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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

GRK2 is directly targeted by miR-K3.(A). Luciferase activity was detected in HEK 293T cells co-transfected by a mimic of miR-K3 (miR-K3) or a negative control nucleotide of miRNA (Neg. Ctrl.) together with pGL3-Control or pGL3-GRK2 3’UTR luciferase reporter (pGL3-GRK2 3’UTR). ** P < 0.01 for Student’s t-test. n.s., not significant. (B). Luciferase assay of 293T cells co-transfected by pGL3-GRK2 3′UTR together with increasing amounts (10, 20, and 50 nM) of miR-K3. (C). Schematic illustration of the putative seed sequences of miR-K3 complementary with GRK2 3’UTR and mutagenesis of binding sites in the 3’UTR of GRK2. (D). The luciferase activity was assayed in 293T cells co-transfected by GRK2 wild type 3’UTR (WT GRK2) or the mutant GRK2 3’UTR construct (mut GRK2) together with miR-K3 or mutant miR-K3 mimic (mut miR-K3). * P < 0.05 and ** P < 0.01 for Student’s t-test. (E). miR-K3 inhibited the expression of exogenous GRK2 protein by targeting its native 3’UTR. Western blotting was performed in HEK 293T cells co-transfected by pcDNA3.1–3×Flag-GRK2-3’UTR together with pEGFP and increasing amounts (10 and 20 nM) mimic of miR-K3. (F). miR-K3 inhibited the expression of endogenous GRK2 protein in HUVEC transfected with increasing amounts (10 and 20 nM) mimic of miR-K3. (G). Mutant miR-K3 failed to target endogenous GRK2. Western blotting was performed in HUVEC transfected by Neg. Ctrl., miR-K3 mimic (20 nM) or mut miR-K3 lacking the seed sequences. (H). Transfection of miR-K3 mimic (20 nM) has the same inhibition level on GRK2 expression as that of KSHV infection.
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ppat.1005171.g003: GRK2 is directly targeted by miR-K3.(A). Luciferase activity was detected in HEK 293T cells co-transfected by a mimic of miR-K3 (miR-K3) or a negative control nucleotide of miRNA (Neg. Ctrl.) together with pGL3-Control or pGL3-GRK2 3’UTR luciferase reporter (pGL3-GRK2 3’UTR). ** P < 0.01 for Student’s t-test. n.s., not significant. (B). Luciferase assay of 293T cells co-transfected by pGL3-GRK2 3′UTR together with increasing amounts (10, 20, and 50 nM) of miR-K3. (C). Schematic illustration of the putative seed sequences of miR-K3 complementary with GRK2 3’UTR and mutagenesis of binding sites in the 3’UTR of GRK2. (D). The luciferase activity was assayed in 293T cells co-transfected by GRK2 wild type 3’UTR (WT GRK2) or the mutant GRK2 3’UTR construct (mut GRK2) together with miR-K3 or mutant miR-K3 mimic (mut miR-K3). * P < 0.05 and ** P < 0.01 for Student’s t-test. (E). miR-K3 inhibited the expression of exogenous GRK2 protein by targeting its native 3’UTR. Western blotting was performed in HEK 293T cells co-transfected by pcDNA3.1–3×Flag-GRK2-3’UTR together with pEGFP and increasing amounts (10 and 20 nM) mimic of miR-K3. (F). miR-K3 inhibited the expression of endogenous GRK2 protein in HUVEC transfected with increasing amounts (10 and 20 nM) mimic of miR-K3. (G). Mutant miR-K3 failed to target endogenous GRK2. Western blotting was performed in HUVEC transfected by Neg. Ctrl., miR-K3 mimic (20 nM) or mut miR-K3 lacking the seed sequences. (H). Transfection of miR-K3 mimic (20 nM) has the same inhibition level on GRK2 expression as that of KSHV infection.

Mentions: To validate GRK2 as a direct target of miR-K3, the full length 3’UTR of GRK2 was amplified and inserted into the downstream of luciferase sequence in the pGL3-Control plasmid (named as pGL3-GRK2 3’UTR). The luciferase reporter assays indicated that miR-K3 significantly inhibited the GRK2 3’UTR reporter activity in a dose-dependent manner but not the pGL3-Control construct (Fig 3A and 3B). We identified a miR-K3 seed sequence in the GRK2 3’UTR (Fig 3C). Mutation of this seed sequence abolished the inhibitory effect of miR-K3 on GRK2 3’UTR (Fig 3D). To directly evaluate the effect of miR-K3 on GRK2 protein expression, miR-K3 mimic was transiently co-transfected with GRK2 expression plasmid, pcDNA3.1–3×Flag-GRK2-3’UTR containing the native 3’UTR, into HEK 293T cells. Western blotting showed that miR-K3 strongly attenuated the expression of GRK2 in a dose-dependent fashion (Fig 3E). Importantly, miR-K3 mimic also markedly suppressed the expression of endogenous GRK2 in HUVEC in a dose-dependent manner (Fig 3F) while the mutant mimic of miR-K3 lacking the seed sequences did not (Fig 3G). The expression level of GRK2 in cells transfected with miR-K3 mimic was similar to that of KSHV infection (Fig 3H). Together these data suggest that GRK2 is a direct target of miR-K3.


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)

GRK2 is directly targeted by miR-K3.(A). Luciferase activity was detected in HEK 293T cells co-transfected by a mimic of miR-K3 (miR-K3) or a negative control nucleotide of miRNA (Neg. Ctrl.) together with pGL3-Control or pGL3-GRK2 3’UTR luciferase reporter (pGL3-GRK2 3’UTR). ** P < 0.01 for Student’s t-test. n.s., not significant. (B). Luciferase assay of 293T cells co-transfected by pGL3-GRK2 3′UTR together with increasing amounts (10, 20, and 50 nM) of miR-K3. (C). Schematic illustration of the putative seed sequences of miR-K3 complementary with GRK2 3’UTR and mutagenesis of binding sites in the 3’UTR of GRK2. (D). The luciferase activity was assayed in 293T cells co-transfected by GRK2 wild type 3’UTR (WT GRK2) or the mutant GRK2 3’UTR construct (mut GRK2) together with miR-K3 or mutant miR-K3 mimic (mut miR-K3). * P < 0.05 and ** P < 0.01 for Student’s t-test. (E). miR-K3 inhibited the expression of exogenous GRK2 protein by targeting its native 3’UTR. Western blotting was performed in HEK 293T cells co-transfected by pcDNA3.1–3×Flag-GRK2-3’UTR together with pEGFP and increasing amounts (10 and 20 nM) mimic of miR-K3. (F). miR-K3 inhibited the expression of endogenous GRK2 protein in HUVEC transfected with increasing amounts (10 and 20 nM) mimic of miR-K3. (G). Mutant miR-K3 failed to target endogenous GRK2. Western blotting was performed in HUVEC transfected by Neg. Ctrl., miR-K3 mimic (20 nM) or mut miR-K3 lacking the seed sequences. (H). Transfection of miR-K3 mimic (20 nM) has the same inhibition level on GRK2 expression as that of KSHV infection.
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Related In: Results  -  Collection

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ppat.1005171.g003: GRK2 is directly targeted by miR-K3.(A). Luciferase activity was detected in HEK 293T cells co-transfected by a mimic of miR-K3 (miR-K3) or a negative control nucleotide of miRNA (Neg. Ctrl.) together with pGL3-Control or pGL3-GRK2 3’UTR luciferase reporter (pGL3-GRK2 3’UTR). ** P < 0.01 for Student’s t-test. n.s., not significant. (B). Luciferase assay of 293T cells co-transfected by pGL3-GRK2 3′UTR together with increasing amounts (10, 20, and 50 nM) of miR-K3. (C). Schematic illustration of the putative seed sequences of miR-K3 complementary with GRK2 3’UTR and mutagenesis of binding sites in the 3’UTR of GRK2. (D). The luciferase activity was assayed in 293T cells co-transfected by GRK2 wild type 3’UTR (WT GRK2) or the mutant GRK2 3’UTR construct (mut GRK2) together with miR-K3 or mutant miR-K3 mimic (mut miR-K3). * P < 0.05 and ** P < 0.01 for Student’s t-test. (E). miR-K3 inhibited the expression of exogenous GRK2 protein by targeting its native 3’UTR. Western blotting was performed in HEK 293T cells co-transfected by pcDNA3.1–3×Flag-GRK2-3’UTR together with pEGFP and increasing amounts (10 and 20 nM) mimic of miR-K3. (F). miR-K3 inhibited the expression of endogenous GRK2 protein in HUVEC transfected with increasing amounts (10 and 20 nM) mimic of miR-K3. (G). Mutant miR-K3 failed to target endogenous GRK2. Western blotting was performed in HUVEC transfected by Neg. Ctrl., miR-K3 mimic (20 nM) or mut miR-K3 lacking the seed sequences. (H). Transfection of miR-K3 mimic (20 nM) has the same inhibition level on GRK2 expression as that of KSHV infection.
Mentions: To validate GRK2 as a direct target of miR-K3, the full length 3’UTR of GRK2 was amplified and inserted into the downstream of luciferase sequence in the pGL3-Control plasmid (named as pGL3-GRK2 3’UTR). The luciferase reporter assays indicated that miR-K3 significantly inhibited the GRK2 3’UTR reporter activity in a dose-dependent manner but not the pGL3-Control construct (Fig 3A and 3B). We identified a miR-K3 seed sequence in the GRK2 3’UTR (Fig 3C). Mutation of this seed sequence abolished the inhibitory effect of miR-K3 on GRK2 3’UTR (Fig 3D). To directly evaluate the effect of miR-K3 on GRK2 protein expression, miR-K3 mimic was transiently co-transfected with GRK2 expression plasmid, pcDNA3.1–3×Flag-GRK2-3’UTR containing the native 3’UTR, into HEK 293T cells. Western blotting showed that miR-K3 strongly attenuated the expression of GRK2 in a dose-dependent fashion (Fig 3E). Importantly, miR-K3 mimic also markedly suppressed the expression of endogenous GRK2 in HUVEC in a dose-dependent manner (Fig 3F) while the mutant mimic of miR-K3 lacking the seed sequences did not (Fig 3G). The expression level of GRK2 in cells transfected with miR-K3 mimic was similar to that of KSHV infection (Fig 3H). Together these data suggest that GRK2 is a direct target of miR-K3.

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