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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-induced angiogenesis both in vitro and in vivo. (A) Matrigel assay analysis of microtubule formation. Tube formation assay was performed with HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both and further transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h, respectively. The photographs of microtubules were captured at 16 h post seeding (original magnification, ×100). (B) Quantification of results in (A). The results represent the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Inhibition of miR-718 suppressed the enhanced effect of Nef on K1-induced angiogenesis. HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both were transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h. The collected cells were mixed with Matrigel and subsequently implanted onto the CAM. Representative photographs of angiogenesis on the CAM are shown. (D) Quantification of results in (C). The number of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Western blot analysis of total PTEN and phosphorylation levels of AKT and mTOR. The tumor tissues from CAM that treated as in (C) were collected and the total proteins of the tissues were extracted for Western blot. Numbers labeled under the bands were the relative intensities of the bands following calibration for loading with house-keeping protein tubulin. The relative value of proteins in K1 + PBS + Neg. Ctrl. group was considered as ‘1’. (F) Inhibition of miR-718 abolished the enhanced effect of Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1, Nef or both were infected with control virus (pCDH; top) or miR-718 sponge (miR-718 sponge; bottom) for 72 h and further re-suspended in serum-free medium. As detailed in the ‘Materials and Methods’ section, the treated cells were injected (s.c.) into nude mice for 10 days and the Matrigel plugs were removed and analyzed. Representative photographs of angiogenesis in the nude mice are shown. (G) The hemoglobin level of the Matrigel plugs treated as in (F) was determined with O.D. value at 540 nm. Data represent mean ± SD, each group with six tumors (n = 6). Three independent experiments were performed and similar results were obtained.
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Figure 7: miR-718 mediates K1- and Nef-induced angiogenesis both in vitro and in vivo. (A) Matrigel assay analysis of microtubule formation. Tube formation assay was performed with HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both and further transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h, respectively. The photographs of microtubules were captured at 16 h post seeding (original magnification, ×100). (B) Quantification of results in (A). The results represent the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Inhibition of miR-718 suppressed the enhanced effect of Nef on K1-induced angiogenesis. HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both were transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h. The collected cells were mixed with Matrigel and subsequently implanted onto the CAM. Representative photographs of angiogenesis on the CAM are shown. (D) Quantification of results in (C). The number of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Western blot analysis of total PTEN and phosphorylation levels of AKT and mTOR. The tumor tissues from CAM that treated as in (C) were collected and the total proteins of the tissues were extracted for Western blot. Numbers labeled under the bands were the relative intensities of the bands following calibration for loading with house-keeping protein tubulin. The relative value of proteins in K1 + PBS + Neg. Ctrl. group was considered as ‘1’. (F) Inhibition of miR-718 abolished the enhanced effect of Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1, Nef or both were infected with control virus (pCDH; top) or miR-718 sponge (miR-718 sponge; bottom) for 72 h and further re-suspended in serum-free medium. As detailed in the ‘Materials and Methods’ section, the treated cells were injected (s.c.) into nude mice for 10 days and the Matrigel plugs were removed and analyzed. Representative photographs of angiogenesis in the nude mice are shown. (G) The hemoglobin level of the Matrigel plugs treated as in (F) was determined with O.D. value at 540 nm. Data represent mean ± SD, each group with six tumors (n = 6). Three independent experiments were performed and similar results were obtained.

Mentions: We further determined whether miR-718 mediated Nef- and K1-induced angiogenesis. Tube formation assay was performed with HUVECs transduced with K1, incubated with soluble Nef protein or subjected to both treatments, and co-transfected with an inhibitor of miR-718 or a scrambled control. As shown in Figure 7A and B, miRNA suppressor of miR-718 blocked tube formation of HUVECs induced by K1, Nef or both.


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-induced angiogenesis both in vitro and in vivo. (A) Matrigel assay analysis of microtubule formation. Tube formation assay was performed with HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both and further transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h, respectively. The photographs of microtubules were captured at 16 h post seeding (original magnification, ×100). (B) Quantification of results in (A). The results represent the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Inhibition of miR-718 suppressed the enhanced effect of Nef on K1-induced angiogenesis. HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both were transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h. The collected cells were mixed with Matrigel and subsequently implanted onto the CAM. Representative photographs of angiogenesis on the CAM are shown. (D) Quantification of results in (C). The number of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Western blot analysis of total PTEN and phosphorylation levels of AKT and mTOR. The tumor tissues from CAM that treated as in (C) were collected and the total proteins of the tissues were extracted for Western blot. Numbers labeled under the bands were the relative intensities of the bands following calibration for loading with house-keeping protein tubulin. The relative value of proteins in K1 + PBS + Neg. Ctrl. group was considered as ‘1’. (F) Inhibition of miR-718 abolished the enhanced effect of Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1, Nef or both were infected with control virus (pCDH; top) or miR-718 sponge (miR-718 sponge; bottom) for 72 h and further re-suspended in serum-free medium. As detailed in the ‘Materials and Methods’ section, the treated cells were injected (s.c.) into nude mice for 10 days and the Matrigel plugs were removed and analyzed. Representative photographs of angiogenesis in the nude mice are shown. (G) The hemoglobin level of the Matrigel plugs treated as in (F) was determined with O.D. value at 540 nm. Data represent mean ± SD, each group with six tumors (n = 6). Three independent experiments were performed and similar results were obtained.
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Figure 7: miR-718 mediates K1- and Nef-induced angiogenesis both in vitro and in vivo. (A) Matrigel assay analysis of microtubule formation. Tube formation assay was performed with HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both and further transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h, respectively. The photographs of microtubules were captured at 16 h post seeding (original magnification, ×100). (B) Quantification of results in (A). The results represent the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (C) Inhibition of miR-718 suppressed the enhanced effect of Nef on K1-induced angiogenesis. HUVECs transduced with K1, incubated with soluble Nef protein for 72 h or both were transfected with negative control nucleotide of miRNA (Neg. Ctrl.; top) or inhibitor of miR-718 (miR-718 inhibitor; bottom) for 48 h. The collected cells were mixed with Matrigel and subsequently implanted onto the CAM. Representative photographs of angiogenesis on the CAM are shown. (D) Quantification of results in (C). The number of blood vessels is expressed as the mean ± SD from three independent experiments (n = 3), each experiment containing six technical replicates. (E) Western blot analysis of total PTEN and phosphorylation levels of AKT and mTOR. The tumor tissues from CAM that treated as in (C) were collected and the total proteins of the tissues were extracted for Western blot. Numbers labeled under the bands were the relative intensities of the bands following calibration for loading with house-keeping protein tubulin. The relative value of proteins in K1 + PBS + Neg. Ctrl. group was considered as ‘1’. (F) Inhibition of miR-718 abolished the enhanced effect of Nef on K1-induced angiogenesis in nude mice. EA.hy926 cells transduced with K1, Nef or both were infected with control virus (pCDH; top) or miR-718 sponge (miR-718 sponge; bottom) for 72 h and further re-suspended in serum-free medium. As detailed in the ‘Materials and Methods’ section, the treated cells were injected (s.c.) into nude mice for 10 days and the Matrigel plugs were removed and analyzed. Representative photographs of angiogenesis in the nude mice are shown. (G) The hemoglobin level of the Matrigel plugs treated as in (F) was determined with O.D. value at 540 nm. Data represent mean ± SD, each group with six tumors (n = 6). Three independent experiments were performed and similar results were obtained.
Mentions: We further determined whether miR-718 mediated Nef- and K1-induced angiogenesis. Tube formation assay was performed with HUVECs transduced with K1, incubated with soluble Nef protein or subjected to both treatments, and co-transfected with an inhibitor of miR-718 or a scrambled control. As shown in Figure 7A and B, miRNA suppressor of miR-718 blocked tube formation of HUVECs induced by K1, Nef or both.

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