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Chemically modified synthetic microRNA-205 inhibits the growth of melanoma cells in vitro and in vivo.

ABSTRACT: microRNA (miR)-205 is downregulated and acts as a tumor suppressor in human melanoma cells. Previously, for clinical application, we added aromatic benzene-pyridine (BP-type) analogs to the 3'-overhang region of the RNA-strand and changed the sequences of the passenger strand in the miR-143 duplex. Here, we demonstrated the antitumor effect in vitro and in vivo of miR-205 that was also chemically modified by BP and had altered passenger sequence. In in vitro experiments, transfection with the synthetic miR-205 (miR-205BP/S3) significantly inhibited the growth of human melanoma cells. Exogenous miR-205BP/S3 suppressed the protein expression levels of E2F1 and VEGF, which are validated targets of miR-205-5p, and BCL2, a transcribed molecule of E2F1, as did Pre-miR-205, used as a miR-205 mimic having the wild-type sequence. On the basis of the results of a luciferase activity assay, miR-205BP/S3 directly targeted E2F1, as did Pre-miR-205. However, miR-205BP/S3 was much more resistant to RNase than Pre-miR-205 in fetal bovine serum and to RNase in mice xenografted with human melanoma tissues. In addition, the intratumoral injection of miR-205BP/S3 exhibited a significant antitumor effect compared with the case of control miRNA or Pre-miR-205 in human melanoma cell-xenografted mice. These findings indicate that miR-205BP/S3 is a possible promising therapeutic modality for melanoma.

SUBMITTER: Noguchi S

PROVIDER: S-EPMC3677302 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Chemically modified synthetic microRNA-205 inhibits the growth of melanoma cells in vitro and in vivo.

Noguchi Shunsuke S Iwasaki Junya J Kumazaki Minami M Mori Takashi T Maruo Kohji K Sakai Hiroki H Yamada Nami N Shimada Kazuyuki K Naoe Tomoki T Kitade Yukio Y Akao Yukihiro Y

Molecular therapy : the journal of the American Society of Gene Therapy 20130430 6

microRNA (miR)-205 is downregulated and acts as a tumor suppressor in human melanoma cells. Previously, for clinical application, we added aromatic benzene-pyridine (BP-type) analogs to the 3'-overhang region of the RNA-strand and changed the sequences of the passenger strand in the miR-143 duplex. Here, we demonstrated the antitumor effect in vitro and in vivo of miR-205 that was also chemically modified by BP and had altered passenger sequence. In in vitro experiments, transfection with the sy ...[more]

PMID: 23629002

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Project description:BackgroundEven though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo.MethodsHuman (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses.ResultsNAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples.ConclusionNAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.

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Chemically modified synthetic microRNA-205 inhibits the growth of melanoma cells in vitro and in vivo.

Publications

Chemically modified synthetic microRNA-205 inhibits the growth of melanoma cells in vitro and in vivo.

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