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USP22 promotes NSCLC tumorigenesis via MDMX up-regulation and subsequent p53 inhibition.

Ding F, Bao C, Tian Y, Xiao H, Wang M, Xie X, Hu F, Mei J - Int J Mol Sci (2014)

Bottom Line: Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology.Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway.Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092, China. drnail@sina.com.

ABSTRACT
Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology. In this study, we investigated the role of USP22 in human NSCLC tumorigenesis along with the underlying mechanisms of action. First, we determined the expression of USP22 in human NSCLC, as well as normal tissues and cell lines. We then studied the effects of USP22 silencing by shRNA on NSCLC cell growth in vitro and tumorigenesis in vivo, along with the effect on the p53 pathway. We found that USP22 is overexpressed in human NSCLC tissues and cell lines. USP22 silencing by shRNA inhibits proliferation, induces apoptosis and arrests cells at the G0/G1 phases in NSCLC cells and curbs human NSCLC tumor growth in a mouse xenograft model. Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway. Our co-immunoprecipitation analysis shows that USP22 interacts with MDMX in NSCLC cells. Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects. Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition. USP22 may represent a novel target for NSCLC treatment.

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Cell proliferation, cell cycle distribution, cell apoptosis and p53 pathway in A549 cells transfected with SCR shRNA, USP22 shRNA, MDMX shRNA or co-transfected with USP22 shRNA and Lv/MDMX. (A) The mRNA and protein expressions of MDMX; (B) levels of p53 pathway proteins in A549 cells by Western blot; (C) cell proliferation by the MTT assay. **p < 0.01; (D,E) Cell cycle distribution by flow cytometry with PI staining (D) and cell apoptosis by flow cytometry with Annexin V-FITC and PI double staining (E). *p < 0.05 vs. SCR shRNA, **p < 0.01 vs. SCR shRNA, #p < 0.05 vs. USP22 shRNA, δp < 0.05 vs. MDMX shRNA. Data are expressed as the means ± SD, n = 3.
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ijms-16-00307-f005: Cell proliferation, cell cycle distribution, cell apoptosis and p53 pathway in A549 cells transfected with SCR shRNA, USP22 shRNA, MDMX shRNA or co-transfected with USP22 shRNA and Lv/MDMX. (A) The mRNA and protein expressions of MDMX; (B) levels of p53 pathway proteins in A549 cells by Western blot; (C) cell proliferation by the MTT assay. **p < 0.01; (D,E) Cell cycle distribution by flow cytometry with PI staining (D) and cell apoptosis by flow cytometry with Annexin V-FITC and PI double staining (E). *p < 0.05 vs. SCR shRNA, **p < 0.01 vs. SCR shRNA, #p < 0.05 vs. USP22 shRNA, δp < 0.05 vs. MDMX shRNA. Data are expressed as the means ± SD, n = 3.

Mentions: Our findings on the relationship between USP22, p53 and MDMX in NSCLC cells suggest that MDMX may be a key mediator of USP22’s regulatory effects in NSCLC. To test this hypothesis, we studied the effects of MDMX silencing by MDMX-specific shRNA transfection in A549 cells (Figure 5A). Indeed, we found that MDMX silencing activated the p53 pathway, inhibited cell proliferation and induced apoptosis and cell cycle arrest at the G0/G1 phases, strikingly similar to that observed with USP22 silencing (Figure 5B–E). Importantly, overexpression of MDMX reversed USP22 silencing, induced p53 activation, growth inhibition, cell cycle arrest and apoptosis in A549 cells (Figure 5B–E). These results demonstrate that MDMX mediates USP22’s regulation effect in the NSCLC cell line.


USP22 promotes NSCLC tumorigenesis via MDMX up-regulation and subsequent p53 inhibition.

Ding F, Bao C, Tian Y, Xiao H, Wang M, Xie X, Hu F, Mei J - Int J Mol Sci (2014)

Cell proliferation, cell cycle distribution, cell apoptosis and p53 pathway in A549 cells transfected with SCR shRNA, USP22 shRNA, MDMX shRNA or co-transfected with USP22 shRNA and Lv/MDMX. (A) The mRNA and protein expressions of MDMX; (B) levels of p53 pathway proteins in A549 cells by Western blot; (C) cell proliferation by the MTT assay. **p < 0.01; (D,E) Cell cycle distribution by flow cytometry with PI staining (D) and cell apoptosis by flow cytometry with Annexin V-FITC and PI double staining (E). *p < 0.05 vs. SCR shRNA, **p < 0.01 vs. SCR shRNA, #p < 0.05 vs. USP22 shRNA, δp < 0.05 vs. MDMX shRNA. Data are expressed as the means ± SD, n = 3.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4307248&req=5

ijms-16-00307-f005: Cell proliferation, cell cycle distribution, cell apoptosis and p53 pathway in A549 cells transfected with SCR shRNA, USP22 shRNA, MDMX shRNA or co-transfected with USP22 shRNA and Lv/MDMX. (A) The mRNA and protein expressions of MDMX; (B) levels of p53 pathway proteins in A549 cells by Western blot; (C) cell proliferation by the MTT assay. **p < 0.01; (D,E) Cell cycle distribution by flow cytometry with PI staining (D) and cell apoptosis by flow cytometry with Annexin V-FITC and PI double staining (E). *p < 0.05 vs. SCR shRNA, **p < 0.01 vs. SCR shRNA, #p < 0.05 vs. USP22 shRNA, δp < 0.05 vs. MDMX shRNA. Data are expressed as the means ± SD, n = 3.
Mentions: Our findings on the relationship between USP22, p53 and MDMX in NSCLC cells suggest that MDMX may be a key mediator of USP22’s regulatory effects in NSCLC. To test this hypothesis, we studied the effects of MDMX silencing by MDMX-specific shRNA transfection in A549 cells (Figure 5A). Indeed, we found that MDMX silencing activated the p53 pathway, inhibited cell proliferation and induced apoptosis and cell cycle arrest at the G0/G1 phases, strikingly similar to that observed with USP22 silencing (Figure 5B–E). Importantly, overexpression of MDMX reversed USP22 silencing, induced p53 activation, growth inhibition, cell cycle arrest and apoptosis in A549 cells (Figure 5B–E). These results demonstrate that MDMX mediates USP22’s regulation effect in the NSCLC cell line.

Bottom Line: Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology.Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway.Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200092, China. drnail@sina.com.

ABSTRACT
Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology. In this study, we investigated the role of USP22 in human NSCLC tumorigenesis along with the underlying mechanisms of action. First, we determined the expression of USP22 in human NSCLC, as well as normal tissues and cell lines. We then studied the effects of USP22 silencing by shRNA on NSCLC cell growth in vitro and tumorigenesis in vivo, along with the effect on the p53 pathway. We found that USP22 is overexpressed in human NSCLC tissues and cell lines. USP22 silencing by shRNA inhibits proliferation, induces apoptosis and arrests cells at the G0/G1 phases in NSCLC cells and curbs human NSCLC tumor growth in a mouse xenograft model. Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway. Our co-immunoprecipitation analysis shows that USP22 interacts with MDMX in NSCLC cells. Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects. Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition. USP22 may represent a novel target for NSCLC treatment.

Show MeSH
Related in: MedlinePlus