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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 expression is reduced in miR-K3-expressing HUVEC and KS lesion samples.(A). The mRNA level of GRK2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were examined by qPCR. (B). The expression of GRK2 proteins in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were detected by Western blotting analysis. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown; same for all of the following Western blotting figures. (C). HUVEC were infected with KSHV, and image captured under light microscope (Phase) and fluorescent microscope (GFP) (Original magnification, ×100). (D). qPCR analysis for GRK2 mRNA in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (E). Western blotting analysis of the expression of GRK2 protein in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (F). Hematoxylin and eosin (H&E) staining of KS lesion (right) and normal (left) tissue sections to show histologic features (left panel; original magnification, ×100) and immunohistochemical staining (IHC) of KSHV LANA and GRK2 (middle and left panels; original magnification, ×200). (G). Quantification of results in (F). *** P < 0.001 for Student’s t-test.
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ppat.1005171.g002: GRK2 expression is reduced in miR-K3-expressing HUVEC and KS lesion samples.(A). The mRNA level of GRK2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were examined by qPCR. (B). The expression of GRK2 proteins in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were detected by Western blotting analysis. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown; same for all of the following Western blotting figures. (C). HUVEC were infected with KSHV, and image captured under light microscope (Phase) and fluorescent microscope (GFP) (Original magnification, ×100). (D). qPCR analysis for GRK2 mRNA in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (E). Western blotting analysis of the expression of GRK2 protein in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (F). Hematoxylin and eosin (H&E) staining of KS lesion (right) and normal (left) tissue sections to show histologic features (left panel; original magnification, ×100) and immunohistochemical staining (IHC) of KSHV LANA and GRK2 (middle and left panels; original magnification, ×200). (G). Quantification of results in (F). *** P < 0.001 for Student’s t-test.

Mentions: Since miRNAs usually exert their functions by binding to their target genes to induce the degradation of the transcripts or inhibit the translation of proteins, bioinformatics analysis with several programs including TargetScan, RNAhybrid, Findtar, and Pita, was performed to predict the putative miR-K3 targets. Based on complementarity with the seed sequences of miR-K3, a putative binding site was predicted in the 3’UTR of G protein-coupled receptor kinase 2 (GRK2), which was also predicted as a potential target of miR-K3 in the previous studies [45,46]. qPCR and Western blotting analyses indeed showed that both mRNA and protein of GRK2 were markedly down-regulated in miR-K3-expressing HUVEC compared to that of the vector control cells (Fig 2A and 2B). To determine whether KSHV infection can decrease GRK2 expression, we examined HUVEC infected with KSHV (Fig 2C). As expected, both mRNA and protein levels of GRK2 were also dramatically reduced in KSHV-infected HUVEC (Fig 2D and 2E). Consistent with these observations, there were less GRK2-postive cells in the KS lesions compared to the normal skin tissues as shown by immunohistochemistry staining (Fig 2F and 2G).


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 expression is reduced in miR-K3-expressing HUVEC and KS lesion samples.(A). The mRNA level of GRK2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were examined by qPCR. (B). The expression of GRK2 proteins in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were detected by Western blotting analysis. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown; same for all of the following Western blotting figures. (C). HUVEC were infected with KSHV, and image captured under light microscope (Phase) and fluorescent microscope (GFP) (Original magnification, ×100). (D). qPCR analysis for GRK2 mRNA in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (E). Western blotting analysis of the expression of GRK2 protein in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (F). Hematoxylin and eosin (H&E) staining of KS lesion (right) and normal (left) tissue sections to show histologic features (left panel; original magnification, ×100) and immunohistochemical staining (IHC) of KSHV LANA and GRK2 (middle and left panels; original magnification, ×200). (G). Quantification of results in (F). *** P < 0.001 for Student’s t-test.
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ppat.1005171.g002: GRK2 expression is reduced in miR-K3-expressing HUVEC and KS lesion samples.(A). The mRNA level of GRK2 in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were examined by qPCR. (B). The expression of GRK2 proteins in HUVEC transduced with lentivirus empty vector (mpCDH) and lentivirus-miR-K3 (miR-K3) were detected by Western blotting analysis. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown; same for all of the following Western blotting figures. (C). HUVEC were infected with KSHV, and image captured under light microscope (Phase) and fluorescent microscope (GFP) (Original magnification, ×100). (D). qPCR analysis for GRK2 mRNA in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (E). Western blotting analysis of the expression of GRK2 protein in KSHV-infected HUVEC as described in (C) (KSHV) or in HUVEC treated with PBS as the negative control (PBS). (F). Hematoxylin and eosin (H&E) staining of KS lesion (right) and normal (left) tissue sections to show histologic features (left panel; original magnification, ×100) and immunohistochemical staining (IHC) of KSHV LANA and GRK2 (middle and left panels; original magnification, ×200). (G). Quantification of results in (F). *** P < 0.001 for Student’s t-test.
Mentions: Since miRNAs usually exert their functions by binding to their target genes to induce the degradation of the transcripts or inhibit the translation of proteins, bioinformatics analysis with several programs including TargetScan, RNAhybrid, Findtar, and Pita, was performed to predict the putative miR-K3 targets. Based on complementarity with the seed sequences of miR-K3, a putative binding site was predicted in the 3’UTR of G protein-coupled receptor kinase 2 (GRK2), which was also predicted as a potential target of miR-K3 in the previous studies [45,46]. qPCR and Western blotting analyses indeed showed that both mRNA and protein of GRK2 were markedly down-regulated in miR-K3-expressing HUVEC compared to that of the vector control cells (Fig 2A and 2B). To determine whether KSHV infection can decrease GRK2 expression, we examined HUVEC infected with KSHV (Fig 2C). As expected, both mRNA and protein levels of GRK2 were also dramatically reduced in KSHV-infected HUVEC (Fig 2D and 2E). Consistent with these observations, there were less GRK2-postive cells in the KS lesions compared to the normal skin tissues as shown by immunohistochemistry staining (Fig 2F and 2G).

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