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HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway.

Xue M, Yao S, Hu M, Li W, Hao T, Zhou F, Zhu X, Lu H, Qin D, Yan Q, Zhu J, Gao SJ, Lu C - Nucleic Acids Res. (2014)

Bottom Line: Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV).Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3' UTR of its mRNA.Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target.

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 210029, P.R. China Department of Physiology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, P.R. China.

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miR-718 mediates K1 and Nef regulation of PTEN-AKT pathway by directly targeting PTEN 3′UTR. (A) Heatmap of top miRNAs with differential expression in EA.hy926 cells transduced with K1, Nef or both. miRNA array analysis was performed and pseudo-color represents intensity scale of K1, Nef and K1 + Nef infection groups versus Mock infection group. Green and red denote low and high expression, respectively. (B) Examination of the inhibition of a PTEN 3′UTR luciferase report by miRNAs selected from miRNA microarray analysis. 293T cells were co-transfected with negative control nucleotide of miRNA (Neg. Ctrl.) or mimics of selected miRNAs together with the pGL3-Luc-PTEN 3′UTR luciferase reporter and assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. *** indicates P < 0.001 for Student's t-test versus Neg. Ctrl. Group. n.s. denotes ‘not significant’. (C) miR-718 expression in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (Soluble Nef in HUVECs; left panel) and in EA.hy926 cells transduced with K1, Nef or both (Ectopic Nef in EA.hy926; right panel), were quantitated by RT-qPCR. Relative quantities of miRNAs expression are represented on the y-axis. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (D) Luciferase assay of pGL3-Luc-PTEN 3′UTR reporter co-transfected with increasing amounts (10, 20 and 50 nM) of miRNA negative control nucleotide (Neg. Ctrl.) or miR-718 mimic (miR-718) for 48 h in 293T cells. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (E) miR-718 targets exogenous PTEN under the control of its native 3′UTR in HEK 293T cells. A genomic PTEN expression vector, 3×His-PTEN-3′UTR, bearing the full 3′UTR sequences was co-transfected with pEGFP and increasing amounts (10 and 50 nM) mimic of miR-718 into HEK 293T cells for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (F) miR-718 targets endogenous PTEN in HUVECs transfected with increasing amounts (10 and 50 nM) mimic of miR-718 for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (G) Mutant miR-718 fails to target endogenous PTEN in HUVECs transfected with miRNA negative control nucleotide, miR-718 mimic or mutant mimic of miR-718 lacking the seed sequences for 48 h in 293T cells. The transfected cells were collected and Western blot was performed with the indicated antibodies. (H) Schematic illustration of the putative seed sequence of miR-718 within PTEN 3′UTR and mutagenesis of binding site in the 3′UTR of PTEN or miR-718 mimic. (I) Effect of seed mutagenesis or mutation of the putative binding site on the PTEN 3′UTR reporter. PTEN wild type (WT PTEN) were co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718) into 293T cells, while mutant 3’UTR construct (mut PTEN) were also co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718). After co-transfection for 48 h, 293T cells were assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. **P < 0.01 and ***P < 0.001 by Student's t-test versus.
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Figure 6: miR-718 mediates K1 and Nef regulation of PTEN-AKT pathway by directly targeting PTEN 3′UTR. (A) Heatmap of top miRNAs with differential expression in EA.hy926 cells transduced with K1, Nef or both. miRNA array analysis was performed and pseudo-color represents intensity scale of K1, Nef and K1 + Nef infection groups versus Mock infection group. Green and red denote low and high expression, respectively. (B) Examination of the inhibition of a PTEN 3′UTR luciferase report by miRNAs selected from miRNA microarray analysis. 293T cells were co-transfected with negative control nucleotide of miRNA (Neg. Ctrl.) or mimics of selected miRNAs together with the pGL3-Luc-PTEN 3′UTR luciferase reporter and assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. *** indicates P < 0.001 for Student's t-test versus Neg. Ctrl. Group. n.s. denotes ‘not significant’. (C) miR-718 expression in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (Soluble Nef in HUVECs; left panel) and in EA.hy926 cells transduced with K1, Nef or both (Ectopic Nef in EA.hy926; right panel), were quantitated by RT-qPCR. Relative quantities of miRNAs expression are represented on the y-axis. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (D) Luciferase assay of pGL3-Luc-PTEN 3′UTR reporter co-transfected with increasing amounts (10, 20 and 50 nM) of miRNA negative control nucleotide (Neg. Ctrl.) or miR-718 mimic (miR-718) for 48 h in 293T cells. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (E) miR-718 targets exogenous PTEN under the control of its native 3′UTR in HEK 293T cells. A genomic PTEN expression vector, 3×His-PTEN-3′UTR, bearing the full 3′UTR sequences was co-transfected with pEGFP and increasing amounts (10 and 50 nM) mimic of miR-718 into HEK 293T cells for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (F) miR-718 targets endogenous PTEN in HUVECs transfected with increasing amounts (10 and 50 nM) mimic of miR-718 for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (G) Mutant miR-718 fails to target endogenous PTEN in HUVECs transfected with miRNA negative control nucleotide, miR-718 mimic or mutant mimic of miR-718 lacking the seed sequences for 48 h in 293T cells. The transfected cells were collected and Western blot was performed with the indicated antibodies. (H) Schematic illustration of the putative seed sequence of miR-718 within PTEN 3′UTR and mutagenesis of binding site in the 3′UTR of PTEN or miR-718 mimic. (I) Effect of seed mutagenesis or mutation of the putative binding site on the PTEN 3′UTR reporter. PTEN wild type (WT PTEN) were co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718) into 293T cells, while mutant 3’UTR construct (mut PTEN) were also co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718). After co-transfection for 48 h, 293T cells were assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. **P < 0.01 and ***P < 0.001 by Student's t-test versus.

Mentions: Because PTEN plays a pivotal role in Nef- and K1-induced angiogenesis, we further investigated the mechanism regulating PTEN expression. We performed a microarray screening to identify miRNAs that were regulated by Nef and K1, and identified a number of cellular miRNAs that were consistently upregulated by K1, Nef or both in HUVECs (Figure 6A). We thus hypothesized that some of these miRNAs might regulate PTEN expression. Bioinformatics prediction for miRNA targets combined with the results of miRNA microarray identified nine miRNAs that had putative targeting sites in PTEN 3′UTR (Figure 6A). Luciferase reporter assay using PTEN 3′UTR luciferase reporter confirmed that, of the nine miRNAs, only cellular hsa-miR-718 (miR-718) significantly inhibited the PTEN 3′UTR reporter activity (Figure 6B). RT-qPCR confirmed that both K1 and Nef indeed increased the expression of miR-718 in both HUVECs and EA.hy926 cells (Figure 6C). In a luciferase reporter assay, miR-718 inhibited the reporter activity of PTEN 3′UTR in a dose-dependent fashion (Figure 6D).


HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway.

Xue M, Yao S, Hu M, Li W, Hao T, Zhou F, Zhu X, Lu H, Qin D, Yan Q, Zhu J, Gao SJ, Lu C - Nucleic Acids Res. (2014)

miR-718 mediates K1 and Nef regulation of PTEN-AKT pathway by directly targeting PTEN 3′UTR. (A) Heatmap of top miRNAs with differential expression in EA.hy926 cells transduced with K1, Nef or both. miRNA array analysis was performed and pseudo-color represents intensity scale of K1, Nef and K1 + Nef infection groups versus Mock infection group. Green and red denote low and high expression, respectively. (B) Examination of the inhibition of a PTEN 3′UTR luciferase report by miRNAs selected from miRNA microarray analysis. 293T cells were co-transfected with negative control nucleotide of miRNA (Neg. Ctrl.) or mimics of selected miRNAs together with the pGL3-Luc-PTEN 3′UTR luciferase reporter and assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. *** indicates P < 0.001 for Student's t-test versus Neg. Ctrl. Group. n.s. denotes ‘not significant’. (C) miR-718 expression in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (Soluble Nef in HUVECs; left panel) and in EA.hy926 cells transduced with K1, Nef or both (Ectopic Nef in EA.hy926; right panel), were quantitated by RT-qPCR. Relative quantities of miRNAs expression are represented on the y-axis. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (D) Luciferase assay of pGL3-Luc-PTEN 3′UTR reporter co-transfected with increasing amounts (10, 20 and 50 nM) of miRNA negative control nucleotide (Neg. Ctrl.) or miR-718 mimic (miR-718) for 48 h in 293T cells. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (E) miR-718 targets exogenous PTEN under the control of its native 3′UTR in HEK 293T cells. A genomic PTEN expression vector, 3×His-PTEN-3′UTR, bearing the full 3′UTR sequences was co-transfected with pEGFP and increasing amounts (10 and 50 nM) mimic of miR-718 into HEK 293T cells for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (F) miR-718 targets endogenous PTEN in HUVECs transfected with increasing amounts (10 and 50 nM) mimic of miR-718 for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (G) Mutant miR-718 fails to target endogenous PTEN in HUVECs transfected with miRNA negative control nucleotide, miR-718 mimic or mutant mimic of miR-718 lacking the seed sequences for 48 h in 293T cells. The transfected cells were collected and Western blot was performed with the indicated antibodies. (H) Schematic illustration of the putative seed sequence of miR-718 within PTEN 3′UTR and mutagenesis of binding site in the 3′UTR of PTEN or miR-718 mimic. (I) Effect of seed mutagenesis or mutation of the putative binding site on the PTEN 3′UTR reporter. PTEN wild type (WT PTEN) were co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718) into 293T cells, while mutant 3’UTR construct (mut PTEN) were also co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718). After co-transfection for 48 h, 293T cells were assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. **P < 0.01 and ***P < 0.001 by Student's t-test versus.
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Figure 6: miR-718 mediates K1 and Nef regulation of PTEN-AKT pathway by directly targeting PTEN 3′UTR. (A) Heatmap of top miRNAs with differential expression in EA.hy926 cells transduced with K1, Nef or both. miRNA array analysis was performed and pseudo-color represents intensity scale of K1, Nef and K1 + Nef infection groups versus Mock infection group. Green and red denote low and high expression, respectively. (B) Examination of the inhibition of a PTEN 3′UTR luciferase report by miRNAs selected from miRNA microarray analysis. 293T cells were co-transfected with negative control nucleotide of miRNA (Neg. Ctrl.) or mimics of selected miRNAs together with the pGL3-Luc-PTEN 3′UTR luciferase reporter and assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. *** indicates P < 0.001 for Student's t-test versus Neg. Ctrl. Group. n.s. denotes ‘not significant’. (C) miR-718 expression in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (Soluble Nef in HUVECs; left panel) and in EA.hy926 cells transduced with K1, Nef or both (Ectopic Nef in EA.hy926; right panel), were quantitated by RT-qPCR. Relative quantities of miRNAs expression are represented on the y-axis. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (D) Luciferase assay of pGL3-Luc-PTEN 3′UTR reporter co-transfected with increasing amounts (10, 20 and 50 nM) of miRNA negative control nucleotide (Neg. Ctrl.) or miR-718 mimic (miR-718) for 48 h in 293T cells. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. (E) miR-718 targets exogenous PTEN under the control of its native 3′UTR in HEK 293T cells. A genomic PTEN expression vector, 3×His-PTEN-3′UTR, bearing the full 3′UTR sequences was co-transfected with pEGFP and increasing amounts (10 and 50 nM) mimic of miR-718 into HEK 293T cells for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (F) miR-718 targets endogenous PTEN in HUVECs transfected with increasing amounts (10 and 50 nM) mimic of miR-718 for 48 h. The transfected cells were collected and Western blot was performed with the indicated antibodies. (G) Mutant miR-718 fails to target endogenous PTEN in HUVECs transfected with miRNA negative control nucleotide, miR-718 mimic or mutant mimic of miR-718 lacking the seed sequences for 48 h in 293T cells. The transfected cells were collected and Western blot was performed with the indicated antibodies. (H) Schematic illustration of the putative seed sequence of miR-718 within PTEN 3′UTR and mutagenesis of binding site in the 3′UTR of PTEN or miR-718 mimic. (I) Effect of seed mutagenesis or mutation of the putative binding site on the PTEN 3′UTR reporter. PTEN wild type (WT PTEN) were co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718) into 293T cells, while mutant 3’UTR construct (mut PTEN) were also co-transfected with miRNA negative control nucleotide (Neg. Ctrl.), natural (miR-718) or mutant miR-718 (mut miR-718). After co-transfection for 48 h, 293T cells were assayed for luciferase activity. The data represent the mean ± SD from three independent experiments (n = 3), each experiment containing four technical replicates. **P < 0.01 and ***P < 0.001 by Student's t-test versus.
Mentions: Because PTEN plays a pivotal role in Nef- and K1-induced angiogenesis, we further investigated the mechanism regulating PTEN expression. We performed a microarray screening to identify miRNAs that were regulated by Nef and K1, and identified a number of cellular miRNAs that were consistently upregulated by K1, Nef or both in HUVECs (Figure 6A). We thus hypothesized that some of these miRNAs might regulate PTEN expression. Bioinformatics prediction for miRNA targets combined with the results of miRNA microarray identified nine miRNAs that had putative targeting sites in PTEN 3′UTR (Figure 6A). Luciferase reporter assay using PTEN 3′UTR luciferase reporter confirmed that, of the nine miRNAs, only cellular hsa-miR-718 (miR-718) significantly inhibited the PTEN 3′UTR reporter activity (Figure 6B). RT-qPCR confirmed that both K1 and Nef indeed increased the expression of miR-718 in both HUVECs and EA.hy926 cells (Figure 6C). In a luciferase reporter assay, miR-718 inhibited the reporter activity of PTEN 3′UTR in a dose-dependent fashion (Figure 6D).

Bottom Line: Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV).Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3' UTR of its mRNA.Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target.

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 210029, P.R. China Department of Physiology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, P.R. China.

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