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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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Nef synergizes with K1 to activate PI3K/AKT/mTOR signaling, and promote cell proliferation and microtubule formation. (A) Western blot analysis of total PTEN and phosphorylation levels of PI3K, AKT and mTOR in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (left panel), and in EA.hy926 cells transduced with K1, Nef or both (right panel), respectively. Numbers labeled under the bands were the relative intensities of the bands after calibrating for loading using house-keeping or nonphosphorylated proteins. The relative value of proteins in Mock + PBS group or Mock group was considered as ‘1’. The amounts of K1 and Nef proteins expressed by lentiviral transduction were examined with their respective antibodies while soluble Nef protein was examined with a His-tag antibody. (B) Cell proliferation examined with a Cell Counting Kit-8 assay. Cell proliferation of HUVECs and EA.hy926 cells treated as described in (A) was determined. Data represent mean ± SD determined from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Plate colony formation assay. EA.hy926 cells treated as described in (A) were examined for colony formation. The photographs depict the colony formation after 2 weeks of seeding (left panel; original magnification, ×100), and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (D) Matrigel assay analysis of tube formation in HUVECs incubated with soluble Nef protein. Tube formation of HUVECs treated as described in (A) was determined. The photographs of microtubules were captured at 16 h post seeding (Soluble Nef + HUVECs; left panel; original magnification, × 100) and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Matrigel assay analysis of tube formation in EA.hy926 cells expressing Nef. Tube formation of EA.hy926 cells treated as described in (A) was determined. The photographs of microtubules were taken at 16 h post incubation (Ectopic Nef + EA.hy926; left panel; original magnification, ×100). Data represent mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates.
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Figure 1: Nef synergizes with K1 to activate PI3K/AKT/mTOR signaling, and promote cell proliferation and microtubule formation. (A) Western blot analysis of total PTEN and phosphorylation levels of PI3K, AKT and mTOR in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (left panel), and in EA.hy926 cells transduced with K1, Nef or both (right panel), respectively. Numbers labeled under the bands were the relative intensities of the bands after calibrating for loading using house-keeping or nonphosphorylated proteins. The relative value of proteins in Mock + PBS group or Mock group was considered as ‘1’. The amounts of K1 and Nef proteins expressed by lentiviral transduction were examined with their respective antibodies while soluble Nef protein was examined with a His-tag antibody. (B) Cell proliferation examined with a Cell Counting Kit-8 assay. Cell proliferation of HUVECs and EA.hy926 cells treated as described in (A) was determined. Data represent mean ± SD determined from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Plate colony formation assay. EA.hy926 cells treated as described in (A) were examined for colony formation. The photographs depict the colony formation after 2 weeks of seeding (left panel; original magnification, ×100), and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (D) Matrigel assay analysis of tube formation in HUVECs incubated with soluble Nef protein. Tube formation of HUVECs treated as described in (A) was determined. The photographs of microtubules were captured at 16 h post seeding (Soluble Nef + HUVECs; left panel; original magnification, × 100) and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Matrigel assay analysis of tube formation in EA.hy926 cells expressing Nef. Tube formation of EA.hy926 cells treated as described in (A) was determined. The photographs of microtubules were taken at 16 h post incubation (Ectopic Nef + EA.hy926; left panel; original magnification, ×100). Data represent mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates.

Mentions: Because K1 is a KSHV oncogene and induces angiogenesis (7,8,63), we determined the synergistic effect of Nef with K1. Both K1 and Nef have been shown to activate the PI3K/AKT pathway (10,32). Indeed, Western blotting showed that soluble Nef protein, or ectopic expression of Nef or K1 alone increased the levels of phosphorylated PI3K, AKT and mTOR in both primary HUVECs and EA.hy926 cells (Figure 1A). Significantly, the presence of both Nef and K1 synergized with each other and further increased the levels of these phosphorylated proteins. Consistent with these results, Nef and K1 decreased the level of PTEN, a dual-specificity protein and lipid phosphatase that counteracts the PI3K activity and functions as a major tumor suppressor (Figure 1A).


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)

Nef synergizes with K1 to activate PI3K/AKT/mTOR signaling, and promote cell proliferation and microtubule formation. (A) Western blot analysis of total PTEN and phosphorylation levels of PI3K, AKT and mTOR in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (left panel), and in EA.hy926 cells transduced with K1, Nef or both (right panel), respectively. Numbers labeled under the bands were the relative intensities of the bands after calibrating for loading using house-keeping or nonphosphorylated proteins. The relative value of proteins in Mock + PBS group or Mock group was considered as ‘1’. The amounts of K1 and Nef proteins expressed by lentiviral transduction were examined with their respective antibodies while soluble Nef protein was examined with a His-tag antibody. (B) Cell proliferation examined with a Cell Counting Kit-8 assay. Cell proliferation of HUVECs and EA.hy926 cells treated as described in (A) was determined. Data represent mean ± SD determined from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Plate colony formation assay. EA.hy926 cells treated as described in (A) were examined for colony formation. The photographs depict the colony formation after 2 weeks of seeding (left panel; original magnification, ×100), and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (D) Matrigel assay analysis of tube formation in HUVECs incubated with soluble Nef protein. Tube formation of HUVECs treated as described in (A) was determined. The photographs of microtubules were captured at 16 h post seeding (Soluble Nef + HUVECs; left panel; original magnification, × 100) and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Matrigel assay analysis of tube formation in EA.hy926 cells expressing Nef. Tube formation of EA.hy926 cells treated as described in (A) was determined. The photographs of microtubules were taken at 16 h post incubation (Ectopic Nef + EA.hy926; left panel; original magnification, ×100). Data represent mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates.
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Figure 1: Nef synergizes with K1 to activate PI3K/AKT/mTOR signaling, and promote cell proliferation and microtubule formation. (A) Western blot analysis of total PTEN and phosphorylation levels of PI3K, AKT and mTOR in HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both (left panel), and in EA.hy926 cells transduced with K1, Nef or both (right panel), respectively. Numbers labeled under the bands were the relative intensities of the bands after calibrating for loading using house-keeping or nonphosphorylated proteins. The relative value of proteins in Mock + PBS group or Mock group was considered as ‘1’. The amounts of K1 and Nef proteins expressed by lentiviral transduction were examined with their respective antibodies while soluble Nef protein was examined with a His-tag antibody. (B) Cell proliferation examined with a Cell Counting Kit-8 assay. Cell proliferation of HUVECs and EA.hy926 cells treated as described in (A) was determined. Data represent mean ± SD determined from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Plate colony formation assay. EA.hy926 cells treated as described in (A) were examined for colony formation. The photographs depict the colony formation after 2 weeks of seeding (left panel; original magnification, ×100), and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (D) Matrigel assay analysis of tube formation in HUVECs incubated with soluble Nef protein. Tube formation of HUVECs treated as described in (A) was determined. The photographs of microtubules were captured at 16 h post seeding (Soluble Nef + HUVECs; left panel; original magnification, × 100) and the graph bars represent the mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Matrigel assay analysis of tube formation in EA.hy926 cells expressing Nef. Tube formation of EA.hy926 cells treated as described in (A) was determined. The photographs of microtubules were taken at 16 h post incubation (Ectopic Nef + EA.hy926; left panel; original magnification, ×100). Data represent mean ± SD (right panel) from three independent experiments (n = 3), each experiment containing six technical replicates.
Mentions: Because K1 is a KSHV oncogene and induces angiogenesis (7,8,63), we determined the synergistic effect of Nef with K1. Both K1 and Nef have been shown to activate the PI3K/AKT pathway (10,32). Indeed, Western blotting showed that soluble Nef protein, or ectopic expression of Nef or K1 alone increased the levels of phosphorylated PI3K, AKT and mTOR in both primary HUVECs and EA.hy926 cells (Figure 1A). Significantly, the presence of both Nef and K1 synergized with each other and further increased the levels of these phosphorylated proteins. Consistent with these results, Nef and K1 decreased the level of PTEN, a dual-specificity protein and lipid phosphatase that counteracts the PI3K activity and functions as a major tumor suppressor (Figure 1A).

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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Related in: MedlinePlus