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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

KSHV infection promotes endothelial cell migration and invasion through miR-K3 by targeting GRK2.(A). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) with the indicated antibodies. (B). MiR-K3 sponge was functional. HEK 293T cells were co-transfected with miR-K3 sensor reporter and miR-K3 mimic, and subsequently transduced with increasing MOI of lentivirus-mediated miR-K3 sponge (miR-K3 sponge) or its control (pCDH). The cells were collected at 48 h post-transduction for luciferase assays. *** P < 0.001 for Student’s t-test. n.s., not significant. (C). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with miR-K3 sponge (miR-K3 sponge) or its control (pCDH) with the indicated antibodies. (D). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (C) at 6 and 12 h post seeding. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH) with the indicated antibodies. (F). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (E) at 6 and 12 h post seeding.
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ppat.1005171.g005: KSHV infection promotes endothelial cell migration and invasion through miR-K3 by targeting GRK2.(A). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) with the indicated antibodies. (B). MiR-K3 sponge was functional. HEK 293T cells were co-transfected with miR-K3 sensor reporter and miR-K3 mimic, and subsequently transduced with increasing MOI of lentivirus-mediated miR-K3 sponge (miR-K3 sponge) or its control (pCDH). The cells were collected at 48 h post-transduction for luciferase assays. *** P < 0.001 for Student’s t-test. n.s., not significant. (C). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with miR-K3 sponge (miR-K3 sponge) or its control (pCDH) with the indicated antibodies. (D). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (C) at 6 and 12 h post seeding. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH) with the indicated antibodies. (F). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (E) at 6 and 12 h post seeding.

Mentions: In addition, overexpression of miR-K3 in KSHV-infected HUVEC reduced the expression of GRK2 (Fig 5A) and further enhanced cell migration and invasion (S2 Fig). To further confirm the role of miR-K3 targeting in KSHV-induced cell migration and invasion, we generated a miR-K3 sponge. In the luciferase reporter assay, transduction of the sponge abolished the inhibitory effect of miR-K3 mimic on its sensor reporter in a dose-dependent manner in HEK 293T cells, indicating that the miR-K3 sponge was functional (Fig 5B). Transduction of the miR-K3 sponge into KSHV-infected HUVEC increased the expression level of GRK2 (Fig 5C) and inhibited cell migration and invasion (Fig 5D). As expected, knock-down of GRK2 by lentivirus-mediated a mixture of short hairpair RNAs in normal HUVEC alone was sufficient to increase cell migration and invasion (Fig 5E and 5F, S3 Fig). Collectively, these results indicated that KSHV-induced cell migration and invasion was mediated by miR-K3 targeting of GRK2.


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)

KSHV infection promotes endothelial cell migration and invasion through miR-K3 by targeting GRK2.(A). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) with the indicated antibodies. (B). MiR-K3 sponge was functional. HEK 293T cells were co-transfected with miR-K3 sensor reporter and miR-K3 mimic, and subsequently transduced with increasing MOI of lentivirus-mediated miR-K3 sponge (miR-K3 sponge) or its control (pCDH). The cells were collected at 48 h post-transduction for luciferase assays. *** P < 0.001 for Student’s t-test. n.s., not significant. (C). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with miR-K3 sponge (miR-K3 sponge) or its control (pCDH) with the indicated antibodies. (D). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (C) at 6 and 12 h post seeding. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH) with the indicated antibodies. (F). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (E) at 6 and 12 h post seeding.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581863&req=5

ppat.1005171.g005: KSHV infection promotes endothelial cell migration and invasion through miR-K3 by targeting GRK2.(A). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) with the indicated antibodies. (B). MiR-K3 sponge was functional. HEK 293T cells were co-transfected with miR-K3 sensor reporter and miR-K3 mimic, and subsequently transduced with increasing MOI of lentivirus-mediated miR-K3 sponge (miR-K3 sponge) or its control (pCDH). The cells were collected at 48 h post-transduction for luciferase assays. *** P < 0.001 for Student’s t-test. n.s., not significant. (C). Western blotting was performed in KSHV-infected HUVEC (KSHV + HUVEC) transduced with miR-K3 sponge (miR-K3 sponge) or its control (pCDH) with the indicated antibodies. (D). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (C) at 6 and 12 h post seeding. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting GRK2 (shGRK2) or the control (mpCDH) with the indicated antibodies. (F). Transwell migration (Left panel) and Matrigel invasion (Right panel) assays for cells treated as in (E) at 6 and 12 h post seeding.
Mentions: In addition, overexpression of miR-K3 in KSHV-infected HUVEC reduced the expression of GRK2 (Fig 5A) and further enhanced cell migration and invasion (S2 Fig). To further confirm the role of miR-K3 targeting in KSHV-induced cell migration and invasion, we generated a miR-K3 sponge. In the luciferase reporter assay, transduction of the sponge abolished the inhibitory effect of miR-K3 mimic on its sensor reporter in a dose-dependent manner in HEK 293T cells, indicating that the miR-K3 sponge was functional (Fig 5B). Transduction of the miR-K3 sponge into KSHV-infected HUVEC increased the expression level of GRK2 (Fig 5C) and inhibited cell migration and invasion (Fig 5D). As expected, knock-down of GRK2 by lentivirus-mediated a mixture of short hairpair RNAs in normal HUVEC alone was sufficient to increase cell migration and invasion (Fig 5E and 5F, S3 Fig). Collectively, these results indicated that KSHV-induced cell migration and invasion was mediated by miR-K3 targeting of GRK2.

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