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MicroRNA-500 sustains nuclear factor-κB activation and induces gastric cancer cell proliferation and resistance to apoptosis.

Zhang L, Ding Y, Yuan Z, Liu J, Sun J, Lei F, Wu S, Li S, Zhang D - Oncotarget (2015)

Bottom Line: Ubiquitin deconjugation of key signalling molecules by deubiquitinases (DUBs) such as cylindromatosis (CYLD), A20, and OTU deubiquitinase 7B (OTUD7B) has emerged as an important regulatory mechanism in the downregulation of NF-κB signalling and homeostasis.Here, we report that the miR-500 directly repressed the expression of CYLD, OTUD7B, and the A20 complex component Tax1-binding protein 1 (TAX1BP1), leading to ubiquitin conjugation of receptor-interacting protein 1 (RIP1) and sustained NF-ĸB activation.Furthermore, we found that miR-500 promoted gastric cancer cell proliferation, survival, and tumorigenicity.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in Southern China, Collaborative Innovation Center for Cancer Medicine, Department of Diagnostic Imaging and Interventional Radiology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China.

ABSTRACT
Ubiquitin deconjugation of key signalling molecules by deubiquitinases (DUBs) such as cylindromatosis (CYLD), A20, and OTU deubiquitinase 7B (OTUD7B) has emerged as an important regulatory mechanism in the downregulation of NF-κB signalling and homeostasis. However, how these serial negative regulations are simultaneously disrupted to result in constitutive activation of NF-κB signalling in cancers remains puzzling. Here, we report that the miR-500 directly repressed the expression of CYLD, OTUD7B, and the A20 complex component Tax1-binding protein 1 (TAX1BP1), leading to ubiquitin conjugation of receptor-interacting protein 1 (RIP1) and sustained NF-ĸB activation. Furthermore, we found that miR-500 promoted gastric cancer cell proliferation, survival, and tumorigenicity. Importantly, miR-500 was upregulated in gastric cancer and was highly correlated with malignant progression and poor survival. Hence, we report the uncovering of a novel mechanism for constitutive NF-κB activation, indicating the potentially pivotal role of miR-500 in the progression of gastric cancer.

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MiR-500 directly suppresses multiple NF-ĸB negative regulatory genes(A) Predicted miR-500 target sequence in 3′ UTRs of CYLD, TAX1BP1, and OTUD7B. (B) Western blots of CYLD, TAX1BP1, and OTUD7B expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-CYLD-3′UTR, pGL3-TAX1BP1-3′UTR, or pGL3-OTUD7B-3′UTR reporter with miR-500 mimic, antagomiR-500, or miR-500-mut mimic. (D) MiRNP IP assay showing the association between miR-500 and CYLD, TAX1BP1, and OTUD7B transcripts. GAPDH served as the negative control. (E) Western blots of K63-linked polyubiquitin levels of RIP1 in miR-500–overexpressing or miR-500–silenced MKN-28 cells treated with TNF-α (10 ng/mL). V, vector; Ctr, control. (F) Western blot analysis of p-IKKβ, total IKKβ, and IκBα expression in cells treated with 10 ng/ml TNF-α. (G)In vitro IKK kinase assay of vector- or miR-500–overexpressing cells, or miR-500–silenced cells treated with 10 ng/mL TNF-α. IKKβ was subjected to IP, and kinase activity was determined by phosphorylation of a recombinant GST-IκBα substrate using a phospho-specific IκαB antibody. The equal IP of IKKβ was shown. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
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Figure 5: MiR-500 directly suppresses multiple NF-ĸB negative regulatory genes(A) Predicted miR-500 target sequence in 3′ UTRs of CYLD, TAX1BP1, and OTUD7B. (B) Western blots of CYLD, TAX1BP1, and OTUD7B expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-CYLD-3′UTR, pGL3-TAX1BP1-3′UTR, or pGL3-OTUD7B-3′UTR reporter with miR-500 mimic, antagomiR-500, or miR-500-mut mimic. (D) MiRNP IP assay showing the association between miR-500 and CYLD, TAX1BP1, and OTUD7B transcripts. GAPDH served as the negative control. (E) Western blots of K63-linked polyubiquitin levels of RIP1 in miR-500–overexpressing or miR-500–silenced MKN-28 cells treated with TNF-α (10 ng/mL). V, vector; Ctr, control. (F) Western blot analysis of p-IKKβ, total IKKβ, and IκBα expression in cells treated with 10 ng/ml TNF-α. (G)In vitro IKK kinase assay of vector- or miR-500–overexpressing cells, or miR-500–silenced cells treated with 10 ng/mL TNF-α. IKKβ was subjected to IP, and kinase activity was determined by phosphorylation of a recombinant GST-IκBα substrate using a phospho-specific IκαB antibody. The equal IP of IKKβ was shown. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.

Mentions: Using the TargetScan program, we found that CYLD, OTUD7B, and the A20 complex component TAX1BP1, which function as critical negative regulatory genes by deconjugating K63-polyubiquitin chains from RIP1 [21, 28], might be potential targets of miR-500 (Figure 5A). Western blot analysis revealed that CYLD, TAX1BP1, and OTUD7B expression levels were significantly decreased in miR-500–transduced cells, but were increased in miR-500–silenced cells (Figure 5B), suggesting that miR-500 negatively regulated these proteins. Furthermore, luciferase assay indicated that miR-500 overexpression decreased the reporter activities linked with the 3′ UTR of their transcripts, but miR-500 silencing increased it (Figure 5C). However, ectopically expressing the miR-500 mutant (miR-500-mut) did not result in repressive effects on the 3′ UTRs (Figure 5C). Importantly, microribonucleoprotein (miRNP) IP showed that miR-500 overexpression enriched the transcripts of CYLD, TAX1BP1, and OTUD7B, but not GAPDH, that assembled into the miRNP complexes, indicating that miR-500 directly targets the mRNA 3′ UTR regions of these transcripts (Figure 5D). Taken together, our results demonstrate that CYLD, TAX1BP1, and OTUD7B are bona fide targets of miR-500.


MicroRNA-500 sustains nuclear factor-κB activation and induces gastric cancer cell proliferation and resistance to apoptosis.

Zhang L, Ding Y, Yuan Z, Liu J, Sun J, Lei F, Wu S, Li S, Zhang D - Oncotarget (2015)

MiR-500 directly suppresses multiple NF-ĸB negative regulatory genes(A) Predicted miR-500 target sequence in 3′ UTRs of CYLD, TAX1BP1, and OTUD7B. (B) Western blots of CYLD, TAX1BP1, and OTUD7B expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-CYLD-3′UTR, pGL3-TAX1BP1-3′UTR, or pGL3-OTUD7B-3′UTR reporter with miR-500 mimic, antagomiR-500, or miR-500-mut mimic. (D) MiRNP IP assay showing the association between miR-500 and CYLD, TAX1BP1, and OTUD7B transcripts. GAPDH served as the negative control. (E) Western blots of K63-linked polyubiquitin levels of RIP1 in miR-500–overexpressing or miR-500–silenced MKN-28 cells treated with TNF-α (10 ng/mL). V, vector; Ctr, control. (F) Western blot analysis of p-IKKβ, total IKKβ, and IκBα expression in cells treated with 10 ng/ml TNF-α. (G)In vitro IKK kinase assay of vector- or miR-500–overexpressing cells, or miR-500–silenced cells treated with 10 ng/mL TNF-α. IKKβ was subjected to IP, and kinase activity was determined by phosphorylation of a recombinant GST-IκBα substrate using a phospho-specific IκαB antibody. The equal IP of IKKβ was shown. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: MiR-500 directly suppresses multiple NF-ĸB negative regulatory genes(A) Predicted miR-500 target sequence in 3′ UTRs of CYLD, TAX1BP1, and OTUD7B. (B) Western blots of CYLD, TAX1BP1, and OTUD7B expression. α-Tubulin served as the loading control. (C) Luciferase assay of cells transfected with pGL3-CYLD-3′UTR, pGL3-TAX1BP1-3′UTR, or pGL3-OTUD7B-3′UTR reporter with miR-500 mimic, antagomiR-500, or miR-500-mut mimic. (D) MiRNP IP assay showing the association between miR-500 and CYLD, TAX1BP1, and OTUD7B transcripts. GAPDH served as the negative control. (E) Western blots of K63-linked polyubiquitin levels of RIP1 in miR-500–overexpressing or miR-500–silenced MKN-28 cells treated with TNF-α (10 ng/mL). V, vector; Ctr, control. (F) Western blot analysis of p-IKKβ, total IKKβ, and IκBα expression in cells treated with 10 ng/ml TNF-α. (G)In vitro IKK kinase assay of vector- or miR-500–overexpressing cells, or miR-500–silenced cells treated with 10 ng/mL TNF-α. IKKβ was subjected to IP, and kinase activity was determined by phosphorylation of a recombinant GST-IκBα substrate using a phospho-specific IκαB antibody. The equal IP of IKKβ was shown. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
Mentions: Using the TargetScan program, we found that CYLD, OTUD7B, and the A20 complex component TAX1BP1, which function as critical negative regulatory genes by deconjugating K63-polyubiquitin chains from RIP1 [21, 28], might be potential targets of miR-500 (Figure 5A). Western blot analysis revealed that CYLD, TAX1BP1, and OTUD7B expression levels were significantly decreased in miR-500–transduced cells, but were increased in miR-500–silenced cells (Figure 5B), suggesting that miR-500 negatively regulated these proteins. Furthermore, luciferase assay indicated that miR-500 overexpression decreased the reporter activities linked with the 3′ UTR of their transcripts, but miR-500 silencing increased it (Figure 5C). However, ectopically expressing the miR-500 mutant (miR-500-mut) did not result in repressive effects on the 3′ UTRs (Figure 5C). Importantly, microribonucleoprotein (miRNP) IP showed that miR-500 overexpression enriched the transcripts of CYLD, TAX1BP1, and OTUD7B, but not GAPDH, that assembled into the miRNP complexes, indicating that miR-500 directly targets the mRNA 3′ UTR regions of these transcripts (Figure 5D). Taken together, our results demonstrate that CYLD, TAX1BP1, and OTUD7B are bona fide targets of miR-500.

Bottom Line: Ubiquitin deconjugation of key signalling molecules by deubiquitinases (DUBs) such as cylindromatosis (CYLD), A20, and OTU deubiquitinase 7B (OTUD7B) has emerged as an important regulatory mechanism in the downregulation of NF-κB signalling and homeostasis.Here, we report that the miR-500 directly repressed the expression of CYLD, OTUD7B, and the A20 complex component Tax1-binding protein 1 (TAX1BP1), leading to ubiquitin conjugation of receptor-interacting protein 1 (RIP1) and sustained NF-ĸB activation.Furthermore, we found that miR-500 promoted gastric cancer cell proliferation, survival, and tumorigenicity.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in Southern China, Collaborative Innovation Center for Cancer Medicine, Department of Diagnostic Imaging and Interventional Radiology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China.

ABSTRACT
Ubiquitin deconjugation of key signalling molecules by deubiquitinases (DUBs) such as cylindromatosis (CYLD), A20, and OTU deubiquitinase 7B (OTUD7B) has emerged as an important regulatory mechanism in the downregulation of NF-κB signalling and homeostasis. However, how these serial negative regulations are simultaneously disrupted to result in constitutive activation of NF-κB signalling in cancers remains puzzling. Here, we report that the miR-500 directly repressed the expression of CYLD, OTUD7B, and the A20 complex component Tax1-binding protein 1 (TAX1BP1), leading to ubiquitin conjugation of receptor-interacting protein 1 (RIP1) and sustained NF-ĸB activation. Furthermore, we found that miR-500 promoted gastric cancer cell proliferation, survival, and tumorigenicity. Importantly, miR-500 was upregulated in gastric cancer and was highly correlated with malignant progression and poor survival. Hence, we report the uncovering of a novel mechanism for constitutive NF-κB activation, indicating the potentially pivotal role of miR-500 in the progression of gastric cancer.

Show MeSH
Related in: MedlinePlus