The Regulatory Mechanism Of USP12 In HeLa Cell Cycle

Ubiquitin-specific proteases(USPs), one of the five families of deubiquitinating enzymes(DUBs), include 54 different types, many of which are closely related to cancer, such as prostate cancer(USP2), malignant adenoma(USP4), leukemia(USP9x) and glioblastoma(USP15). Therefore, numbers of current studies focus on analysis of tumor-associated USP, to explore potentially targeted therapies for cancer.Ubiquitin-specific protease 12(USP12), a member of USPs family, contains 1113 bp nucleotides, and 370 amino acids, with molecular weight of 43 KDa. Currently, USP12 has been found to serve as tumor promoters or tumor suppressor in prostate cancer, colon cancer or human epithelial carcinoma tumor, due to its deubiquitination function. Cervical cancer is the second most common cancer among women worldwide. Several studies have confirmed that ubiquitination or deubiquitination, such as P53 ubiquitination, ubiquitination of eukaryotic translation initiation complex 3(e IF3I) protein and USP19 deubiquitination, is related to cervical cancer. However, the effect of USP12 on human cervical carcinoma as well as its regulatory mechanism has not been reported yet. This study demonstrated that USP12 could regulate He La(cervical cancer) cell cycle progression, which depends on its deubiquitinating enzyme activity. In addition, we further explored protein regions that affect USP12 activity, thereby providing more evidence for molecular targeted therapy against cervical cancer. Part one: Effect of USP12 depletion on apoptosis and cell cycle of Hela cellObjective: In addition to its association with intracellular localization of substrate proteins, deubiquitination of histone H2 A and H2 B as well as the development of Xenopus laevis, USP12 is involved in the regulation of apoptosis or cell cycle in tumor cells, including prostate cancer cells, colon cancer cells, C33 A cells(HPV-negative human cervical carcinoma cells). Nevertheless, its relationship with He La cell(cervical cancer cell) remains an open issue. In this study, USP12 gene in He La cells was knocked down, to observe its effect on apoptosis and cell cycle of He La cell, thus further exploring the relationship between USP12 and cervical cancer.Methods:1 Three specific USP12 si RNA was transfected into He La cells. Then total RNA and total protein were extracted after 72 h. The effects of USP12 si RNAs were detected using real-time quantitative PCR(q RT-PCR) and Western blot analysis. Subsequently, USP12 si RNA-3 was transfected into He La cells, and cells were collected at different time points(at 24 h, 48 h and 72 h, respectively), to extract total RNA and total protein. q RT-PCR and Western blot analysis was used to measure m RNA or protein levels of USP12, thus determining the optimal time point for USP12 si RNA silencing.2 To ascertain the impact of USP12 knockdown on He La cells, USP12 si RNA-3 and negative control-si RNA were transfected into He La cells. The cells were harvested after 48 h, and fixed with 70% ethanol for incubation overnight. Flow cytometry was used to measure cell apoptosis and cell cycle distribution.3 The effect of USP12 depletion on cell cycle related factors: USP12 si RNA-3 and negative control-si RNA were transfected into He La cells, and total RNA were extracted after 48 h. Transcription of BMI-1, c-Myc and cyclin D2 were measured by q RT-PCR.Results:1 q RT-PCR results revealed that USP12 m RNA levels of He La cells in three si RNA transfection groups were decreased significantly, 72 h after transfection, as compared with the control group(*P<0.05). USP12 si RNA-3 was the most efficient in silencing(#P<0.05), and inhibited USP12 expression most greatly 48 h after transfection. Detection results by Western blot was consistent with those by q RT-PCR.2 We measured the number of apoptotic cells and cell cycle distribution by PI staining using flow cytometry. 48 h after knock-down of USP12 in He La cells, cell apoptosis rates in the control group and USP12 si RNA group were 4.03%±0.330% and 4.32%±0.324%, respectively, showing no statistical difference. The detection result of cell cycle revealed that, compared with the control group, the number of cells at G0/G1 phase in USP12 si RNA group was increased, rising from 60.7% ± 0.839% to 72.1% ± 2.61%, showing significant difference. This suggested that depletion of USP12 induce He La cell cycle arrest at G0/G1 phase, thereby inhibiting cell proliferation.3 Real-time PCR showed that, BMI-1, c-Myc and cyclin D2 m RNA levels in USP12 si RNA group decreased, as compared with the control group.Conclusions:1 In He La cells, si RNA-3 inhibited USP12 expression most greatly 48 h after transfection.2 FCM results showed that depletion of USP12 impaired cell growth by inhibiting cell cycle progression without causing apoptosis in He La cells.3 USP12 plays an important role in promoting He La cell cycle and involves the regulation of c-Myc, cyclin D2 and BMI-1 expression. Part two: The impact of USP12 missense mutation on its deubiquitinating enzyme activityObjective: In part one, USP12 gene was knocked-down in He La cells, confirming regulatory role of USP12 in He La cell cycle. However, it remains unknown whether USP12 deubiquitinating enzyme activity will affect He La cell apoptosis and cell cycle. Therefore, in addition to previously constructed USP12(C48S) and USP12(H173D) mutants, we plan to constructe seven additional mutants of USP12 with reference to the NCBI database and assess deubiquitinating enzymatic activity of these mutants. This would help gain indepth insight into the impact of USP12 enzyme activity on He La cell apoptosis and cell cycle.Methods:1 The previously constructed p GEX-USP12(WT) plasmid served as a template. Site-directed mutagenesis PCR was used to obtain plasmids, such as p GEX-USP12 V7 I, R61 H, A69 G, R152 H, L153 S, R237 C and N314 S, which were then confirmed by DNA sequencing. The above plasmids and previously constructed plasmids p GEX-USP12(C48S) and p GEX-USP12(H173D) served as templates. p AC-T7-USP12 wild-type and mutant plasmids were constructed by inserting the DNA fragments encoding the corresponding ORFs at the Bam H I site of plasmid p AC-T7.2 With Ub-Met-β-gal and GST-Ub52 as model substrates, we conducted the USP cleavage assay to assess deubiquitinating enzymatic activity of USP12 wild type and its nine mutants. The results were detected by Odyssey Infrared Imaging System.Results:1 DNA sequencing confirmed successful site-directed mutagenesis at the expected locis in each plasmid of p GEX-USP12: 19 G>A, 182 G>A, 206 C>G, 455 G>A, 458 T>C,709 C>T and 941 A> G mutations resulted in amino acid changes in V7 I, R61 H, A69 G, R152 H, L153 S, R237 C and N314 S. Furthermore, using restriction enzymes identified the constructed recombinant plasmids of p AC-T7-USP12. The results indicated that we successfully constructed p AC-T7-USP12 recombinant plasmid.2 Impact of missense mutations of USP12 on its deubiquitinating enzyme activity: In Ub-Met-β-gal detection system, USP12(C48S) and USP12(H173D) were inactivated mutants. This result was consistent with our preliminary findings. USP12 wild-type and other mutants have deubiquitinating enzyme activity, which can produce the desired Met-β-gal bands. Further semi-quantitative analysis showed deubiquitinating enzyme activity of USP12(L153S) and USP12(R237C) mutant was increased to 275% ± 32.6% and 158% ± 11.2% of wild-type respectively. There was no change in the activity of several mutants, including USP12(V7I), USP12(R61H), USP12(A69G), USP12(R152H) and USP12(N314S), as compared to the wild type.In GST-Ub52 detection system, USP12(C48S) remained inactivated. This was in accordance with findings by Ub-Met-β-gal detection system. Due to deubiquitinating enzyme activity, wild-type USP12 and other mutants can cleave the model substrate GST-Ub52. Further semi-quantitative analysis found that compared to its wild type, mutants of USP12 had similar deubiquitinating enzyme activity.Conclusions:1 In this part, USP12 V7 I, R61 H, A69 G, R152 H, L153 S, R237 C and N314 S mutants were constructed successfully.2 The deubiquitinating enzyme activity of USP12(C48S) was abrogated. Activity of USP12(H173D) in Ub-Met-β-gal detection system disappeared, but no change was found in GST-Ub52 detection system. In addition, USP12(L153S) and R237 C could improve USP12 enzyme activity, using Ub-Met-β-gal as a substrate. The remaining five SNP mutations of USP12 had no impact on its deubiquitinating enzyme activity. Part three: Effect of deubiquitinating enzyme activity of USP12 mutants on Hela cell apoptosis and cell cycleObjective: Our previous studies have demonstrated that USP12 can affect cell cycle progression in He La cells. To further investigate the impact of deubiquitinating enzyme activity of USP12 on He La cell apoptosis and cell cycle, p EGFP-USP12(WT), p EGFP-USP12(C48S), p EGFP-USP12(L153S) and p EGFP-USP12(R237C) plasmids were constructed, to observe their effect on He La cell apoptosis and cell cycle.Methods:1 USP12 mutants with active changes were selected. p GEX-USP12(WT), p GEX-USP12(C48S), p GEX-USP12(L153S) and p GEX-USP12(R237C) served as templates. Fusions of USP12 including WT, C48 S, L153 S and R237 C to green fluorescent protein(GFP) were constructed by inserting the DNA fragments encoding the corresponding ORFs at the Ecolr I site of plasmid p EGFP-C1.2 p EGFP-USP12 plasmids constructed in the step one and control plasmid p EGFP-C1 were transfected into He La cells, respectively. The cells were collected after 48 h, and subsequently fixed in 70% ethanol for incubation overnight. Cell apoptosis and cell cycle distribution were examined by flow cytometric analysis.3 p EGFP-USP12 plasmids constructed in the first step and control plasmid p EGFP-C1 were transfected into He La cells. After 48 h, cells were collected and the endogenous m RNA levels of BMI-1, c-Myc and cyclin D2 were analyzed by q RT-PCR.Results:1 Using restriction enzymes identified the constructed recombinant plasmids of p EGFP-USP12. The results indicated that we successfully constructed p EGFP-USP12 recombinant plasmid, including p EGFP-USP12(WT), p EGFP-USP12(C48S), p EGFP-USP12(L153S) and p EGFP-USP12(R237C).2 FCM analysis showed, after transfection of p EGFP-C1, p EGFP-USP12(WT), p EGFP-USP12(C48S), p EGFP-USP12(L153S) and p EGFP-USP12(R237C) into He La cell, analyses for the number of apoptotic cells demonstrated no significant differences among the p EGFP-USP transfected groups and the control group(P>0.05). In terms of cell cycle distribution, cell cycle progression was promoted by USP12(WT) and USP12(L153S), compared to the control group. Cells undergoing G0/G1 phase in both groups were reduced, while cells undergoing S phase were increased, with more marked change in L153 S group. The role of USP12(R237C) in cell cycle was similar to that of the wild type. Conversely, we did not observe any variation in the C48 S group compared to the control group.3 The q RT-PCR results showed that compared to the control group, the transcript levels of BMI-1, c-Myc and cyclin D2 genes increased in the wildtype group but did not change in the C48 S group. Similar to the wild-type, USP12(R237C) played a similar role in regulating c-Myc and cyclin D2 but not BMI-1. Furthermore, cells transfected with USP12(L153S) showed higher gene transcriptional activity compared to the wild-type group.Conclusions:1 In this part, p EGFP-USP12(WT), p EGFP-USP12(C48S), p EGFP-USP12(L153S) and p EGFP-USP12(R237C) were constructed successfully.2 He La cell apoptosis was not affected by the deubiquitinating enzyme activity of USP12, but the role of USP12 in He La cell cycle was in a deubiquitinating enzyme activity-dependent manner.3 The cell cycle promoting roles of USP12 WT, L153 S and R237 C in He La cells are mechanistically linked with the c-Myc/cyclin D2 pathway and/or BMI-1. Part four: The impact of USP12 multisite mutation on its deubiquitinating enzyme activityObjective: Our previous findings showed that, in addition to three conserved domains(Cys, Asp and His region) of USP12, site-directed mutagenesis in other regions could also affect the deubiquitinating enzymes activity of USP12. Recent studies have confirmed that some domains of USPs that outside the core catalytic domain, were associated with USP deubiquitinating enzyme activity, such as UBL(ubiquitin-like) region of USP4 and USP7, and 717-781 amino acid region of USP13. The question remains, except for the core catalytic domain of USP12, were there other domains that regulated its deubiquitinating enzyme activity? The sequence comparison of USP12 and USP46 showed highly homology, but the enzyme activity of USP46 is higher than USP12. We generated USP12 multisite mutant in each of which a fragment in USP12 was replaced by a corresponding segment in USP46. Subsequently, deubiquitinating enzyme activity of this mutant was detected, in order to explore other domains that played key roles in USP12 activity. This may contribute to the understanding of the genetic mechanisms that underlie cervical cancer.Methods:1 With p GEX-USP12(WT) or p GEX-USP46(WT) plasmid as a template, we designed specific primers for multisite mutagenesis. Chimeras(USP12(152-169) and USP46(148-165)) in which the peptides(from R152 to P169 for USP12 and K148 to L165 for USP46) were exchanged between USP12 and USP46 were constructed with PCR. The above multisite mutants were verified by DNA sequencing.2 The USP cleavage assay was performed to assess deubiquitinating enzyme activity of USP12(152-169) and USP46(148-165) mutants, using Ub-Met-β-gal as model substrate.Results:1 DNA sequencing confirmed successful multisite mutagenesis at the expected locis in plasmid of p GEX-USP12(152-169). This multisite mutation resulted in amino acid changes of 152-169 from RLPNGNIDNENNNSTPDP to KLKNGNMNEPAENNKPEL(which was consistent with 148-165 amino acid sequence of USP46). In addition, p GEX-USP46(148-165) plasmid also experienced successful multisite mutagenesis at the expected locis. The multisite mutation resulted in amino acid changes of 148-165 from KLKNGNMNEPAENNKPEL to RLPNGNIDNENNNSTPDP(which was consistent with 152-169 amino acid sequence of USP12).2 The USP cleavage assay showed that USP12(152-169) mutant has deubiquitinating enzyme activity, could cleave Ub-Met-β-gal model substrate into Met-β-gal. Moreover, the relative deubiquitinating enzyme activity increased to 331% ± 28.9% in USP12(152-169) compared with wild-type(***P<0.001), while similar with USP46(WT). Deubiquitinating enzyme activity of USP46(148-165) mutant did not change, as compared to USP46(WT).Conclusions:1 In this part, p GEX-USP12(152-169) and p GEX-USP46(148-165) multisite mutant plasmids were constructed successfully.2 In Ub-Met-β-gal enzyme activity detection system, after multisite mutations of USP12, its deubiquitinating enzyme activity was significantly enhanced, which was similar to that of USP46 wild-type, suggesting the effect on USP12 deubiquitinating enzyme activity is related to sequence R152 to P169.