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A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling.

Hu M, Wang C, Li W, Lu W, Bai Z, Qin D, Yan Q, Zhu J, Krueger BJ, Renne R, Gao SJ, Lu C - PLoS Pathog. (2015)

Bottom Line: Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion.Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT.Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, P. R. China; Key Laboratory Of Pathogen Biology Of Jiangsu Province, Nanjing Medical University, Nanjing, P. R. China; Department of Microbiology, Nanjing Medical University, Nanjing, P. R. China.

ABSTRACT
Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

No MeSH data available.


Related in: MedlinePlus

Activation of AKT is necessary to miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for HUVEC which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further with lentivirus-AKT-DN (AKT-DN) or its control (pCDH). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Western blotting analysis of phosphorylated AKT in HUVEC treated as in (A). The antibody against HA-tag was used to detect the transduction of AKT-DN. (C). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting AKT (shAKT) or its control (pCDH). * P < 0.05 and *** P < 0.001 for Student’s t-test. (D). Western blotting analysis of phosphorylated AKT levels in HUVEC treated as in (C). (E). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC, which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO), were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (F). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in KSHV-infected HUVEC treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO) were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
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ppat.1005171.g008: Activation of AKT is necessary to miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for HUVEC which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further with lentivirus-AKT-DN (AKT-DN) or its control (pCDH). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Western blotting analysis of phosphorylated AKT in HUVEC treated as in (A). The antibody against HA-tag was used to detect the transduction of AKT-DN. (C). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting AKT (shAKT) or its control (pCDH). * P < 0.05 and *** P < 0.001 for Student’s t-test. (D). Western blotting analysis of phosphorylated AKT levels in HUVEC treated as in (C). (E). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC, which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO), were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (F). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in KSHV-infected HUVEC treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO) were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.

Mentions: Our results showed that both GRK2/CXCR2/AKT signaling and GRK2/AKT interaction could lead to higher level of AKT activation. To determine if miR-K3-induced AKT activation mediated the enhanced cell migration and invasion, we transduced miR-K3-expressing HUVEC with a dominant negative mutant of AKT (AKT-DN). Expression of AKT-DN inhibited cell migration and invasion in miR-K3-expressing HUVEC (Fig 8A). Western blotting confirmed the decreased level of phosphorylated AKT (Fig 8B). Similar inhibition of cell migration and invasion was also observed in KSHV-infected HUVEC following knock down of AKT (Fig 8C and 8D, S6 Fig). To further confirm these observations, MK-2206, an AKT inhibitor, was used to treat miR-K3-transduced or KSHV-infected HUVEC. Consistently, treatment of MK-2206 not only inhibited cell migration and invasion (S7A and S7C Fig), but also decreased the level of phosphorylated AKT (S7B and S7D Fig). Importantly, inhibition of AKT with MK-2206 also blocked the induction of MMP1, 9 and 10, and IL-6, IL-8 by miR-K3 transduction or KSHV infection in HUVEC (Fig 8E and 8F).


A KSHV microRNA Directly Targets G Protein-Coupled Receptor Kinase 2 to Promote the Migration and Invasion of Endothelial Cells by Inducing CXCR2 and Activating AKT Signaling.

Hu M, Wang C, Li W, Lu W, Bai Z, Qin D, Yan Q, Zhu J, Krueger BJ, Renne R, Gao SJ, Lu C - PLoS Pathog. (2015)

Activation of AKT is necessary to miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for HUVEC which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further with lentivirus-AKT-DN (AKT-DN) or its control (pCDH). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Western blotting analysis of phosphorylated AKT in HUVEC treated as in (A). The antibody against HA-tag was used to detect the transduction of AKT-DN. (C). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting AKT (shAKT) or its control (pCDH). * P < 0.05 and *** P < 0.001 for Student’s t-test. (D). Western blotting analysis of phosphorylated AKT levels in HUVEC treated as in (C). (E). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC, which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO), were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (F). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in KSHV-infected HUVEC treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO) were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
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ppat.1005171.g008: Activation of AKT is necessary to miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for HUVEC which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further with lentivirus-AKT-DN (AKT-DN) or its control (pCDH). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Western blotting analysis of phosphorylated AKT in HUVEC treated as in (A). The antibody against HA-tag was used to detect the transduction of AKT-DN. (C). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for KSHV-infected HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting AKT (shAKT) or its control (pCDH). * P < 0.05 and *** P < 0.001 for Student’s t-test. (D). Western blotting analysis of phosphorylated AKT levels in HUVEC treated as in (C). (E). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC, which were transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3) expression and further treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO), were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (F). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in KSHV-infected HUVEC treated with the AKT inhibitor, MK-2206 (MK-2206) or its control (DMSO) were determined by qPCR. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
Mentions: Our results showed that both GRK2/CXCR2/AKT signaling and GRK2/AKT interaction could lead to higher level of AKT activation. To determine if miR-K3-induced AKT activation mediated the enhanced cell migration and invasion, we transduced miR-K3-expressing HUVEC with a dominant negative mutant of AKT (AKT-DN). Expression of AKT-DN inhibited cell migration and invasion in miR-K3-expressing HUVEC (Fig 8A). Western blotting confirmed the decreased level of phosphorylated AKT (Fig 8B). Similar inhibition of cell migration and invasion was also observed in KSHV-infected HUVEC following knock down of AKT (Fig 8C and 8D, S6 Fig). To further confirm these observations, MK-2206, an AKT inhibitor, was used to treat miR-K3-transduced or KSHV-infected HUVEC. Consistently, treatment of MK-2206 not only inhibited cell migration and invasion (S7A and S7C Fig), but also decreased the level of phosphorylated AKT (S7B and S7D Fig). Importantly, inhibition of AKT with MK-2206 also blocked the induction of MMP1, 9 and 10, and IL-6, IL-8 by miR-K3 transduction or KSHV infection in HUVEC (Fig 8E and 8F).

Bottom Line: Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion.Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT.Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, P. R. China; Key Laboratory Of Pathogen Biology Of Jiangsu Province, Nanjing Medical University, Nanjing, P. R. China; Department of Microbiology, Nanjing Medical University, Nanjing, P. R. China.

ABSTRACT
Kaposi's sarcoma (KS) is a highly disseminated angiogenic tumor of endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV encodes more than two dozens of miRNAs but their roles in KSHV-induced tumor dissemination and metastasis remain unknown. Here, we found that ectopic expression of miR-K12-3 (miR-K3) promoted endothelial cell migration and invasion. Bioinformatics and luciferase reporter analyses showed that miR-K3 directly targeted G protein-coupled receptor (GPCR) kinase 2 (GRK2, official gene symbol ADRBK1). Importantly, overexpression of GRK2 reversed miR-K3 induction of cell migration and invasion. Furthermore, the chemokine receptor CXCR2, which was negatively regulated by GRK2, was upregulated in miR-K3-transduced endothelial cells. Knock down of CXCR2 abolished miR-K3-induced cell migration and invasion. Moreover, miR-K3 downregulation of GRK2 relieved its direct inhibitory effect on AKT. Both CXCR2 induction and the release of AKT from GRK2 were required for miR-K3 maximum activation of AKT and induction of cell migration and invasion. Finally, deletion of miR-K3 from the KSHV genome abrogated its effect on the GRK2/CXCR2/AKT pathway and KSHV-induced migration and invasion. Our data provide the first-line evidence that, by repressing GRK2, miR-K3 facilitates cell migration and invasion via activation of CXCR2/AKT signaling, which likely contribute to the dissemination of KSHV-induced tumors.

No MeSH data available.


Related in: MedlinePlus