- Here, we report that ubiquitin specific peptidase 3 (USP3) promotes proliferation and metastasis of esophageal squamous cell carcinoma (ESCC) cells by mediating deubiquitination of Aurora A. - In this mechanism, USP3 leads to suppression of Aurora A ubiquitination, resulting less proteasome degradation. - deubiquitinated mimetic K143R of Aurora A and found that K143R significantly promoted the proliferation and invasion of ESCC cells and was not regulated by the deubiquitination of USP3. - Moreover, Aurora A K143R potentiated the kinase activity of Aurora A in ESCC cells. - Thus, our findings demonstrate that the tumorigenic feature of ESCC is in part mediated by USP3- facilitated deubiquitination of Aurora A.. - USP3 is able to regulate monoubiquitinated H2A and H2B levels, and USP3 knockdown in U2OS cells leads to delay in the S-phase of the cell cycle as well as increased accumulation of ionizing radiation-induced DNA damage [11]. - Aurora A and USP3 gene knockdown were performed by siRNA in this study. - E Expression of Aurora A mRNA in 16 human ESCC specimens. - G Aurora A and USP3 protein expression in 3 typical ESCC specimens. - High expression of Aurora A and USP3 in human ESCC We first studied human ESCC samples. - 1A), and that high expression of Aurora A correlated to a high ESCC grade (Fig. - RT-PCR results showed that mRNA levels of Aurora A and USP3 were highly expressed in ESCC compared to adjacent tissues (Fig. - The tissue samples were then lysed and protein content extracted in order to determine the protein levels of Aurora A and USP3. - We found that Aurora A and USP3 proteins were highly expressed in ESCC tis- sues (Fig. - In conclusion, these findings indicate that Aurora A and USP3 are highly expressed in human ESCCtissues and are associated with poor tumor grade.. - Increased proliferation of ESCC cell lines by Aurora A and USP3. - Next, we investigated the function of Aurora A and USP3 in ESCC cell lines. - We selected ESCC cell line ECa109 as a research model and performed siRNA knockdown and overexpression of Aurora A and USP3 in the ECa109 cells. - Knockdown and overexpression of Aurora A (Fig. - 2A, C and E) and USP3 (Fig. - In ECa109 cells, knockdown of Aurora A and USP3 using siRNA significantly slowed the proliferation of ECa109 cells (Fig. - Overexpression of Aur- ora A and USP3 increased the rate ECa109 cell prolifera- tion (Fig. - Similarly, colony formation assays confirmed a decrease in the number of clones in Aurora A and USP3 knockdown ECa109 cells (Fig. - 2K and L), and an increase in the number of Aurora A and USP3 overexpressed ECa109 cell clones (Fig. - Our data confirms that Aurora A and USP3 were able to pro- mote proliferation of ESCC cells.. - A-B Aurora A and USP3 were knocked down by siRNA in ESCCcell line ECa109 cells, respectively, and knockdown efficiency was measured by qPCR. - C-D Aurora A and USP3 were knocked down by siRNA in ECa109 cells, and the protein was knocked out in the cells by western blot. - E-F Overexpression of Aurora A and USP3 in ECa109 cells, and Western blot analysis confirmed that proteins were highly expressed in cells. - G-J CCK8 confirmed that Aurora A and USP3 promoted the proliferation of ECa109 cells. - K-L After Aurora A and USP3 knockdown by siRNA in ECa109 cells, cells were plated in 12-well plates, cultured for 14 days, and then stained with crystal violet. - M-N After over-expressing Flag-Aurora A and HA-USP3 in ECa109 cells, cells were plated in 12-well plates, cultured for 14 days, and then stained with crystal violet. - Aurora A and USP3 promote invasion and metastasis of ESCC cells. - We then studied the role of Aurora A and USP3 in the invasion and metastasis of ESCC cells. - ECa109 cells were plated in 6-well plates. - The wound healing assays exam- ined the influences of Aurora A and USP3 knockdown on migration ability. - The migration distances of Aurora A (Fig. - 3A) and USP3 (Fig. - 3C) and USP3 (Fig. - 3E) and USP3 (Fig. - After overexpression of Aurora A (Fig. - 3G) and USP3 (Fig. - Our data indicates that Aurora A and USP3 upregulation enhances the abil- ity of ESCC cells to invade and metastasize.. - After overexpression of. - A-B ECa109 cells were plated in 6-well plates. - After siRNA knocked out Aurora A and USP3, wound healing experiments were performed to observe the degree of scratch healing. - C-D After overexpression of Flag- Aurora A and HA-USP3, wound healing experiments were performed to observe the degree of healing of the scratches. - E-F ECa109 cells were plated in 6-well plates, and after knocking down Aurora A and USP3 with siRNA, transwell experiments were performed and the number of cells passing through transwell was counted. - G-H After overexpression of Flag-Aurora A and HA-USP3, transwell experiments were performed and the number of cells passing through transwell was counted. - USP3, the protein stability of Aurora A in CHX-treated ESCC cells was significantly increased (Fig. - Therefore, USP3 enhances the stability of Aurora A protein by inhibiting the proteasome deg- radation pathway of Aurora A protein.. - USP3 interacts with Aurora A and reduces ubiquitination of Aurora A. - Proteasome degradation of Aurora A is dependent on the ubiquitination of Aurora A. - The ubiquitination site of Aurora A was predicted to be at the lysine molecule. - Similarly, immunoprecipitation of Aurora A protein showed that Aurora A binds USP3 instead of USP7 (Fig. - We mutated the predicted Aurora A ly- sine ubiquitination site to arginine (K143R), and the co- immunoprecipitation experiment demonstrated a de- crease in the interaction between Aurora A K143R and USP3 (Fig. - The level of ubi- quitination of Aurora A K143R was significantly lower. - 5 USP3 interacts with Aurora-A to inhibit ubiquitination of Aurora A K143. - A Aurora A ubiquitinated mutant Aurora A K143R was constructed and expressed in ECa109 cells. - G The ubiquitination of Aurora A was detected after overexpression of USP3. - H Compare the ubiquitination levels of Aurora A K143R and Aurora A. - than that of Aurora A (Fig. - Our data confirm that USP3 interacts with Aurora A and mediates deubiquiti- nation of the Aurora A K143 locus.. - After knockdown of USP3 in ECa109 cells, overexpression of Aurora A K143R reversed the inhibitory effect of USP3- siRNA on ESCC cells (Fig. - Similarly, in ECa109 cells overexpressing both Aurora A K143R and USP3 ex- perienced attenuation in degree of cell proliferation (Fig. - On the other hand, overexpression of Aurora A K143R significantly promoted invasion and metastasis (Fig. - 6F and G) of ECa109 cells. - After overexpression of Aurora A K143R, USP3-siRNA inhibited cell invasion (Fig. - 6H and J) and USP3 significantly attenuated cell in- vasion (Fig. - We examined mRNA and protein expression of EMT-related markers and found that over- expression of Aurora A K143R significantly promoted the process of EMT (Fig. - After overexpression of Aurora A K143R in ECa109 cells, mRNA expressions of Aurora A-dependent cell cycle progression-associated genes were significantly increased (Fig. - Thus, USP3-dependent deubiquitination of Aurora A in ESCC cells enhances the kinase activity of Aurora A.. - showed that after treatment with Aurora kinase inhibitor VX-680, the ex- pression of Aurora A decreased and the expression of E- cadherin increased with the increase of VX-680 concen- tration [13]. - The above studies suggest that high expres- sion of Aurora-A may affect the adhesion of tumor cells to cells by affecting E-cadherin. - F-G After overexpression of Aurora A K143R by ECa109 cells, cell invasion and metastasis increased. - H-K After overexpression of the Aurora A K143R mutant, USP3 had a reduced effect on cell invasion and metastasis. - mRNA and protein assays demonstrated that Aurora A and Aurora A K143R promoted the EMT process. - After overexpression of the Aurora A K143R mutant, USP3 attenuated changes in the EMT process. - In our study, we found that USP3 interacted with Aurora A to reduce the ubiqui- tination level of Aurora A and inhibited the proteasome degradation pathway of Aurora A. - We constructed the deubiquitinated mimetic K143R of Aurora A and found that K143R significantly promoted the proliferation and invasion of ESCC cells, and was not regulated by the deu- biquitination of USP3. - Moreover, Aurora A K143R poten- tiated the kinase activity of Aurora A in ESCC cells. - https://doi.org . - https://doi.org/10.1016/j.bbrc . - https://doi.org/10.1074/jbc . - https://doi.org/10.1016/j.bbagen.2016.. - https://doi.org/10.3322/caac.20107.. - https://doi.org/10.1084/jem.20131436.. - A The mRNA expression of cell cycle-related downstream genes in ECa109 cells overexpressed Aurora A and Aurora A K143R was detected. - B The mRNA expression of AP-2 α -related downstream genes under Aurora A and Aurora A K143R overexpression was compared. - D Overexpression of Aurora A and Aurora A K143R was performed to detect the expression of AKT-RAS-related downstream gene mRNA. - E-F Compare the effects of Aurora A and Aurora A K143R on cell cycle changes. - https://doi.org/10.1016/j.. - https://doi.org/10.2478/pjs-2014-0028.. - https://doi.org/10.1007/s . - https://doi.org/10.4161/cc.26814.. - https://doi.org/10.1016/j.intimp . - Overexpression of Aurora-A enhances invasion and matrix metalloproteinase-2 expression in esophageal squamous cell carcinoma cells. - https://doi.org/10.1038/s . - https://doi.org/10.1002/med.21399.. - doi.org/10.1002/ijc.22154.. - Overexpression of Aurora-A promotes laryngeal cancer progression by enhancing invasive ability and
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