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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 miR-K3 promotes endothelial cell migration and invasion.(A). KSHV miR-K3 expression in HUVEC infected with KSHV BAC16 virus induced from iSLK-BAC16 cells or transduced by the different MOI of lentiviral miR-K3 were determined by qPCR. The miR-K3 level in KSHV group was set as ‘‘1” for comparison. (B). HUVEC were transduced with 2 MOI of lentivirus empty vector (mpCDH; left) and lentivirus-miR-K3 (miR-K3; right), and representative images were taken under light microscope (Phase; top) and fluorescent microscope (RFP; bottom) (Original magnification, ×100). (C). Cells treated as in (B) were analyzed for RFP expression by flow cytometry to determine transduction efficiency. y axis units are numbers of cells. (D). Luciferase activity was detected in 2 MOI of lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) transduced HUVEC transfected by the pGL3-Control (Control) or the pGL3-miR-K3 sensor reporter (miR-K3-Sensor). *** P < 0.001 for Student’s t-test. n.s., not significant. (E). Transwell migration (left panel) and Matrigel invasion (right panel) assays for HUVEC transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3). The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (F). The quantification results of Transwell migration assay in (E). (G). The quantification results of Matrigel invasion assay in (E). (H). The quantification results of wound healing assay in S1 Fig. (I). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (B) were determined by qPCR.
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ppat.1005171.g001: Ectopic expression of miR-K3 promotes endothelial cell migration and invasion.(A). KSHV miR-K3 expression in HUVEC infected with KSHV BAC16 virus induced from iSLK-BAC16 cells or transduced by the different MOI of lentiviral miR-K3 were determined by qPCR. The miR-K3 level in KSHV group was set as ‘‘1” for comparison. (B). HUVEC were transduced with 2 MOI of lentivirus empty vector (mpCDH; left) and lentivirus-miR-K3 (miR-K3; right), and representative images were taken under light microscope (Phase; top) and fluorescent microscope (RFP; bottom) (Original magnification, ×100). (C). Cells treated as in (B) were analyzed for RFP expression by flow cytometry to determine transduction efficiency. y axis units are numbers of cells. (D). Luciferase activity was detected in 2 MOI of lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) transduced HUVEC transfected by the pGL3-Control (Control) or the pGL3-miR-K3 sensor reporter (miR-K3-Sensor). *** P < 0.001 for Student’s t-test. n.s., not significant. (E). Transwell migration (left panel) and Matrigel invasion (right panel) assays for HUVEC transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3). The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (F). The quantification results of Transwell migration assay in (E). (G). The quantification results of Matrigel invasion assay in (E). (H). The quantification results of wound healing assay in S1 Fig. (I). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (B) were determined by qPCR.

Mentions: It has been reported that KSHV infection facilitates the migration and invasion of endothelial cells, and several KSHV-encoded genes and cellular miRNAs have been shown to participate in this process [6,43,44]. To determine whether KSHV-encoded miRNAs were also involved in regulating the migration and invasion of endothelial cells, HUVEC were transduced with the different MOI of lentivirus expressing miR-K3. At 2 MOI, miR-K3-transduced HUVEC exhibited a miR-K3 expression level similar to that of KSHV (BAC16)-infected HUVEC (Fig 1A). Thus, we chose 2 MOI for the following transduction experiments. Under this condition, over 95% cells were RFP-positive at day 3 or 4 post-transduction, indicating the successful lentivirus transduction (Fig 1B and 1C). Expectedly, miR-K3 markedly inhibited the reporter activity of pGL3-miR-K3 sensor reporter, indicating that the miR-K3 expression construct in HUVEC was functional (Fig 1D). In Transwell migration and Matrigel invasion assays, HUVEC transduced with miR-K3 exhibited increased levels of migration and invasion when compared with those transduced with the vector control (Fig 1E, 1F and 1G). Consistently, wound healing assays showed that HUVEC transduced with miR-K3 had increased level of motility compared to those transduced with the vector control (Fig 1H and S1 Fig). Besides migration and invasion, we also have screened many other phenotypes including cell proliferation, cell cycle, and plate colony formation, however, we found that miR-K3 did not affect these phenotypes’ changes. Furthermore, qPCR was performed to detect several cytokines that are related to cell migration and invasion. We found that miR-K3 upregulated the levels of transcripts of matrix metalloproteinases (MMPs) 1, 9 and 10, and inflammatory cytokines IL-6 and IL-8 by 2–9 folds compared to the control vector (Fig 1I).


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 miR-K3 promotes endothelial cell migration and invasion.(A). KSHV miR-K3 expression in HUVEC infected with KSHV BAC16 virus induced from iSLK-BAC16 cells or transduced by the different MOI of lentiviral miR-K3 were determined by qPCR. The miR-K3 level in KSHV group was set as ‘‘1” for comparison. (B). HUVEC were transduced with 2 MOI of lentivirus empty vector (mpCDH; left) and lentivirus-miR-K3 (miR-K3; right), and representative images were taken under light microscope (Phase; top) and fluorescent microscope (RFP; bottom) (Original magnification, ×100). (C). Cells treated as in (B) were analyzed for RFP expression by flow cytometry to determine transduction efficiency. y axis units are numbers of cells. (D). Luciferase activity was detected in 2 MOI of lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) transduced HUVEC transfected by the pGL3-Control (Control) or the pGL3-miR-K3 sensor reporter (miR-K3-Sensor). *** P < 0.001 for Student’s t-test. n.s., not significant. (E). Transwell migration (left panel) and Matrigel invasion (right panel) assays for HUVEC transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3). The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (F). The quantification results of Transwell migration assay in (E). (G). The quantification results of Matrigel invasion assay in (E). (H). The quantification results of wound healing assay in S1 Fig. (I). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (B) were determined by qPCR.
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ppat.1005171.g001: Ectopic expression of miR-K3 promotes endothelial cell migration and invasion.(A). KSHV miR-K3 expression in HUVEC infected with KSHV BAC16 virus induced from iSLK-BAC16 cells or transduced by the different MOI of lentiviral miR-K3 were determined by qPCR. The miR-K3 level in KSHV group was set as ‘‘1” for comparison. (B). HUVEC were transduced with 2 MOI of lentivirus empty vector (mpCDH; left) and lentivirus-miR-K3 (miR-K3; right), and representative images were taken under light microscope (Phase; top) and fluorescent microscope (RFP; bottom) (Original magnification, ×100). (C). Cells treated as in (B) were analyzed for RFP expression by flow cytometry to determine transduction efficiency. y axis units are numbers of cells. (D). Luciferase activity was detected in 2 MOI of lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3) transduced HUVEC transfected by the pGL3-Control (Control) or the pGL3-miR-K3 sensor reporter (miR-K3-Sensor). *** P < 0.001 for Student’s t-test. n.s., not significant. (E). Transwell migration (left panel) and Matrigel invasion (right panel) assays for HUVEC transduced with lentivirus empty vector (mpCDH) or lentivirus-miR-K3 (miR-K3). The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). (F). The quantification results of Transwell migration assay in (E). (G). The quantification results of Matrigel invasion assay in (E). (H). The quantification results of wound healing assay in S1 Fig. (I). The mRNA expression of MMP1, 9, 10 and IL-6, 8 in HUVEC treated as in (B) were determined by qPCR.
Mentions: It has been reported that KSHV infection facilitates the migration and invasion of endothelial cells, and several KSHV-encoded genes and cellular miRNAs have been shown to participate in this process [6,43,44]. To determine whether KSHV-encoded miRNAs were also involved in regulating the migration and invasion of endothelial cells, HUVEC were transduced with the different MOI of lentivirus expressing miR-K3. At 2 MOI, miR-K3-transduced HUVEC exhibited a miR-K3 expression level similar to that of KSHV (BAC16)-infected HUVEC (Fig 1A). Thus, we chose 2 MOI for the following transduction experiments. Under this condition, over 95% cells were RFP-positive at day 3 or 4 post-transduction, indicating the successful lentivirus transduction (Fig 1B and 1C). Expectedly, miR-K3 markedly inhibited the reporter activity of pGL3-miR-K3 sensor reporter, indicating that the miR-K3 expression construct in HUVEC was functional (Fig 1D). In Transwell migration and Matrigel invasion assays, HUVEC transduced with miR-K3 exhibited increased levels of migration and invasion when compared with those transduced with the vector control (Fig 1E, 1F and 1G). Consistently, wound healing assays showed that HUVEC transduced with miR-K3 had increased level of motility compared to those transduced with the vector control (Fig 1H and S1 Fig). Besides migration and invasion, we also have screened many other phenotypes including cell proliferation, cell cycle, and plate colony formation, however, we found that miR-K3 did not affect these phenotypes’ changes. Furthermore, qPCR was performed to detect several cytokines that are related to cell migration and invasion. We found that miR-K3 upregulated the levels of transcripts of matrix metalloproteinases (MMPs) 1, 9 and 10, and inflammatory cytokines IL-6 and IL-8 by 2–9 folds compared to the control vector (Fig 1I).

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