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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

Deletion of miR-K3 from the KSHV genome attenuates KSHV induction of endothelial cell migration and invasion.(A). Transwell migration assay for HUVEC infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K3 deletion mutant virus (miR-K3_Mut). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Matrigel invasion assay for HUVEC treated as in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (A) were determined by RT-qPCR. (D). Western blotting analysis of expression of GRK2, CXCR2, phosphorylated AKT, and KSHV RTA in HUVEC treated as in (A) with the indicated antibodies. (E). Schematic representation of the mechanism by which miR-K3 facilitates endothelial cell migration and invasion.
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ppat.1005171.g009: Deletion of miR-K3 from the KSHV genome attenuates KSHV induction of endothelial cell migration and invasion.(A). Transwell migration assay for HUVEC infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K3 deletion mutant virus (miR-K3_Mut). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Matrigel invasion assay for HUVEC treated as in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (A) were determined by RT-qPCR. (D). Western blotting analysis of expression of GRK2, CXCR2, phosphorylated AKT, and KSHV RTA in HUVEC treated as in (A) with the indicated antibodies. (E). Schematic representation of the mechanism by which miR-K3 facilitates endothelial cell migration and invasion.

Mentions: Since inhibition of miR-K3 function with a specific sponge in KSHV infected HUVEC decreased cell migration and invasion (Fig 5D), we wished to further confirm these results by genetic knock out of miR-K3 from the KSHV genome. We next infected HUVEC with wild type recombinant KSHV BAC16 and a mutant with miR-K3 deleted (BAC16 miR-K3_mut virus). We ensured that the expression of miR-K3 was abrogated in miR-K3_mut virus and the deletion of miR-K3 from the KSHV genome didn’t affect the expression of other miRNAs by qPCR analysis (S8 Fig). As shown in Fig 9A and 9B, the levels of cell migration and invasion in HUVEC infected by the mutant virus were significantly lower than those infected by the wild type virus. Consistent with these observations, cells infected by the mutant virus had decreased levels of MMP1, 9, and 10 and IL-6 and IL-8 mRNAs transcripts than those infected by the wild type virus (Fig 9C). Importantly, mutant cells had a higher level of GRK2, and lower levels of CXCR2 and activated AKT than those of wild type cells (Fig 9D), while RTA was dramatically elevated in mutant cells (Fig 9D), which was consistent with previous reports [20,22]. To further confirm the role of AKT in miR-K3 regulation of mRNAs transcripts of MMPs and inflammatory cytokines, we transfected AKT cDNA into HUVEC infected by the mutant virus. As expected, transfection of AKT cDNA in miR-K3_mut-infected HUVEC increased the levels of MMP1, 9, and 10 and IL-6 and IL-8 transcripts compared to cells transfected with control vector (S9 Fig). These data collectively suggest that during promotion of cell migration and invasion, miR-K3 up-regulates the levels of transcripts of MMPs and inflammatory cytokines through the activation of AKT signaling.


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)

Deletion of miR-K3 from the KSHV genome attenuates KSHV induction of endothelial cell migration and invasion.(A). Transwell migration assay for HUVEC infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K3 deletion mutant virus (miR-K3_Mut). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Matrigel invasion assay for HUVEC treated as in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (A) were determined by RT-qPCR. (D). Western blotting analysis of expression of GRK2, CXCR2, phosphorylated AKT, and KSHV RTA in HUVEC treated as in (A) with the indicated antibodies. (E). Schematic representation of the mechanism by which miR-K3 facilitates endothelial cell migration and invasion.
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Related In: Results  -  Collection

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

ppat.1005171.g009: Deletion of miR-K3 from the KSHV genome attenuates KSHV induction of endothelial cell migration and invasion.(A). Transwell migration assay for HUVEC infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K3 deletion mutant virus (miR-K3_Mut). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (B). Matrigel invasion assay for HUVEC treated as in (A). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test. (C). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (A) were determined by RT-qPCR. (D). Western blotting analysis of expression of GRK2, CXCR2, phosphorylated AKT, and KSHV RTA in HUVEC treated as in (A) with the indicated antibodies. (E). Schematic representation of the mechanism by which miR-K3 facilitates endothelial cell migration and invasion.
Mentions: Since inhibition of miR-K3 function with a specific sponge in KSHV infected HUVEC decreased cell migration and invasion (Fig 5D), we wished to further confirm these results by genetic knock out of miR-K3 from the KSHV genome. We next infected HUVEC with wild type recombinant KSHV BAC16 and a mutant with miR-K3 deleted (BAC16 miR-K3_mut virus). We ensured that the expression of miR-K3 was abrogated in miR-K3_mut virus and the deletion of miR-K3 from the KSHV genome didn’t affect the expression of other miRNAs by qPCR analysis (S8 Fig). As shown in Fig 9A and 9B, the levels of cell migration and invasion in HUVEC infected by the mutant virus were significantly lower than those infected by the wild type virus. Consistent with these observations, cells infected by the mutant virus had decreased levels of MMP1, 9, and 10 and IL-6 and IL-8 mRNAs transcripts than those infected by the wild type virus (Fig 9C). Importantly, mutant cells had a higher level of GRK2, and lower levels of CXCR2 and activated AKT than those of wild type cells (Fig 9D), while RTA was dramatically elevated in mutant cells (Fig 9D), which was consistent with previous reports [20,22]. To further confirm the role of AKT in miR-K3 regulation of mRNAs transcripts of MMPs and inflammatory cytokines, we transfected AKT cDNA into HUVEC infected by the mutant virus. As expected, transfection of AKT cDNA in miR-K3_mut-infected HUVEC increased the levels of MMP1, 9, and 10 and IL-6 and IL-8 transcripts compared to cells transfected with control vector (S9 Fig). These data collectively suggest that during promotion of cell migration and invasion, miR-K3 up-regulates the levels of transcripts of MMPs and inflammatory cytokines through the activation of AKT signaling.

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