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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

Ectopic expression of GRK2 inhibits miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (top) and Matrigel invasion (bottom) assays for HUVEC transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3), which were subsequently co-transduced with lentivirus-mediated empty vector (pHAGE) and lentivirus-GRK2 (GRK2), respectively. The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (B). The quantification results of Transwell migration assay in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The quantification results of Matrigel invasion assay in (A). ** P < 0.01 and *** P < 0.001 for Student’s t-test. (D). Western blotting was performed in HUVEC treated as in (A) with the indicated antibodies. The antibody against His-tag was used to detect the exogenous expression of GRK2. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE), or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE) with the indicated antibodies. The antibody against His-tag was used to examine the exogenous expression of GRK2. (F). Transwell migration assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (G). Matrigel invasion assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
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ppat.1005171.g004: Ectopic expression of GRK2 inhibits miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (top) and Matrigel invasion (bottom) assays for HUVEC transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3), which were subsequently co-transduced with lentivirus-mediated empty vector (pHAGE) and lentivirus-GRK2 (GRK2), respectively. The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (B). The quantification results of Transwell migration assay in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The quantification results of Matrigel invasion assay in (A). ** P < 0.01 and *** P < 0.001 for Student’s t-test. (D). Western blotting was performed in HUVEC treated as in (A) with the indicated antibodies. The antibody against His-tag was used to detect the exogenous expression of GRK2. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE), or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE) with the indicated antibodies. The antibody against His-tag was used to examine the exogenous expression of GRK2. (F). Transwell migration assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (G). Matrigel invasion assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.

Mentions: Decrease of GRK2 has been shown to augment the migratory response of polymorphonuclear leukocytes (PMNs) [47]. To characterize the role of GRK2 in miR-K3-induced migration and invasion of HUVEC, miR-K3-expressing HUVEC were transduced with lentivirus-GRK2. Overexpression of GRK2 significantly abolished miR-K3-induced cell migration and invasion at 6 h and 12 h post-seeding (Fig 4A, 4B and 4C), Western-blotting confirmed the suppression of endogenous GRK2 by miR-K3 (Lane 3 vs lane 1 in Fig 4D). Transduction with lentivirus-GRK2 increased the expression level of GRK2 but was reduced by miR-K3 (Lane 2 vs lane 4 in Fig 4D). As expected, KSHV infection also downregulated the expression of endogenous GRK2 (Lane 3 vs lane 1 in Fig 4E). Again, transduction with lentivirus-GRK2 increased the expression level of GRK2 but was reduced by KSHV infection (Lane 2 vs lane 4 in Fig 4E). Consistent with these results, while KSHV infection enhanced cell migration and invasion, overexpression of GRK2 inhibited cell migration and invasion of both HUVEC and KSHV-infected HUVEC (Fig 4F and 4G).


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)

Ectopic expression of GRK2 inhibits miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (top) and Matrigel invasion (bottom) assays for HUVEC transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3), which were subsequently co-transduced with lentivirus-mediated empty vector (pHAGE) and lentivirus-GRK2 (GRK2), respectively. The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (B). The quantification results of Transwell migration assay in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The quantification results of Matrigel invasion assay in (A). ** P < 0.01 and *** P < 0.001 for Student’s t-test. (D). Western blotting was performed in HUVEC treated as in (A) with the indicated antibodies. The antibody against His-tag was used to detect the exogenous expression of GRK2. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE), or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE) with the indicated antibodies. The antibody against His-tag was used to examine the exogenous expression of GRK2. (F). Transwell migration assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (G). Matrigel invasion assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
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Related In: Results  -  Collection

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Show All Figures
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ppat.1005171.g004: Ectopic expression of GRK2 inhibits miR-K3-induced endothelial cell migration and invasion.(A). Transwell migration (top) and Matrigel invasion (bottom) assays for HUVEC transduced with lentivirus-mediated empty vector (mpCDH) or miR-K3 (miR-K3), which were subsequently co-transduced with lentivirus-mediated empty vector (pHAGE) and lentivirus-GRK2 (GRK2), respectively. The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (B). The quantification results of Transwell migration assay in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The quantification results of Matrigel invasion assay in (A). ** P < 0.01 and *** P < 0.001 for Student’s t-test. (D). Western blotting was performed in HUVEC treated as in (A) with the indicated antibodies. The antibody against His-tag was used to detect the exogenous expression of GRK2. (E). Western blotting was performed in normal HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE), or KSHV-infected HUVEC transduced with lentivirus-GRK2 (GRK2) and its control (pHAGE) with the indicated antibodies. The antibody against His-tag was used to examine the exogenous expression of GRK2. (F). Transwell migration assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (G). Matrigel invasion assay for HUVEC treated as in (E) at 6 and 12 h post seeding. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
Mentions: Decrease of GRK2 has been shown to augment the migratory response of polymorphonuclear leukocytes (PMNs) [47]. To characterize the role of GRK2 in miR-K3-induced migration and invasion of HUVEC, miR-K3-expressing HUVEC were transduced with lentivirus-GRK2. Overexpression of GRK2 significantly abolished miR-K3-induced cell migration and invasion at 6 h and 12 h post-seeding (Fig 4A, 4B and 4C), Western-blotting confirmed the suppression of endogenous GRK2 by miR-K3 (Lane 3 vs lane 1 in Fig 4D). Transduction with lentivirus-GRK2 increased the expression level of GRK2 but was reduced by miR-K3 (Lane 2 vs lane 4 in Fig 4D). As expected, KSHV infection also downregulated the expression of endogenous GRK2 (Lane 3 vs lane 1 in Fig 4E). Again, transduction with lentivirus-GRK2 increased the expression level of GRK2 but was reduced by KSHV infection (Lane 2 vs lane 4 in Fig 4E). Consistent with these results, while KSHV infection enhanced cell migration and invasion, overexpression of GRK2 inhibited cell migration and invasion of both HUVEC and KSHV-infected HUVEC (Fig 4F and 4G).

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