| Ubiquitin-specific protease 22(USP22) was belonging to Ubiquitinspecific protease(USP), a subfamily of deubiquitinating enzymes(DUBs). USP22 consists of 525 amino acids and contains an N-terminal UBP-type zinc finger and a C-terminal USP domain. USP22 is a key subunit of the human Spt-Ada-Gcn5 acetyltransferase(h SAGA) transcriptional cofactor complex. Within h SAGA, USP22 regulates the transcription of downstream genes related to epigenetic alteration and cancer progression by removing ubiquitin from histone H2 A and H2 B. USP22 is involved in oncogenesis due to its role in a large variety of cancers. USP22 expression is increased in laryngeal squamous cell carcinoma, salivary adenoid cystic carcinoma, cervical cancer, salivary duct carcinoma, colorectal carcinoma. The overexpression of USP22 is related to poor prognosis and may be used as a biomarker during diagnosis. USP22 can regulate the growth and prognosis of many cancers, but little is known about the effect of the enzyme activity of USP22 on regulating the growth of tumors. In this study, we used si RNA to specifically suppress expression of USP22 and observed that the knock-down of USP22 could effectively inhibit He La 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. Part one: Knock-down of USP22 by small interfering RNA affects onHe La cell cycle progressionObjective:USP22 can regulate the occurrence of tumor, recurrence, distant metastasis, treatment-resistant and poor prognosis via its functions in some cell signaling pathways. 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. So in this study, we used si RNA to specifically suppress expression of USP22 in He La cell to explore the association between the apoptosis and cell cycle of He La cell and USP22.Methods:1 The specific sequence for silencing USP22 was transfected in He La cell, and the total m RNA and protein were extracted after 24 h, 48 h, 72 h respectively. The effect of RNA interference was analyzed by q RT-PCR and western blot analyses.2 72 hours after transfected with USP22 si RNA, He La cells were collected for flow cytometric analysis to determine the impact on apoptosis and the cell cycle.3 He La cells were transfected with si RNA, negative control si RNA. After incubation for 72 hours, total RNA was extracted from the cells. The m RNA levels of BMI-1, p53, c-Myc and cyclin D2 were measured by q RT-PCR.Results:1 The results of our study showed that the USP22 si RNA-1 was the most efficient in silencing the expression of USP22, and it was optimally effective 72 h post-transfection. Therefore, USP22 si RNA-1 was used for all subsequent experiments.2 In the USP22 si RNA-1-transfected group, significant increases in the numbers of cells undergoing G1 cell cycle arrest were observed compared with the control si RNA-transfected group, but the number of apoptotic cells was not changed significantly. These results indicate that USP22 may facilitate tumor cell growth by regulating cell cycle progression in He La cells.3 Downregulation of USP22 by si RNA in the He La cell line resulted in the decreased expression of BMI-1, c-MYC andcyclin D2 and increased expression of p53. 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 He La cell proliferation via its effects on p53.Conclusions:1 The specific sequence for silencing USP22 was transfected in He La cell and the results showed that the USP22 si RNA-1 was the most efficient.2 The results of flow cytometric analysis indicate that USP22 may facilitate tumor cell growth by regulating cell cycle progression in He La cells.3 USP22 promotes cell proliferation via the c-Myc/cyclin D2 pathway and BMI-1 pathway. USP22 can also regulate the He La cell proliferation via its effects on p53. Part two: The USP22 deubiquitinating enzyme activity is necessary forUSP22-mediated regulation of the cell cycle in He La cellsObjective:In part one, the results suggest that USP22 may facilitate tumor cell growth by regulating cell cycle progression in He La cells, but the relationship between the USP22 deubiquitinating enzyme activity and this effect is not known. To investigate whether the deubiquitinating enzyme activity of USP22 affected the He La cell cycle, we constructed the C185S(a mutant that had no deubiquitinating enzyme activity). The USP cleavage assay, using GST-ub52 and ub-Met-β-gal as model substrates, was conducted to assess enzymatic activity of the mutant. The effects on apoptosis and cell cycle of the mutant was examined by flow cytometric analysis.Methods:1 Using pc DNA3.1-V5-FLAG-HUSP22 as a template, p GEX-USP22 plasmid was produced by inserting the complete coding sequence of USP22 from pc DNA3.1-V5-FLAG-HUSP22 into p GEX-6p-1 plasmid, and confirmed by restriction enzyme digestion and DNA sequencing. Plasmid construction of p AC-T7-USP22. p AC-T7-USP22 plasmid was produced by inserting the complete coding sequence of USP22 from p GEX-USP22 into p AC-T7 plasmid, and confirmed by by restriction enzyme digestion and DNA sequencing.2 Using p GEX-USP22 as a template, site-directed mutagenese was carried out to generate p GEX-USP22(C185S) plasmids. Mutations were confirmed by DNA sequencing. p AC-T7-USP22(C185S) plasmid was produced by inserting the complete coding sequence of USP22(C185S) from p GEX-USP22(C185S) into p AC-T7 plasmid, and confirmed by DNA sequencing.3 Using GST-ub52 and ub-Met-β-gal as model substrates, the USP cleavage assay were conducted to assess enzymatic activity of the USP22(WT) and USP22(C185S). Then the results were detected by Odyssey Infrared Imaging System.4 72 hours after transfected with USP22(WT) and USP22(C185S), He La cells were collected for flow cytometric analysis to determine the impact on apoptosis and the cell cycle. The m RNA levels of BMI-1, p53, c-Myc and cyclin D2 were measured by q RT-PCR.Results:1 USP22(WT) and USP22(C185S) was constructed as expected and the results of USP cleavage assay suggested that the USP22(C185S) had lost the deubiquitinating enzyme activity.2 We examined apoptosis and cell cycle distribution by flow cytometric analysis. In the USP22(WT) and USP22(C185S) transfected groups, there were no significant change in the number of apoptotic cells compared with the GFP-transfected group. There was a marked decrease cells in the G1 phase in USP22 WT-transfected group compared with USP22 CS-transfected group.3 We transfected He La cells with USP22(WT) and USP22(C185S), and observed that that upregulation of USP22(WT) in the He La cell line resulted in increased expression of BMI-1, c-MYC and cyclin D2, but decreased expression of p53. USP22(C185S) did not have these effects.Conclusions:1 These results suggested that the effects of USP22 on He La cell cycle depended on the deubiquitinating enzyme activity.2 When the deubiquitinating enzyme activity disappeared, the regulation effects on cell cycle would disappear.3 USP22(C185S) can not regulate BMI-1, c-MYC, cyclin D2 and p53.Part three: The impact of four USP22 mutations on enzymatic activity and HeLa cell cycleObjective:In part two, the results have suggested that the USP22 deubiquitinating enzyme activity is necessary for USP22-mediated regulation of the cell cycle in He La cells. This part of study aims to evaluate the impact of USP22 SNPs on enzymatic activity of the USP22 in order to provide new clues on the physiologic function of USP22 and its role in He La cell.Methods:1 Using p GEX-USP22 as a template, site-directed mutagenese was carried out to generate USP22(R98W), USP22(N283S), USP22(P290L), and USP22(Y513C) mutations. p GEX-USP22(R98W), p GEX-USP22(N283S), p GEX-USP22(P290L), p GEX-USP22(Y513C), p AC-T7-USP22(R98W), p AC-T7-USP22(N283S), p AC-T7-USP22(P290L) and p AC-T7-USP22(Y513C) plasmids were constructed.2 USP cleavage assay. Using GST-ub52 and ub-Met-β-gal as model substrates, the USP cleavage assay were conducted to assess enzymatic activity of the USP22(R98W, N283 S, P290 L and Y513C). Then the results were detected by Odyssey Infrared Imaging System.3 72 hours after transfected with USP22(WT), USP22(C185S) and USP22(Y513C), He La cells were collected for flow cytometric analysis to determine the impact on apoptosis and the cell cycle. The m RNA levels of BMI-1, p53, c-Myc and cyclin D2 were measured by q RT-PCR.Results:1 USP22(R98W, N283 S, P290 L and Y513C) were constructed as expected and the results of USP cleavage assay suggested that USP22(R98W, N283 S, P290L) have the same activity compared to the wild type, detected by USP cleavage assay using both GST-Ub52 and Ub-Met-β-gal as substrates, but the deubiquitinating enzyme activity of USP22(Y513C) mutant was significantly deseased.2 We examined apoptosis and cell cycle distribution by flow cytometric analysis. In the USP22(Y513C) transfected groups, there were no significant change in the apoptosis and cell cycle compared with the GFP-C1-transfected group.3 We transfected He La cells with USP22(Y513C) and observed that upregulation of USP22(Y513C) in the He La cell line resulted in no change in the expression of BMI-1, c-MYC and cyclin D2 and p53.Conclusions:1 The effects of USP22 on He La cell cycle depended on the deubiquitinating enzyme activity. When the mutation lost the deubiquitinating enzyme activity, the regulation effects on cell cycle would disappear.2 USP22(Y513C) can not regulate BMI-1, c-MYC, cyclin D2 and p53. These results also suggested that the USP22 deubiquitinating enzyme activity is necessary for USP22-mediated regulation of the cell cycle in He La cells. |
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