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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 activates the NF-ĸB signalling pathway(A) NF-κB luciferase reporter activities were analysed in miR-500 or miR-500 plus IκBα-mut cells. (B) NF-κB luciferase reporter activities were analysed in miR-500–inhibited cells. (C) Real-time PCR analysis indicating an apparent overlap between NF-κB–dependent gene expression and miR-500–regulated gene expression. The pseudocolour represents the intensity scale of miR-500 versus Vector (V) or antagomiR-500 versus Control (Ctr), generated by log2 transformation. (D) Western blotting of nuclear p65 expression. The nuclear protein p84 was used as a nuclear protein marker. (E) Immunofluorescence staining of subcellular localisation of NF-κB/p65. (F) Cell cycle analysis of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23). (G) TUNEL of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23), and treated with cisplatin (20 μM) for 36 h. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
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Figure 4: MiR-500 activates the NF-ĸB signalling pathway(A) NF-κB luciferase reporter activities were analysed in miR-500 or miR-500 plus IκBα-mut cells. (B) NF-κB luciferase reporter activities were analysed in miR-500–inhibited cells. (C) Real-time PCR analysis indicating an apparent overlap between NF-κB–dependent gene expression and miR-500–regulated gene expression. The pseudocolour represents the intensity scale of miR-500 versus Vector (V) or antagomiR-500 versus Control (Ctr), generated by log2 transformation. (D) Western blotting of nuclear p65 expression. The nuclear protein p84 was used as a nuclear protein marker. (E) Immunofluorescence staining of subcellular localisation of NF-κB/p65. (F) Cell cycle analysis of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23). (G) TUNEL of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23), and treated with cisplatin (20 μM) for 36 h. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.

Mentions: We next explored the underlying molecular mechanism that might be responsible for the oncogenic roles of miR-500. Since NF-ĸB signaling pathway is one of the key signalling pathways that contributes to cell proliferation and apoptosis [29], and has been found frequently hyperactivated in gastric cancers [4], we then examined whether miR-500 regulated the NF-κB activity. As expected, overexpression of miR-500 significantly increased, but silencing of miR-500 reduced, the NF-ĸB luciferase activity and the expression levels of numerous NF-ĸB downstream target genes in gastric cancer cells (Figure 4A–4C). And transfection of a IκBα dominant-negative mutant (IκBα-mut) abrogated NF-ĸB activation induced by miR-500 overexpression (Figure 4A). Moreover, cellular fractionation and immunofluorescence staining assays showed that miR-500 overexpression promoted nuclear accumulation of NF-κB/p65, while miR-500 silencing reduced nuclear NF-κB/p65 expression but not total p65 level (Figure 4D, 4E and Supplementary Figure 5), indicating that miR-500 activates the NF-κB signalling pathway by promoting nuclear NF-κB/p65 accumulation. Importantly, the stimulatory effects of miR-500 on gastric cancer cell proliferation and survival were markedly reduced upon NF-κB inhibition by transfection of IκBα-mut or treatment with a NF-κB inhibitor (Figure 4F and 4G). Thus, these results demonstrate that functional activation of the NF-κB signalling pathway is vital to the biological effects of miR-500 in gastric cancer progression.


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 activates the NF-ĸB signalling pathway(A) NF-κB luciferase reporter activities were analysed in miR-500 or miR-500 plus IκBα-mut cells. (B) NF-κB luciferase reporter activities were analysed in miR-500–inhibited cells. (C) Real-time PCR analysis indicating an apparent overlap between NF-κB–dependent gene expression and miR-500–regulated gene expression. The pseudocolour represents the intensity scale of miR-500 versus Vector (V) or antagomiR-500 versus Control (Ctr), generated by log2 transformation. (D) Western blotting of nuclear p65 expression. The nuclear protein p84 was used as a nuclear protein marker. (E) Immunofluorescence staining of subcellular localisation of NF-κB/p65. (F) Cell cycle analysis of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23). (G) TUNEL of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23), and treated with cisplatin (20 μM) for 36 h. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
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Related In: Results  -  Collection

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Figure 4: MiR-500 activates the NF-ĸB signalling pathway(A) NF-κB luciferase reporter activities were analysed in miR-500 or miR-500 plus IκBα-mut cells. (B) NF-κB luciferase reporter activities were analysed in miR-500–inhibited cells. (C) Real-time PCR analysis indicating an apparent overlap between NF-κB–dependent gene expression and miR-500–regulated gene expression. The pseudocolour represents the intensity scale of miR-500 versus Vector (V) or antagomiR-500 versus Control (Ctr), generated by log2 transformation. (D) Western blotting of nuclear p65 expression. The nuclear protein p84 was used as a nuclear protein marker. (E) Immunofluorescence staining of subcellular localisation of NF-κB/p65. (F) Cell cycle analysis of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23). (G) TUNEL of MKN-28 cells transfected with miR-500 or miR-500 plus IκBα-mut, or miR-500 plus NF-κB inhibitor (JSH-23), and treated with cisplatin (20 μM) for 36 h. Each bar represents the mean ± SD of three independent experiments. *p < 0.05.
Mentions: We next explored the underlying molecular mechanism that might be responsible for the oncogenic roles of miR-500. Since NF-ĸB signaling pathway is one of the key signalling pathways that contributes to cell proliferation and apoptosis [29], and has been found frequently hyperactivated in gastric cancers [4], we then examined whether miR-500 regulated the NF-κB activity. As expected, overexpression of miR-500 significantly increased, but silencing of miR-500 reduced, the NF-ĸB luciferase activity and the expression levels of numerous NF-ĸB downstream target genes in gastric cancer cells (Figure 4A–4C). And transfection of a IκBα dominant-negative mutant (IκBα-mut) abrogated NF-ĸB activation induced by miR-500 overexpression (Figure 4A). Moreover, cellular fractionation and immunofluorescence staining assays showed that miR-500 overexpression promoted nuclear accumulation of NF-κB/p65, while miR-500 silencing reduced nuclear NF-κB/p65 expression but not total p65 level (Figure 4D, 4E and Supplementary Figure 5), indicating that miR-500 activates the NF-κB signalling pathway by promoting nuclear NF-κB/p65 accumulation. Importantly, the stimulatory effects of miR-500 on gastric cancer cell proliferation and survival were markedly reduced upon NF-κB inhibition by transfection of IκBα-mut or treatment with a NF-κB inhibitor (Figure 4F and 4G). Thus, these results demonstrate that functional activation of the NF-κB signalling pathway is vital to the biological effects of miR-500 in gastric cancer progression.

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