure and poor prognosis via its functions in some cell signaling pathways: USP22 regulates the cell cycle via the c-Myc/cyclin D2 pathway and down-regulation of p15 and p21 expression in HepG2 cells (Ling et al., 2012); USP22 silencing was also found to lead to reduced expression of cell cycle proteins, including CDK1, CDK2 and Cyclin B1 in human brain glioma cells (Li et al., 2013); USP22 may act as an oncogene in human colorectal cancer as it positively regulates the cell cycle via both the BMI-1-mediated INK4a/ARF pathway and the Akt signaling pathway (Liu et al., 2012); USP22 antagonizes p53 transcriptional activation by deubiquitinating sirt1 to suppress cell apoptosis (Lin et al., 2012). The siRNA-mediated knock-down of USP22 could effectively induce cell cycle arrest by regulating target molecules, such as cyclin D2, p21, p15 and p53, and could also inhibit cell growth. Elevated expression of USP22 can predict shorter intervals of tumor recurrence, distant metastasis, therapeutic failure and poor prognosis in patients with many types of cancer such as colorectal cancer (Liu et al., 2010, 2011), breast cancer (Y. Zhang et al., 2011), gastric cancer (Yang et al., 2011) and others (Ueda et al., 2015). USP22 can regulate the growth and prognosis of many cancers, but little is known about the impact of USP22 on the growth of human cervical cancer cell lines, and whether the deubiquitinating enzyme activity of USP22 is necessary for these effects is not clear. In this
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Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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study, we used siRNA to specifically suppress expression of USP22, and we observed that the knock-down of USP22 could effectively inhibit HeLa cell proliferation and induce cell cycle arrest. We also suggest that USP22 may regulate cell cycle progression by down-regulating p53 expression and up-regulating cyclin D2, BMI-1, c-MYC expression. The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth.
were analyzed by western blotting, and total protein extracts of the pGEX-Ub52 group were purified by glutathione agarose beads and detected by Coomassie Brilliant Blue. The pGEX-6p-1-USP46 and pACT7-USP46 plasmids expressing USP46 were used as positive controls for the USP cleavage assay. The relative intensity of the resulting bands was analyzed by the Odyssey V3.0 software. 2.5. Site-directed mutagenesis and deubiquitinating enzyme activity assay
2. Materials and methods 2.1. Cell culture antibodies and reagents The human HeLa cell line was purchased from American Type Culture Collection (ATCC). The cell line was cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% fetal calf serum (FBS, GIBCO) and 1% penicillin–streptomycin at 37 °C in a 5% CO2 atmosphere. The USP22 antibody (1:500) was from Abcam (UK). β-actin (1:5000) was from Proteintech (USA). Lipofectamine 2000 was from Invitrogen Corp (USA). The reverse-transcription polymerase chain reaction (RT-PCR) kit and primers were from Takara (Japan). 2.2. Transfection of siRNA HeLa cells were seeded in 6-well plates at a concentration of 4 × 105 cells/2 ml in DMEM supplemented with 10% FBS and 1% penicillin– streptomycin. After 24 h, the medium was replaced with fresh medium without antibiotics. Meanwhile, the cells were transfected with siRNA or a negative control oligonucleotide using Lipofectamine RNAiMAX according to the manufacturer's instructions. After incubation for 4–6 h, the medium containing the Lipofectamine RNAiMAX complexes was replaced with fresh DMEM containing 10% FBS. Then, the cells were cultured for subsequent experiments. All siRNAs were obtained from Invitrogen Corp (USA), and the three specific sequences for silencing were: human USP22 siRNA-1, sense 5′-GCU GUU UCA CAA AGA AGC AUA UUC A-3′, and anti-sense 5′-UGA AUA UGC UUC UUU GUG AAA CAG C-3′; siRNA-2, sense 5′-CAG CAG CCC ACG GAC AGU CUC AAC A -3′, and anti-sense 5′-UGU UGA GAC UGU CCG UGG GCU GCU G 3′; siRNA-3, sense 5′-GCC AAG UCC UGU AUC UGC CAU GUC U-3′, and anti-sense 5′-AGA CAU GGC AGA UAC AGG ACU UGG C-3′. The transfection efficiency was assessed by transfection of negative control siRNA. The negative control siRNA sequence was: 5′-UUC UCC GAA CGU GUC ACG UTT ACG UGA CAC GUU CGG AGA ATT-3′. The effect of RNA interference was analyzed by qRT-PCR and western blot analyses. 2.3. Molecular cloning of USP22 The pcDNA3.1-USP22-V5-FLAG plasmid was a gift from Boyko S. Atanassov. The pcDNA3.1-USP22-V5-FLAG plasmid was digested by NotI and XhoI, and then USP22 was inserted via blunt-end ligation. The expression vector pGEX-6p-1 (Amersham Pharmacia Biotech) was digested by SalI and filled in to generate blunt ends. Then, the pGEX6p-1-USP22 plasmid was produced by inserting the complete coding sequence of USP22 into the pGEX-6p-1 plasmid. The pGEX-USP22 plasmid was digested by NotI and BamHI, and then USP22 was inserted via bluntend ligation. The expression vector pAC-T7 plasmid was digested by BamHI and filled in to generate blunt ends. Then, a pAC-T7- USP22 plasmid was produced by inserting the complete coding sequence of USP22 into the pAC-T7 plasmid. 2.4. Deubiquitinating enzyme activity assay of hUSP22 The USP cleavage assays used ubiquitin-met-β-galactosidase (Ub-Met-β-gal) and pGEX-Ub52 (GST-Ub52) (W. Zhang et al., 2011) as model substrates. Total protein extracts of the Ub-Met-β-gal group
Cys 185, which is located within the conserved Cys-box of USP22, was mutated to serine. Arg 98 was mutated to tryptophan, Asn 283 was mutated to serine, Pro 290 was mutated to leucine, and Tyr 513 was mutated to cysteine. The resulting plasmids were pGEX-USP22 (C185S), pGEX-USP22 (R98W), pGEX-USP22 (N283S), pGEX-USP22 (P290L) and pGEX-USP22 (Y513C), respectively. Mutations were confirmed by DNA sequencing. The deubiquitinating enzyme activity assay method was consistent with the method used for USP22 wild type (WT). 2.5.1. Flow cytometry analysis HeLa cells were seeded in 6-well plates at a concentration of 4 × 10 5 cells/2 ml in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. After 24 h, the medium was replaced with fresh medium without antibiotics. Meanwhile, the cells were transfected with siRNA, negative control siRNA, pEGFP-C1, pEGFP-USP22 (WT), pEGFP-USP22 (C185S) and pEGFP-USP22 (Y513C) using Lipofectamine RNAiMAX and Lipofectamine 2000 according to the manufacturer's instructions. After incubation for 48 h, the cells were collected for flow cytometric analysis. For the apoptosis and cell-cycle analyses, 1–2 × 106 cells were stained with propidium iodide and analyzed with a Becton–Dickinson FACSort analyzer and Cell Quest software (BD Biosciences). 2.5.2. Transfection of USP22 and SNPs, RNA extraction and qRT-PCR analysis HeLa cells were seeded in 6-well plates at a concentration of 4 × 10 5 cells/2 ml in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. After 24 h, the medium was replaced with fresh medium without antibiotics. Meanwhile, the cells were transfected with siRNA, negative control siRNA, pEGFP-C1, pEGFP-USP22 (WT), pEGFP-USP22 (C185S) and pEGFP-USP22 (Y513C). After incubation for 48 h, total RNA was extracted from the cells using Trizol (Invitrogen, USA) and reverse transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The Trans Start Top Green qPCR Super Mix (TransGen Biotech, Beijing, China) was used for realtime quantitative PCR. The mRNA levels of BMI-1, p53, c-Myc and cyclin D2, as well as that of the internal standard β-actin were measured by qRT-PCR in triplicate using the Rotor Gene 6000 real-time detection system (Bio-Rad). 2.6. Statistical analysis All statistical analyses were performed by using the SPSS software version 17.0. Statistical analysis was performed using Student's t-test and Wilcoxon rank sum test. Differences were considered statistically significant when p values were b0.05. 3. Results 3.1. Suppression of USP22 Expression in HeLa cells by siRNA transfection Three siRNAs (siRNA-1, siRNA-2 and siRNA-3) were used to silence the expression of USP22 in HeLa cells. The effect of the transfection was determined by qRT-PCR and western blot analysis (Fig. 1). The results of our study showed that the USP22 siRNA-1 was the most efficient in silencing the expression of USP22 (Fig. 1A, B), and it was optimally
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 1. Suppression of USP22 expression in HeLa cells by siRNA transfection. (A, B) HeLa cells were transfected with siRNA-1, -2 and -3 (20 nM) for 24 h, respectively; subsequently, the protein levels were quantified by western blot and the USP22 mRNA levels were quantified by qRT-PCR. These results demonstrate that siRNA-1 most efficiently inhibited the expression of USP22. (C, D) The effect of interference could last at least 72 h, as determined by western blot analysis and qRT-PCR.
effective 72 h post-transfection (Fig. 1C, D). Therefore, USP22 siRNA-1 was used for all subsequent experiments. 3.2. Inhibition of the cell cycle in HeLa cells after USP22 siRNA transfection After transfected with USP22 siRNA-1, HeLa cells were collected for flow cytometric analysis to determine the impact on apoptosis and the cell cycle. In the USP22 siRNA-1-transfected group, significant increases in the numbers of cells undergoing G1 cell cycle arrest were observed compared with the control siRNA-transfected group (Fig. 2B), but the number of apoptotic cells was not changed significantly (Fig. 2A). These results indicate that USP22 may facilitate tumor cell growth by regulating cell cycle progression in HeLa cells. 3.3. Knock down of USP22 by USP22 siRNA can regulate BMI-1, c-Myc, cyclin D2 and p53 in HeLa cells USP22 regulates the cell cycle via the c-Myc/cyclin D2 pathway (Ling et al., 2012), BMI-1-mediated INK4a/ARF pathway and Akt signaling pathway (Liu et al., 2012). USP22 expression also leads to reduced expression of cell cycle proteins, including CDK1, CDK2, p53 and Cyclin B1 (Li et al., 2013) in many cancer cells. The mechanism through which it regulates the cell cycle in HeLa cells is not known. Therefore, we examined the transcriptional relationship between USP22 and BMI-1, p53, c-Myc and cyclin D2 expression by qRT-PCR in HeLa cells (Fig. 3). Downregulation of USP22 by siRNA in the HeLa cell line resulted in the decreased expression of BMI-1, c-MYC and cyclin D2 (Fig. 3B, C, D) and increased expression of p53 (Fig. 3A). This novel finding suggests that USP22 promotes cell proliferation via the c-Myc/cyclin
D2 pathway and BMI-1 pathway. USP22 can also regulate the HeLa cell proliferation via its effects on p53.
3.4. Deubiquitinating enzyme activity of USP22 (wild type) and SNPs To investigate whether the deubiquitinating enzyme activity of USP22 affected the HeLa cell cycle, we constructed the C185S (a mutant that had no deubiquitinating enzyme activity) (Quesada et al., 2004). R98W, N283S, P290L and Y513C mutants (these four SNPs were searched in NCBI:http://www.ncbi.nlm.nih.gov/projects/SNP) were generated by site-directed mutagenesis to determine whether USP22 SNPs affect the deubiquitinating enzyme activity of USP22 (Fig. 4A). We performed an USP cleavage assay using Ub-Met-β-gal and GSTUb52 as model substrates. Cys 185 is within the conserved Cys-box of USP22, and when it was mutated to serine, the activity of USP22 was abolished as expected (Fig. 4B; C). Thus, USP22 WT has deubiquitinating enzyme activity, and the C185S mutant is inactive. The deubiquitinating enzyme activity of USP22 (R98W), USP22 (N283S) and USP22 (P290L) were not changed significantly compared with wild-type (Fig. 4B, C) as detected by an USP cleavage assay using both GST-Ub52 and UbMet-β-gal as substrates. However, we found that the deubiquitinating enzyme activity of USP22 (Y513C) was deceased significantly compared with wild-type as detected by an USP cleavage assay using both UbMet-β-gal and GST-Ub52 as substrates (Fig. 4D, E, F). The results from three independent experiments showed that the deubiquitinating enzyme activity of USP22 (Y513C) was abolished using Ub-Met-β-gal as a substrate (Fig. 4D), and the relative deubiquitinating enzyme activity decreased to 1.7% in the USP22 (Y513C) mutant compared with wild-type (p b 0.001) using GST-Ub52 as a substrate (Fig. 3E, F).
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 2. Knock-down of USP22 facilitates apoptosis and induces G1 cell cycle arrest in HeLa cells. (A) After transfection with siRNA for 72 h, the distribution of apoptotic cells was measured by flow cytometric analysis, and the apoptosis rates in different groups are shown in the bar graph (*p b 0.05); (B) After transfection with siRNA for 72 h, the cell cycle distribution of each group was determined by flow cytometric analysis (*p b 0.05).
Fig. 3. USP22 regulates the p53, BMI-1 and c-Myc/cyclin D2 signaling pathway in HeLa cells. Relative p53, BMI-1, c-Myc and cyclin D2 mRNA expression in HeLa cells transiently transfected with USP22 siRNA for 72 h. β-actin mRNA levels were used as an internal normalization control. Each independent qRT-PCR experiment was performed 3 times. (A) the level of p53; (B) the level of BMI-1; (C) the level of c-Myc; (D) the level of cyclin D2 (*p b 0.05).
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 4. Deubiquitinating enzyme activity of USP22 (wild type) and SNPs. (A) Constructs of USP22 wild-type (WT) and mutants USP22 (C185S, R98W, N283S, P290L and Y513C) for the USP cleavage assay; (B) cleavage of model substrate, Ub-Met-β-gal, by USP22. Ub-Met-β-gal was coexpressed with USP22 WT and mutants in Escherichia coli BL21. The results were detected by western blot analysis. USP46 and pGEX-6P-1 (an empty plasmid vector) are used as positive and negative controls, respectively; (C) cleavage of model substrate, GST-Ub52, by USP22. GST-Ub52 was coexpressed with USP22 WT and mutants in E. coli BL21. GST-Ub52 and its cleavage products, GST-Ub, were purified by glutathione agarose beads and detected by Coomassie Brilliant Blue. USP46 and pAC-T7 (an empty plasmid vector) are used as positive and negative controls, respectively; (D) cleavage of model substrate, Ub-Met-β-gal, by USP22 (Y513C) was indicated by three independent experiments in lanes 4, 5, and 6; (E) cleavage of model substrate, GST-Ub52, by USP22 (Y513C) was indicated by three independent experiments in lanes 4, 5, and 6; (F) the quantification of enzymatic activity in the cleavage of model substrate, GST-Ub52, by USP22 (Y513C) is represented by the bar graph. The deubiquitinating enzyme activity of wild-type was defined as 100%. The relative deubiquitinating enzyme activity of USP22 (Y513C) was measured compared to that of wild-type. Values that differ significantly from wild-type (Student's t-test) are indicated with an asterisk (*p b 0.05).
These results suggest that the USP22 (Y513C) mutant significantly impact the deubiquitinating enzyme activity of USP22. 3.5. The USP22 deubiquitinating enzyme activity is necessary for USP22mediated regulation of the cell cycle in HeLa cells We had known that USP22 could regulate the cell cycle in HeLa cells, but it was not clear whether the deubiquitinating enzyme activity was necessary for this effect. To address this question, HeLa cells were transfected with USP22 WT, C185S and Y513C. We examined apoptosis and cell cycle distribution by flow cytometric analysis. In the USP22 WT, CS and Y513C transfected groups, there were no significant change in the number of apoptotic cells compared with the GFP-transfected group; (Fig. 5A). There was a total of 49.3% of USP22 WT-transfected cells in the G1 phase; this was a marked decrease from the 60.7% in GFP-transfected cells. The USP22 CS- and USP22 Y513C-transfected groups did not have a significant impact on HeLa cell apoptosis and cell cycle compared with the GFP-transfected group (Fig. 5B). These results indicate that USP22 may facilitate tumor cell growth by regulating cell cycle progression in HeLa cells, and the deubiquitinating enzyme activity is necessary for these effects. 3.6. USP22 acts as an oncogene by regulating the BMI-1 pathway, c-Myc pathway and p53 in HeLa cells Fig. 3 shows that USP22 promoted cell proliferation via the c-Myc/ cyclin D2 pathway, BMI-1 pathway and p53, as evident by the results of knocking down USP22 in HeLa cells. However, the relationship between these effects and the deubiquitinating enzyme activity of USP22 was not clear. Therefore, we transfected HeLa cells with USP22 WT, C185S and Y513C and observed the levels of BMI-1, p53, c-Myc and
cyclin D2 by qRT-PCR. The results showed that upregulation of USP22 in the HeLa cell line resulted in increased expression of BMI-1, c-MYC and cyclin D2 (Fig. 6B, C, D), but decreased expression of p53 (Fig. 6A). This novel finding suggests that USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway and BMI-1 pathway. USP22 can also regulate HeLa cell proliferation via p53, and these effects were dependent on the deubiquitinating enzyme activity of USP22.
4. Discussion In the present study, we report that expression of USP22 is observed in the HeLa cell line, and USP22 expression is effectively depleted from HeLa cells by siRNA as detected by RT-PCR and western blot analyses. USP22 depletion results in cell cycle arrest at the G1 phase, indicating that the USP22 gene is involved in the regulation of HeLa cell proliferation. Downregulation of USP22 by siRNA in the HeLa cell line results in the decreased expression of BMI-1, c-MYC and cyclin D2, but increased expression of p53. This work indicates that USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway, BMI-1 pathway and p53. In addition, to investigate whether the deubiquitinating enzyme activity of USP22 affects the HeLa cell cycle, we constructed the C185S mutant (a mutant that had no deubiquitinating enzyme activity) and overexpressed it in HeLa cells to observe its effects on the cell cycle. The results show that USP22 C185S could not regulate apoptosis and the cell cycle. We also assessed the effects of an enzyme activitydecreased SNP (Y513C) on apoptosis and the cell cycle in HeLa cells. The results were consistent with those observed for the C185S mutant. Taken together, these data indicate that USP22 can regulate HeLa cell cycle, and these effects were dependent on the deubiquitinating enzyme activity of USP22.
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 5. The deubiquitinating enzyme activity is necessary for USP22 to regulate the cell cycle in HeLa cells. After transfection with GFP, USP22 WT, C185S and Y513C for 48 h, the apoptosis and cell cycle distribution of each group were determined by flow cytometric analysis. (A) After transfection with siRNA for 48 h, the distribution of apoptotic cells was measured by flow cytometric analysis, and the apoptosis rates in different groups are shown in the bar graph (*p b 0.05); (B) after transfection with GFP, USP22 WT, C185S and Y513C for 48 h, the cell cycle distribution of each group was determined by flow cytometric analysis (*p b 0.05).
Our data reveal that USP22 can regulate HeLa cell and cell cycle. Our findings are in good agreement with previous studies (Lv et al., 2011; Hu et al., 2012; Li et al., 2012; Liu et al., 2012; Xu et al., 2012). In our study, USP22 expression was effectively knocked down by siRNA as
detected by RT-PCR and western blot analyses. USP22 depletion resulted in cell cycle arrest at the G1 phase, and the number of cells in the G1 phase was 73.2% in the USP22 siRNA group; this was a marked increase from the 59.5% in the control siRNA group. The data indicate that the
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 6. The deubiquitinating enzyme activity is necessary for USP22 to regulate the p53, BMI-1 and c-Myc/cyclin D2 signaling pathways in HeLa cells. Relative p53, BMI-1, c-Myc and cyclin D2 mRNA expression in HeLa cells transiently transfected with USP22 (WT, C185S, Y513C) for 48 h, respectively. β-actin mRNA levels were used as an internal normalization control. Each independent qRT-PCR experiment was performed 3 times.(A) the level of p53; (B) the level of BMI-1; (C) the level of c-Myc; (D) the level of cyclin D2 (*p b 0.05).
USP22 gene is involved in the regulation of HeLa cell a cell cycle, and USP22 was demonstrated to play an important role in the regulation of human HeLa cell proliferation. In the present study, we find that downregulation of USP22 by siRNA in the HeLa cell line results in the decreased expression of BMI-1, c-MYC and cyclin D2, but increased expression of p53. This work indicates that USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway, BMI-1 pathway and p53. Our results are consistent with many studies (Berezovska et al., 2006; Liu et al., 2012; Ling et al., 2012). p53 has been thought to be a negative regulator of the cell cycle and proliferation (Vogelstein et al., 2000). In HCT116 cells, downregulation of USP22 could up-regulate the expression of p53 (Lv et al., 2011). In HeLa cells, knock-down of USP22 could upregulate the expression of p53, and overexpression of USP22 could down-regulate the expression of p53. Our data showed that USP22 regulated the cell cycle via p53 in HeLa cells, consistent with the observations in HCT116 cells. To investigate whether USP22's deubiquitinating enzyme activity affected the HeLa cell cycle and apoptosis, we constructed the C185S (a mutant which had no deubiquitinating enzyme activity) and Y513C (one SNP that had minimal deubiquitinating enzyme activity) mutants. We then expressed these two mutants in HeLa cells. The results showed that these mutants could not regulate human HeLa cell proliferation. Thus, these results indicate that the deubiquitinating enzyme activity is necessary for USP22 to facilitate tumor cell growth by regulating cell cycle progression in HeLa cells. We also assessed the relationship between the deubiquitinating enzyme activity of USP22 and the c-Myc/cyclin D2 pathway, the BMI-1 pathway and p53. The data showed the ability of USP22 to regulate these pathways depended on its deubiquitinating enzyme activity. In summary, USP22 could effectively regulate the cell cycle and apoptosis by regulating target molecules, such as BMI-1, c-MYC, cyclin D2, and p53, and it could also regulate HeLa cell growth. Importantly, its deubiquitinating enzyme activity is necessary for USP22 to regulate the cell cycle in HeLa cells. Consequently, USP22 may be an attractive therapeutic target for the treatment of cervical cancer. Further investigations should explore the exact role of USP22 in tumor progression, as well as the specific regulators and effectors of USP22.
Acknowledgments This study was supported by Key Medical Guidance Topics of Health Department (20130459).
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Peng, J., Hu, Q., Liu, W., He, X., Cui, L., Chen, X., Yang, M., Liu, H., Wei, W., Liu, S., Wang, H., 2013. USP9X expression correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma. Diagn. Pathol. 8, 177. Quesada, V., Diaz-Perales, A., Gutierrez-Fernandez, A., Garabaya, C., Cal, S., Lopez-Otin, C., 2004. Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochem. Biophys. Res. Commun. 314, 54–62. Reyes-Turcu, F.E., Wilkinson, K.D., 2009. Polyubiquitin binding and disassembly by deubiquitinating enzymes. Chem. Rev. 109, 1495–1508. Takayama, Y., Toda, T., 2010. Coupling histone homeostasis to centromere integrity via the ubiquitin–proteasome system. Cell Div. 5, 18. Ueda, Y., Sobue, T., Morimoto, A., Egawa-Takata, T., Hashizume, C., Kishida, H., Okamoto, S., Yoshino, K., Fujita, M., Enomoto, T., Tomine, Y., Fukuyoshi, J., Kimura, T., 2015. Evaluation of a free-coupon program for cervical cancer screening among the young: a nationally funded program conducted by a local government in Japan. J. Epidemiol. 25, 50–56. Vogelstein, B., Lane, D., Levine, A.J., 2000. Surfing the p53 network. Nature 408, 307–310. Xu, H., Liu, Y.L., Yang, Y.M., Dong, X.S., 2012. Knock-down of ubiquitin-specific protease 22 by micro-RNA interference inhibits colorectal cancer growth. Int. J. Color. Dis. 27, 21–30.
Yang, D.D., Cui, B.B., Sun, L.Y., Zheng, H.Q., Huang, Q., Tong, J.X., Zhang, Q.F., 2011. The coexpression of USP22 and BMI-1 may promote cancer progression and predict therapy failure in gastric carcinoma. Cell Biochem. Biophys. 61, 703–710. Zhang, X.Y., Pfeiffer, H.K., Thorne, A.W., McMahon, S.B., 2008a. USP22, an hSAGA subunit and potential cancer stem cell marker, reverses the polycomb-catalyzed ubiquitylation of histone H2A. Cell Cycle 7, 1522–1524. Zhang, X.Y., Varthi, M., Sykes, S.M., Phillips, C., Warzecha, C., Zhu, W., Wyce, A., Thorne, A.W., Berger, S.L., McMahon, S.B., 2008b. The putative cancer stem cell marker USP22 is a subunit of the human SAGA complex required for activated transcription and cell-cycle progression. Mol. Cell 29, 102–111. Zhang, W., Tian, Q.B., Li, Q.K., Wang, J.M., Wang, C.N., Liu, T., Liu, D.W., Wang, M.W., 2011a. Lysine 92 amino acid residue of USP46, a gene associated with ‘behavioral despair’ in mice, influences the deubiquitinating enzyme activity. PLoS One 6, e26297. Zhang, Y., Yao, L., Zhang, X., Ji, H., Wang, L., Sun, S., Pang, D., 2011b. Elevated expression of USP22 in correlation with poor prognosis in patients with invasive breast cancer. J. Cancer Res. Clin. Oncol. 137, 1245–1253.
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Research paper
The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth Ying-Li Liu a, Jie Zheng b, Li-Juan Tang a, Wei Han a, Jian-Min Wang a, Dian-Wu Liu a, Qing-Bao Tian a,⁎ a b
Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, China Department of Functional Neurosurgery, Hebei General Hospital, Shijiazhuang, China
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 10 June 2015 Accepted 28 June 2015 Available online xxxx Keywords: USP22 Deubiquitinating enzyme activity HeLa cell Small interfering RNA Cell cycle Apoptosis
a b s t r a c t Ubiquitin-specific protease 22 (USP22) can regulate the cell cycle and apoptosis in many cancer cell types, while it is still unclear whether the deubiquitinating enzyme activity of USP22 is necessary for these processes. As little is known about the impact of USP22 on the growth of HeLa cell, we observed whether USP22 can effectively regulate HeLa cell growth as well as the necessity of deubiquitinating enzyme activity for these processes in HeLa cell. In this study, we demonstrate that USP22 can regulate cell cycle but not apoptosis in HeLa cell. The deubiquitinating enzyme activity of USP22 is necessary for this process as confirmed by an activity-deleted mutant (C185S) and an activity-decreased mutant (Y513C). In addition, the deubiquitinating enzyme activity of USP22 is related to the levels of BMI-1, c-Myc, cyclin D2 and p53. Our findings indicate that the deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, and it promotes cell proliferation via the c-Myc/cyclin D2, BMI-1 and p53 pathways in HeLa cell. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ubiquitin-specific protease (USP), a subfamily of deubiquitinating enzymes (DUBs) (D'Andrea and Pellman, 1998; Reyes-Turcu and Wilkinson, 2009), is responsible for the removal of ubiquitin or polyubiquitin from target proteins, the processing of ubiquitin precursors, and the disassembly of unanchored polyubiquitin by catalyzing the hydrolysis of isopeptide bonds in ubiquitin-protein conjugates (Takayama and Toda, 2010). The growth of many cancers is regulated by USPs: USP8 is related to non-small cell lung cancer (Baykara et al., 2013); USP28 is a potential prognostic marker for bladder cancer (Guo et al., 2014); and USP9X expression correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma (Peng et al., 2013). Therefore, USPs are important factors for cancer. Recent observations have identified an 11-gene poly-comb/cancer stem cell signature that could predict the likelihood of treatment failure in cancer patients (Glinsky, 2005). Ubiquitin-specific protease 22 (USP22) is a new putative cancer stem cell marker involved in the 11-gene polycomb/cancer stem cell signature (Zhang et al., 2008a,b). It belongs to a large family of proteins with ubiquitin hydrolase activity. USP22 is a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA)
Abbreviations: USP, ubiquitin-specific protease; DUBs, deubiquitinating enzymes; USP22, ubiquitin-specific protease 22; qRT-PCR, quantitative real-time polymerase chain reaction. ⁎ Corresponding author. E-mail address: [email protected] (Q.-B. Tian).
transcriptional cofactor complex. Within hSAGA, USP22 regulates the transcription of downstream genes related to epigenetic alteration and cancer progression by removing ubiquitin from histone H2A and H2B (Zhang et al., 2008b). Moreover, USP22 can regulate tumor recurrence, distant metastasis, therapeutic failure and poor prognosis via its functions in some cell signaling pathways: USP22 regulates the cell cycle via the c-Myc/cyclin D2 pathway and down-regulation of p15 and p21 expression in HepG2 cells (Ling et al., 2012); USP22 silencing was also found to lead to reduced expression of cell cycle proteins, including CDK1, CDK2 and Cyclin B1 in human brain glioma cells (Li et al., 2013); USP22 may act as an oncogene in human colorectal cancer as it positively regulates the cell cycle via both the BMI-1-mediated INK4a/ARF pathway and the Akt signaling pathway (Liu et al., 2012); USP22 antagonizes p53 transcriptional activation by deubiquitinating sirt1 to suppress cell apoptosis (Lin et al., 2012). The siRNA-mediated knock-down of USP22 could effectively induce cell cycle arrest by regulating target molecules, such as cyclin D2, p21, p15 and p53, and could also inhibit cell growth. Elevated expression of USP22 can predict shorter intervals of tumor recurrence, distant metastasis, therapeutic failure and poor prognosis in patients with many types of cancer such as colorectal cancer (Liu et al., 2010, 2011), breast cancer (Y. Zhang et al., 2011), gastric cancer (Yang et al., 2011) and others (Ueda et al., 2015). USP22 can regulate the growth and prognosis of many cancers, but little is known about the impact of USP22 on the growth of human cervical cancer cell lines, and whether the deubiquitinating enzyme activity of USP22 is necessary for these effects is not clear. In this
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study, we used siRNA to specifically suppress expression of USP22, and we observed that the knock-down of USP22 could effectively inhibit HeLa cell proliferation and induce cell cycle arrest. We also suggest that USP22 may regulate cell cycle progression by down-regulating p53 expression and up-regulating cyclin D2, BMI-1, c-MYC expression. The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth.
were analyzed by western blotting, and total protein extracts of the pGEX-Ub52 group were purified by glutathione agarose beads and detected by Coomassie Brilliant Blue. The pGEX-6p-1-USP46 and pACT7-USP46 plasmids expressing USP46 were used as positive controls for the USP cleavage assay. The relative intensity of the resulting bands was analyzed by the Odyssey V3.0 software. 2.5. Site-directed mutagenesis and deubiquitinating enzyme activity assay
2. Materials and methods 2.1. Cell culture antibodies and reagents The human HeLa cell line was purchased from American Type Culture Collection (ATCC). The cell line was cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% fetal calf serum (FBS, GIBCO) and 1% penicillin–streptomycin at 37 °C in a 5% CO2 atmosphere. The USP22 antibody (1:500) was from Abcam (UK). β-actin (1:5000) was from Proteintech (USA). Lipofectamine 2000 was from Invitrogen Corp (USA). The reverse-transcription polymerase chain reaction (RT-PCR) kit and primers were from Takara (Japan). 2.2. Transfection of siRNA HeLa cells were seeded in 6-well plates at a concentration of 4 × 105 cells/2 ml in DMEM supplemented with 10% FBS and 1% penicillin– streptomycin. After 24 h, the medium was replaced with fresh medium without antibiotics. Meanwhile, the cells were transfected with siRNA or a negative control oligonucleotide using Lipofectamine RNAiMAX according to the manufacturer's instructions. After incubation for 4–6 h, the medium containing the Lipofectamine RNAiMAX complexes was replaced with fresh DMEM containing 10% FBS. Then, the cells were cultured for subsequent experiments. All siRNAs were obtained from Invitrogen Corp (USA), and the three specific sequences for silencing were: human USP22 siRNA-1, sense 5′-GCU GUU UCA CAA AGA AGC AUA UUC A-3′, and anti-sense 5′-UGA AUA UGC UUC UUU GUG AAA CAG C-3′; siRNA-2, sense 5′-CAG CAG CCC ACG GAC AGU CUC AAC A -3′, and anti-sense 5′-UGU UGA GAC UGU CCG UGG GCU GCU G 3′; siRNA-3, sense 5′-GCC AAG UCC UGU AUC UGC CAU GUC U-3′, and anti-sense 5′-AGA CAU GGC AGA UAC AGG ACU UGG C-3′. The transfection efficiency was assessed by transfection of negative control siRNA. The negative control siRNA sequence was: 5′-UUC UCC GAA CGU GUC ACG UTT ACG UGA CAC GUU CGG AGA ATT-3′. The effect of RNA interference was analyzed by qRT-PCR and western blot analyses. 2.3. Molecular cloning of USP22 The pcDNA3.1-USP22-V5-FLAG plasmid was a gift from Boyko S. Atanassov. The pcDNA3.1-USP22-V5-FLAG plasmid was digested by NotI and XhoI, and then USP22 was inserted via blunt-end ligation. The expression vector pGEX-6p-1 (Amersham Pharmacia Biotech) was digested by SalI and filled in to generate blunt ends. Then, the pGEX6p-1-USP22 plasmid was produced by inserting the complete coding sequence of USP22 into the pGEX-6p-1 plasmid. The pGEX-USP22 plasmid was digested by NotI and BamHI, and then USP22 was inserted via bluntend ligation. The expression vector pAC-T7 plasmid was digested by BamHI and filled in to generate blunt ends. Then, a pAC-T7- USP22 plasmid was produced by inserting the complete coding sequence of USP22 into the pAC-T7 plasmid. 2.4. Deubiquitinating enzyme activity assay of hUSP22 The USP cleavage assays used ubiquitin-met-β-galactosidase (Ub-Met-β-gal) and pGEX-Ub52 (GST-Ub52) (W. Zhang et al., 2011) as model substrates. Total protein extracts of the Ub-Met-β-gal group
Cys 185, which is located within the conserved Cys-box of USP22, was mutated to serine. Arg 98 was mutated to tryptophan, Asn 283 was mutated to serine, Pro 290 was mutated to leucine, and Tyr 513 was mutated to cysteine. The resulting plasmids were pGEX-USP22 (C185S), pGEX-USP22 (R98W), pGEX-USP22 (N283S), pGEX-USP22 (P290L) and pGEX-USP22 (Y513C), respectively. Mutations were confirmed by DNA sequencing. The deubiquitinating enzyme activity assay method was consistent with the method used for USP22 wild type (WT). 2.5.1. Flow cytometry analysis HeLa cells were seeded in 6-well plates at a concentration of 4 × 10 5 cells/2 ml in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. After 24 h, the medium was replaced with fresh medium without antibiotics. Meanwhile, the cells were transfected with siRNA, negative control siRNA, pEGFP-C1, pEGFP-USP22 (WT), pEGFP-USP22 (C185S) and pEGFP-USP22 (Y513C) using Lipofectamine RNAiMAX and Lipofectamine 2000 according to the manufacturer's instructions. After incubation for 48 h, the cells were collected for flow cytometric analysis. For the apoptosis and cell-cycle analyses, 1–2 × 106 cells were stained with propidium iodide and analyzed with a Becton–Dickinson FACSort analyzer and Cell Quest software (BD Biosciences). 2.5.2. Transfection of USP22 and SNPs, RNA extraction and qRT-PCR analysis HeLa cells were seeded in 6-well plates at a concentration of 4 × 10 5 cells/2 ml in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. After 24 h, the medium was replaced with fresh medium without antibiotics. Meanwhile, the cells were transfected with siRNA, negative control siRNA, pEGFP-C1, pEGFP-USP22 (WT), pEGFP-USP22 (C185S) and pEGFP-USP22 (Y513C). After incubation for 48 h, total RNA was extracted from the cells using Trizol (Invitrogen, USA) and reverse transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The Trans Start Top Green qPCR Super Mix (TransGen Biotech, Beijing, China) was used for realtime quantitative PCR. The mRNA levels of BMI-1, p53, c-Myc and cyclin D2, as well as that of the internal standard β-actin were measured by qRT-PCR in triplicate using the Rotor Gene 6000 real-time detection system (Bio-Rad). 2.6. Statistical analysis All statistical analyses were performed by using the SPSS software version 17.0. Statistical analysis was performed using Student's t-test and Wilcoxon rank sum test. Differences were considered statistically significant when p values were b0.05. 3. Results 3.1. Suppression of USP22 Expression in HeLa cells by siRNA transfection Three siRNAs (siRNA-1, siRNA-2 and siRNA-3) were used to silence the expression of USP22 in HeLa cells. The effect of the transfection was determined by qRT-PCR and western blot analysis (Fig. 1). The results of our study showed that the USP22 siRNA-1 was the most efficient in silencing the expression of USP22 (Fig. 1A, B), and it was optimally
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 1. Suppression of USP22 expression in HeLa cells by siRNA transfection. (A, B) HeLa cells were transfected with siRNA-1, -2 and -3 (20 nM) for 24 h, respectively; subsequently, the protein levels were quantified by western blot and the USP22 mRNA levels were quantified by qRT-PCR. These results demonstrate that siRNA-1 most efficiently inhibited the expression of USP22. (C, D) The effect of interference could last at least 72 h, as determined by western blot analysis and qRT-PCR.
effective 72 h post-transfection (Fig. 1C, D). Therefore, USP22 siRNA-1 was used for all subsequent experiments. 3.2. Inhibition of the cell cycle in HeLa cells after USP22 siRNA transfection After transfected with USP22 siRNA-1, HeLa cells were collected for flow cytometric analysis to determine the impact on apoptosis and the cell cycle. In the USP22 siRNA-1-transfected group, significant increases in the numbers of cells undergoing G1 cell cycle arrest were observed compared with the control siRNA-transfected group (Fig. 2B), but the number of apoptotic cells was not changed significantly (Fig. 2A). These results indicate that USP22 may facilitate tumor cell growth by regulating cell cycle progression in HeLa cells. 3.3. Knock down of USP22 by USP22 siRNA can regulate BMI-1, c-Myc, cyclin D2 and p53 in HeLa cells USP22 regulates the cell cycle via the c-Myc/cyclin D2 pathway (Ling et al., 2012), BMI-1-mediated INK4a/ARF pathway and Akt signaling pathway (Liu et al., 2012). USP22 expression also leads to reduced expression of cell cycle proteins, including CDK1, CDK2, p53 and Cyclin B1 (Li et al., 2013) in many cancer cells. The mechanism through which it regulates the cell cycle in HeLa cells is not known. Therefore, we examined the transcriptional relationship between USP22 and BMI-1, p53, c-Myc and cyclin D2 expression by qRT-PCR in HeLa cells (Fig. 3). Downregulation of USP22 by siRNA in the HeLa cell line resulted in the decreased expression of BMI-1, c-MYC and cyclin D2 (Fig. 3B, C, D) and increased expression of p53 (Fig. 3A). This novel finding suggests that USP22 promotes cell proliferation via the c-Myc/cyclin
D2 pathway and BMI-1 pathway. USP22 can also regulate the HeLa cell proliferation via its effects on p53.
3.4. Deubiquitinating enzyme activity of USP22 (wild type) and SNPs To investigate whether the deubiquitinating enzyme activity of USP22 affected the HeLa cell cycle, we constructed the C185S (a mutant that had no deubiquitinating enzyme activity) (Quesada et al., 2004). R98W, N283S, P290L and Y513C mutants (these four SNPs were searched in NCBI:http://www.ncbi.nlm.nih.gov/projects/SNP) were generated by site-directed mutagenesis to determine whether USP22 SNPs affect the deubiquitinating enzyme activity of USP22 (Fig. 4A). We performed an USP cleavage assay using Ub-Met-β-gal and GSTUb52 as model substrates. Cys 185 is within the conserved Cys-box of USP22, and when it was mutated to serine, the activity of USP22 was abolished as expected (Fig. 4B; C). Thus, USP22 WT has deubiquitinating enzyme activity, and the C185S mutant is inactive. The deubiquitinating enzyme activity of USP22 (R98W), USP22 (N283S) and USP22 (P290L) were not changed significantly compared with wild-type (Fig. 4B, C) as detected by an USP cleavage assay using both GST-Ub52 and UbMet-β-gal as substrates. However, we found that the deubiquitinating enzyme activity of USP22 (Y513C) was deceased significantly compared with wild-type as detected by an USP cleavage assay using both UbMet-β-gal and GST-Ub52 as substrates (Fig. 4D, E, F). The results from three independent experiments showed that the deubiquitinating enzyme activity of USP22 (Y513C) was abolished using Ub-Met-β-gal as a substrate (Fig. 4D), and the relative deubiquitinating enzyme activity decreased to 1.7% in the USP22 (Y513C) mutant compared with wild-type (p b 0.001) using GST-Ub52 as a substrate (Fig. 3E, F).
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Fig. 2. Knock-down of USP22 facilitates apoptosis and induces G1 cell cycle arrest in HeLa cells. (A) After transfection with siRNA for 72 h, the distribution of apoptotic cells was measured by flow cytometric analysis, and the apoptosis rates in different groups are shown in the bar graph (*p b 0.05); (B) After transfection with siRNA for 72 h, the cell cycle distribution of each group was determined by flow cytometric analysis (*p b 0.05).
Fig. 3. USP22 regulates the p53, BMI-1 and c-Myc/cyclin D2 signaling pathway in HeLa cells. Relative p53, BMI-1, c-Myc and cyclin D2 mRNA expression in HeLa cells transiently transfected with USP22 siRNA for 72 h. β-actin mRNA levels were used as an internal normalization control. Each independent qRT-PCR experiment was performed 3 times. (A) the level of p53; (B) the level of BMI-1; (C) the level of c-Myc; (D) the level of cyclin D2 (*p b 0.05).
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Fig. 4. Deubiquitinating enzyme activity of USP22 (wild type) and SNPs. (A) Constructs of USP22 wild-type (WT) and mutants USP22 (C185S, R98W, N283S, P290L and Y513C) for the USP cleavage assay; (B) cleavage of model substrate, Ub-Met-β-gal, by USP22. Ub-Met-β-gal was coexpressed with USP22 WT and mutants in Escherichia coli BL21. The results were detected by western blot analysis. USP46 and pGEX-6P-1 (an empty plasmid vector) are used as positive and negative controls, respectively; (C) cleavage of model substrate, GST-Ub52, by USP22. GST-Ub52 was coexpressed with USP22 WT and mutants in E. coli BL21. GST-Ub52 and its cleavage products, GST-Ub, were purified by glutathione agarose beads and detected by Coomassie Brilliant Blue. USP46 and pAC-T7 (an empty plasmid vector) are used as positive and negative controls, respectively; (D) cleavage of model substrate, Ub-Met-β-gal, by USP22 (Y513C) was indicated by three independent experiments in lanes 4, 5, and 6; (E) cleavage of model substrate, GST-Ub52, by USP22 (Y513C) was indicated by three independent experiments in lanes 4, 5, and 6; (F) the quantification of enzymatic activity in the cleavage of model substrate, GST-Ub52, by USP22 (Y513C) is represented by the bar graph. The deubiquitinating enzyme activity of wild-type was defined as 100%. The relative deubiquitinating enzyme activity of USP22 (Y513C) was measured compared to that of wild-type. Values that differ significantly from wild-type (Student's t-test) are indicated with an asterisk (*p b 0.05).
These results suggest that the USP22 (Y513C) mutant significantly impact the deubiquitinating enzyme activity of USP22. 3.5. The USP22 deubiquitinating enzyme activity is necessary for USP22mediated regulation of the cell cycle in HeLa cells We had known that USP22 could regulate the cell cycle in HeLa cells, but it was not clear whether the deubiquitinating enzyme activity was necessary for this effect. To address this question, HeLa cells were transfected with USP22 WT, C185S and Y513C. We examined apoptosis and cell cycle distribution by flow cytometric analysis. In the USP22 WT, CS and Y513C transfected groups, there were no significant change in the number of apoptotic cells compared with the GFP-transfected group; (Fig. 5A). There was a total of 49.3% of USP22 WT-transfected cells in the G1 phase; this was a marked decrease from the 60.7% in GFP-transfected cells. The USP22 CS- and USP22 Y513C-transfected groups did not have a significant impact on HeLa cell apoptosis and cell cycle compared with the GFP-transfected group (Fig. 5B). These results indicate that USP22 may facilitate tumor cell growth by regulating cell cycle progression in HeLa cells, and the deubiquitinating enzyme activity is necessary for these effects. 3.6. USP22 acts as an oncogene by regulating the BMI-1 pathway, c-Myc pathway and p53 in HeLa cells Fig. 3 shows that USP22 promoted cell proliferation via the c-Myc/ cyclin D2 pathway, BMI-1 pathway and p53, as evident by the results of knocking down USP22 in HeLa cells. However, the relationship between these effects and the deubiquitinating enzyme activity of USP22 was not clear. Therefore, we transfected HeLa cells with USP22 WT, C185S and Y513C and observed the levels of BMI-1, p53, c-Myc and
cyclin D2 by qRT-PCR. The results showed that upregulation of USP22 in the HeLa cell line resulted in increased expression of BMI-1, c-MYC and cyclin D2 (Fig. 6B, C, D), but decreased expression of p53 (Fig. 6A). This novel finding suggests that USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway and BMI-1 pathway. USP22 can also regulate HeLa cell proliferation via p53, and these effects were dependent on the deubiquitinating enzyme activity of USP22.
4. Discussion In the present study, we report that expression of USP22 is observed in the HeLa cell line, and USP22 expression is effectively depleted from HeLa cells by siRNA as detected by RT-PCR and western blot analyses. USP22 depletion results in cell cycle arrest at the G1 phase, indicating that the USP22 gene is involved in the regulation of HeLa cell proliferation. Downregulation of USP22 by siRNA in the HeLa cell line results in the decreased expression of BMI-1, c-MYC and cyclin D2, but increased expression of p53. This work indicates that USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway, BMI-1 pathway and p53. In addition, to investigate whether the deubiquitinating enzyme activity of USP22 affects the HeLa cell cycle, we constructed the C185S mutant (a mutant that had no deubiquitinating enzyme activity) and overexpressed it in HeLa cells to observe its effects on the cell cycle. The results show that USP22 C185S could not regulate apoptosis and the cell cycle. We also assessed the effects of an enzyme activitydecreased SNP (Y513C) on apoptosis and the cell cycle in HeLa cells. The results were consistent with those observed for the C185S mutant. Taken together, these data indicate that USP22 can regulate HeLa cell cycle, and these effects were dependent on the deubiquitinating enzyme activity of USP22.
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Fig. 5. The deubiquitinating enzyme activity is necessary for USP22 to regulate the cell cycle in HeLa cells. After transfection with GFP, USP22 WT, C185S and Y513C for 48 h, the apoptosis and cell cycle distribution of each group were determined by flow cytometric analysis. (A) After transfection with siRNA for 48 h, the distribution of apoptotic cells was measured by flow cytometric analysis, and the apoptosis rates in different groups are shown in the bar graph (*p b 0.05); (B) after transfection with GFP, USP22 WT, C185S and Y513C for 48 h, the cell cycle distribution of each group was determined by flow cytometric analysis (*p b 0.05).
Our data reveal that USP22 can regulate HeLa cell and cell cycle. Our findings are in good agreement with previous studies (Lv et al., 2011; Hu et al., 2012; Li et al., 2012; Liu et al., 2012; Xu et al., 2012). In our study, USP22 expression was effectively knocked down by siRNA as
detected by RT-PCR and western blot analyses. USP22 depletion resulted in cell cycle arrest at the G1 phase, and the number of cells in the G1 phase was 73.2% in the USP22 siRNA group; this was a marked increase from the 59.5% in the control siRNA group. The data indicate that the
Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075
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Fig. 6. The deubiquitinating enzyme activity is necessary for USP22 to regulate the p53, BMI-1 and c-Myc/cyclin D2 signaling pathways in HeLa cells. Relative p53, BMI-1, c-Myc and cyclin D2 mRNA expression in HeLa cells transiently transfected with USP22 (WT, C185S, Y513C) for 48 h, respectively. β-actin mRNA levels were used as an internal normalization control. Each independent qRT-PCR experiment was performed 3 times.(A) the level of p53; (B) the level of BMI-1; (C) the level of c-Myc; (D) the level of cyclin D2 (*p b 0.05).
USP22 gene is involved in the regulation of HeLa cell a cell cycle, and USP22 was demonstrated to play an important role in the regulation of human HeLa cell proliferation. In the present study, we find that downregulation of USP22 by siRNA in the HeLa cell line results in the decreased expression of BMI-1, c-MYC and cyclin D2, but increased expression of p53. This work indicates that USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway, BMI-1 pathway and p53. Our results are consistent with many studies (Berezovska et al., 2006; Liu et al., 2012; Ling et al., 2012). p53 has been thought to be a negative regulator of the cell cycle and proliferation (Vogelstein et al., 2000). In HCT116 cells, downregulation of USP22 could up-regulate the expression of p53 (Lv et al., 2011). In HeLa cells, knock-down of USP22 could upregulate the expression of p53, and overexpression of USP22 could down-regulate the expression of p53. Our data showed that USP22 regulated the cell cycle via p53 in HeLa cells, consistent with the observations in HCT116 cells. To investigate whether USP22's deubiquitinating enzyme activity affected the HeLa cell cycle and apoptosis, we constructed the C185S (a mutant which had no deubiquitinating enzyme activity) and Y513C (one SNP that had minimal deubiquitinating enzyme activity) mutants. We then expressed these two mutants in HeLa cells. The results showed that these mutants could not regulate human HeLa cell proliferation. Thus, these results indicate that the deubiquitinating enzyme activity is necessary for USP22 to facilitate tumor cell growth by regulating cell cycle progression in HeLa cells. We also assessed the relationship between the deubiquitinating enzyme activity of USP22 and the c-Myc/cyclin D2 pathway, the BMI-1 pathway and p53. The data showed the ability of USP22 to regulate these pathways depended on its deubiquitinating enzyme activity. In summary, USP22 could effectively regulate the cell cycle and apoptosis by regulating target molecules, such as BMI-1, c-MYC, cyclin D2, and p53, and it could also regulate HeLa cell growth. Importantly, its deubiquitinating enzyme activity is necessary for USP22 to regulate the cell cycle in HeLa cells. Consequently, USP22 may be an attractive therapeutic target for the treatment of cervical cancer. Further investigations should explore the exact role of USP22 in tumor progression, as well as the specific regulators and effectors of USP22.
Acknowledgments This study was supported by Key Medical Guidance Topics of Health Department (20130459).
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Please cite this article as: Liu, Y.-L., et al., The deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.06.075