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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 CXCR2, which was negatively regulated by GRK2, contributes to miR-K3-induced endothelial cell migration and invasion.(A). The mRNA level of CXCR2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) or HUVEC treated with PBS (PBS) and infected with KSHV (KSHV) were examined by qPCR. (B). The expressions of CXCR2 protein in HUVEC treated as in (A). (C). Confocal microscopy of HUVEC treated as in (A), then stained for red fluorescence protein (refers to CXCR2; red). 4’, 6’-diamidino-2-phenylindole (DAPI) (blue) stains nuclei. (D). Representative flow cytometry histograms for CXCR2 expression on the surface of HUVEC treated as in (A). Cells were stained with anti-CXCR2 MAb and fluorescein isothiocyanate-labeled IgG was used as an isotype control antibody. (E). Immunohistochemical (IHC) staining of CXCR2 in normal skin (Normal Skin; top) and KS lesions (Skin KS; bottom). (F). Quantification of the results in (E). *** P < 0.001 for Student’s t-test. (G). Western blotting was performed using HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and co-transduced with GRK2 (GRK2) or its control (pHAGE), respectively, with the indicated antibodies. The antibody against His-tag was used to detect the exogenous GRK2. (H). Western blotting was performed in HUVEC transduced with miR-K3 (miR-K3) or empty vector (mpCDH), which were further transduced with a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH), respectively. (I). Transwell migration (left) and Matrigel invasion (right) assays were performed in HUVEC treated as in (E). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
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ppat.1005171.g006: Activation of CXCR2, which was negatively regulated by GRK2, contributes to miR-K3-induced endothelial cell migration and invasion.(A). The mRNA level of CXCR2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) or HUVEC treated with PBS (PBS) and infected with KSHV (KSHV) were examined by qPCR. (B). The expressions of CXCR2 protein in HUVEC treated as in (A). (C). Confocal microscopy of HUVEC treated as in (A), then stained for red fluorescence protein (refers to CXCR2; red). 4’, 6’-diamidino-2-phenylindole (DAPI) (blue) stains nuclei. (D). Representative flow cytometry histograms for CXCR2 expression on the surface of HUVEC treated as in (A). Cells were stained with anti-CXCR2 MAb and fluorescein isothiocyanate-labeled IgG was used as an isotype control antibody. (E). Immunohistochemical (IHC) staining of CXCR2 in normal skin (Normal Skin; top) and KS lesions (Skin KS; bottom). (F). Quantification of the results in (E). *** P < 0.001 for Student’s t-test. (G). Western blotting was performed using HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and co-transduced with GRK2 (GRK2) or its control (pHAGE), respectively, with the indicated antibodies. The antibody against His-tag was used to detect the exogenous GRK2. (H). Western blotting was performed in HUVEC transduced with miR-K3 (miR-K3) or empty vector (mpCDH), which were further transduced with a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH), respectively. (I). Transwell migration (left) and Matrigel invasion (right) assays were performed in HUVEC treated as in (E). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.

Mentions: It has been reported that GRK2 was negatively correlated with the expression of the chemokine receptor CXCR2 in neutrophils, and increased expression of GRK2 down-regulated CXCR2, leading to impairment of neutrophil migration into an infectious focus in vivo [48,49]. Given these findings, we reasoned that CXCR2 may also be involved in GRK2 mediation of miR-K3-induced cell migration and invasion. Indeed, both mRNA and protein levels of CXCR2 were elevated in miR-K3-expressing and KSHV-infected HUVEC compared to the respective control cells (Fig 6A and 6B). In agreement with its membrane localization, we observed a higher level of CXCR2 on the membrane of KSHV-infected HUVEC than mock infected control cells (Fig 6C). Similar results were also observed on the surface of HUVEC transected with a miR-K3 mimic (S4 Fig). As expected, flow cytometry analysis showed a higher level of CXCR2 surface expression on miR-K3-transduced HUVEC than on the cells transduced with the control vector (Fig 6D). Importantly, we observed a higher level of CXCR2 expression in KS lesions than the normal skin tissues by immunohistochemistry staining (Fig 6E and 6F). To determine whether the increased expression of CXCR2 in the miR-K3-expressing cells was due to the downregulation of GRK2, we overexpressed GRK2 in the miR-K3-expressing HUVEC. As shown in Fig 6G, overexpression of GRK2 dramatically down-regulated CXCR2 expression in both normal and miR-K3-expressing HUVEC. To determine the role of CXCR2 in miR-K3-mediated cell migration and invasion, we performed knock-down of CXCR2 with lentivirus-mediated a mixture of short hairpair RNAs (shCXCR2) (Fig 6H and S5 Fig). Knock-down of CXCR2 significantly inhibited miR-K3-induced cell migration and invasion (Fig 6I). These data indicated that CXCR2 mediated miR-K3 induced cell migration and invasion as a result of 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)

Activation of CXCR2, which was negatively regulated by GRK2, contributes to miR-K3-induced endothelial cell migration and invasion.(A). The mRNA level of CXCR2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) or HUVEC treated with PBS (PBS) and infected with KSHV (KSHV) were examined by qPCR. (B). The expressions of CXCR2 protein in HUVEC treated as in (A). (C). Confocal microscopy of HUVEC treated as in (A), then stained for red fluorescence protein (refers to CXCR2; red). 4’, 6’-diamidino-2-phenylindole (DAPI) (blue) stains nuclei. (D). Representative flow cytometry histograms for CXCR2 expression on the surface of HUVEC treated as in (A). Cells were stained with anti-CXCR2 MAb and fluorescein isothiocyanate-labeled IgG was used as an isotype control antibody. (E). Immunohistochemical (IHC) staining of CXCR2 in normal skin (Normal Skin; top) and KS lesions (Skin KS; bottom). (F). Quantification of the results in (E). *** P < 0.001 for Student’s t-test. (G). Western blotting was performed using HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and co-transduced with GRK2 (GRK2) or its control (pHAGE), respectively, with the indicated antibodies. The antibody against His-tag was used to detect the exogenous GRK2. (H). Western blotting was performed in HUVEC transduced with miR-K3 (miR-K3) or empty vector (mpCDH), which were further transduced with a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH), respectively. (I). Transwell migration (left) and Matrigel invasion (right) assays were performed in HUVEC treated as in (E). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
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ppat.1005171.g006: Activation of CXCR2, which was negatively regulated by GRK2, contributes to miR-K3-induced endothelial cell migration and invasion.(A). The mRNA level of CXCR2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) or HUVEC treated with PBS (PBS) and infected with KSHV (KSHV) were examined by qPCR. (B). The expressions of CXCR2 protein in HUVEC treated as in (A). (C). Confocal microscopy of HUVEC treated as in (A), then stained for red fluorescence protein (refers to CXCR2; red). 4’, 6’-diamidino-2-phenylindole (DAPI) (blue) stains nuclei. (D). Representative flow cytometry histograms for CXCR2 expression on the surface of HUVEC treated as in (A). Cells were stained with anti-CXCR2 MAb and fluorescein isothiocyanate-labeled IgG was used as an isotype control antibody. (E). Immunohistochemical (IHC) staining of CXCR2 in normal skin (Normal Skin; top) and KS lesions (Skin KS; bottom). (F). Quantification of the results in (E). *** P < 0.001 for Student’s t-test. (G). Western blotting was performed using HUVEC transduced with lentivirus-mediated miR-K3 (miR-K3) or empty vector (mpCDH), and co-transduced with GRK2 (GRK2) or its control (pHAGE), respectively, with the indicated antibodies. The antibody against His-tag was used to detect the exogenous GRK2. (H). Western blotting was performed in HUVEC transduced with miR-K3 (miR-K3) or empty vector (mpCDH), which were further transduced with a mixture of short hairpin RNAs targeting CXCR2 (shCXCR2) or its control (mpCDH), respectively. (I). Transwell migration (left) and Matrigel invasion (right) assays were performed in HUVEC treated as in (E). * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.
Mentions: It has been reported that GRK2 was negatively correlated with the expression of the chemokine receptor CXCR2 in neutrophils, and increased expression of GRK2 down-regulated CXCR2, leading to impairment of neutrophil migration into an infectious focus in vivo [48,49]. Given these findings, we reasoned that CXCR2 may also be involved in GRK2 mediation of miR-K3-induced cell migration and invasion. Indeed, both mRNA and protein levels of CXCR2 were elevated in miR-K3-expressing and KSHV-infected HUVEC compared to the respective control cells (Fig 6A and 6B). In agreement with its membrane localization, we observed a higher level of CXCR2 on the membrane of KSHV-infected HUVEC than mock infected control cells (Fig 6C). Similar results were also observed on the surface of HUVEC transected with a miR-K3 mimic (S4 Fig). As expected, flow cytometry analysis showed a higher level of CXCR2 surface expression on miR-K3-transduced HUVEC than on the cells transduced with the control vector (Fig 6D). Importantly, we observed a higher level of CXCR2 expression in KS lesions than the normal skin tissues by immunohistochemistry staining (Fig 6E and 6F). To determine whether the increased expression of CXCR2 in the miR-K3-expressing cells was due to the downregulation of GRK2, we overexpressed GRK2 in the miR-K3-expressing HUVEC. As shown in Fig 6G, overexpression of GRK2 dramatically down-regulated CXCR2 expression in both normal and miR-K3-expressing HUVEC. To determine the role of CXCR2 in miR-K3-mediated cell migration and invasion, we performed knock-down of CXCR2 with lentivirus-mediated a mixture of short hairpair RNAs (shCXCR2) (Fig 6H and S5 Fig). Knock-down of CXCR2 significantly inhibited miR-K3-induced cell migration and invasion (Fig 6I). These data indicated that CXCR2 mediated miR-K3 induced cell migration and invasion as a result of 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