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Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole-imidazole polyamide, which targets an E-box motif.

Taniguchi M, Fujiwara K, Nakai Y, Ozaki T, Koshikawa N, Toshio K, Kataba M, Oguni A, Matsuda H, Yoshida Y, Tokuhashi Y, Fukuda N, Ueno T, Soma M, Nagase H - FEBS Open Bio (2014)

Bottom Line: Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors.In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo.Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

View Article: PubMed Central - PubMed

Affiliation: Division of Orthopedic Surgery, Nihon University School of Medicine, 30-1 Oyaguchi Kami-Cho, Itabashi, Tokyo 173-8610, Japan.

ABSTRACT
Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors. In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

No MeSH data available.


Related in: MedlinePlus

MALAT1 is one of possible target genes of Myc-6. (A) Sequence of 5′-upstream region of human MALAT1 gene. The positions relative to the first nucleotide of MALAT1 exon 1 (+1) are indicated. The putative Myc-6-target sequence is underlined, and E-box-like sequence is highlighted in gray. (B and C) Gel retardation assay. FITC-labeled specific (MALAT1) or non-specific (Mismatch) oligonucleotide (1 μM) was incubated with water or Myc-6 (5 μM) for 1 h at 37 °C. The reaction mixtures were separated by 4–20% gradient polyacrylamide gel electrophoresis and then analyzed by LAS4000 (FUJIFILM) (B). FITC-labeled specific oligonucleotide (0.5 μM) was incubated with Myc-6 (0.5 μM) in the presence or absence of the indicated amounts of unlabeled specific (upper) or non-specific (lower) oligonucleotides. The reaction mixtures were analyzed as in (B) (C). (D) Myc-6-mediated down-regulation of MALAT1. MG63 cells were treated with or without the indicated concentrations of Myc-6. Twenty-four hours after treatment, the expression level of MALAT1 was analyzed by real-time PCR. p < 0.05 was considered statistically significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. The columns represent means ± SD. (E) Knockdown of MALAT1 suppresses MG63 cell growth. MG63 cells were transfected with control siRNA or with siRNA against MALAT1. The cell viability was assessed by WST-8 assay. ∗∗p < 0.001.
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f0020: MALAT1 is one of possible target genes of Myc-6. (A) Sequence of 5′-upstream region of human MALAT1 gene. The positions relative to the first nucleotide of MALAT1 exon 1 (+1) are indicated. The putative Myc-6-target sequence is underlined, and E-box-like sequence is highlighted in gray. (B and C) Gel retardation assay. FITC-labeled specific (MALAT1) or non-specific (Mismatch) oligonucleotide (1 μM) was incubated with water or Myc-6 (5 μM) for 1 h at 37 °C. The reaction mixtures were separated by 4–20% gradient polyacrylamide gel electrophoresis and then analyzed by LAS4000 (FUJIFILM) (B). FITC-labeled specific oligonucleotide (0.5 μM) was incubated with Myc-6 (0.5 μM) in the presence or absence of the indicated amounts of unlabeled specific (upper) or non-specific (lower) oligonucleotides. The reaction mixtures were analyzed as in (B) (C). (D) Myc-6-mediated down-regulation of MALAT1. MG63 cells were treated with or without the indicated concentrations of Myc-6. Twenty-four hours after treatment, the expression level of MALAT1 was analyzed by real-time PCR. p < 0.05 was considered statistically significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. The columns represent means ± SD. (E) Knockdown of MALAT1 suppresses MG63 cell growth. MG63 cells were transfected with control siRNA or with siRNA against MALAT1. The cell viability was assessed by WST-8 assay. ∗∗p < 0.001.

Mentions: To elucidate the precise molecular mechanism(s) behind Myc-6-mediated induction of apoptosis and suppression of tumor growth, we sought to identify a putative Myc-6-target gene(s) by using DNA microarray-based analysis. After the extensive screening, we have finally obtained 14 genes whose expression levels were significantly lower in Myc-6-treated MG63 cells relative to those in untreated cells (Table 1). The close inspection of their 5′-upstream regions (up to 1 kb) revealed that MALAT1, which encodes a long noncoding RNA [21], contains a theoretical Myc-6-target sequence including E-box-like motif (at positions −258 to −251) (Fig. 4A). To verify whether Myc-6 could specifically bind to this sequence, we have performed gel retardation assay. FITC-labeled double-strand oligonucleotide corresponding to the above-mentioned MALAT1 sequence or the unrelated sequence (Mismatch, Supplementary Table 2) was generated, incubated with or without Myc-6 and the reaction mixtures were separated by polyacrylamide gel electrophoresis. Unlike the non-specific oligonucleotide, a definite mobility shift of the specific oligonucleotide was observed in the presence of Myc-6 compared to that in the absence of Myc-6 (Fig. 4B). Furthermore, this mobility shift was clearly inhibited by the excess amount of specific oligonucleotide but not by non-specific one (Fig. 4C), indicating that Myc-6 specifically binds to its theoretical target site located within 5′-upstream region of MALAT1.


Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole-imidazole polyamide, which targets an E-box motif.

Taniguchi M, Fujiwara K, Nakai Y, Ozaki T, Koshikawa N, Toshio K, Kataba M, Oguni A, Matsuda H, Yoshida Y, Tokuhashi Y, Fukuda N, Ueno T, Soma M, Nagase H - FEBS Open Bio (2014)

MALAT1 is one of possible target genes of Myc-6. (A) Sequence of 5′-upstream region of human MALAT1 gene. The positions relative to the first nucleotide of MALAT1 exon 1 (+1) are indicated. The putative Myc-6-target sequence is underlined, and E-box-like sequence is highlighted in gray. (B and C) Gel retardation assay. FITC-labeled specific (MALAT1) or non-specific (Mismatch) oligonucleotide (1 μM) was incubated with water or Myc-6 (5 μM) for 1 h at 37 °C. The reaction mixtures were separated by 4–20% gradient polyacrylamide gel electrophoresis and then analyzed by LAS4000 (FUJIFILM) (B). FITC-labeled specific oligonucleotide (0.5 μM) was incubated with Myc-6 (0.5 μM) in the presence or absence of the indicated amounts of unlabeled specific (upper) or non-specific (lower) oligonucleotides. The reaction mixtures were analyzed as in (B) (C). (D) Myc-6-mediated down-regulation of MALAT1. MG63 cells were treated with or without the indicated concentrations of Myc-6. Twenty-four hours after treatment, the expression level of MALAT1 was analyzed by real-time PCR. p < 0.05 was considered statistically significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. The columns represent means ± SD. (E) Knockdown of MALAT1 suppresses MG63 cell growth. MG63 cells were transfected with control siRNA or with siRNA against MALAT1. The cell viability was assessed by WST-8 assay. ∗∗p < 0.001.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048845&req=5

f0020: MALAT1 is one of possible target genes of Myc-6. (A) Sequence of 5′-upstream region of human MALAT1 gene. The positions relative to the first nucleotide of MALAT1 exon 1 (+1) are indicated. The putative Myc-6-target sequence is underlined, and E-box-like sequence is highlighted in gray. (B and C) Gel retardation assay. FITC-labeled specific (MALAT1) or non-specific (Mismatch) oligonucleotide (1 μM) was incubated with water or Myc-6 (5 μM) for 1 h at 37 °C. The reaction mixtures were separated by 4–20% gradient polyacrylamide gel electrophoresis and then analyzed by LAS4000 (FUJIFILM) (B). FITC-labeled specific oligonucleotide (0.5 μM) was incubated with Myc-6 (0.5 μM) in the presence or absence of the indicated amounts of unlabeled specific (upper) or non-specific (lower) oligonucleotides. The reaction mixtures were analyzed as in (B) (C). (D) Myc-6-mediated down-regulation of MALAT1. MG63 cells were treated with or without the indicated concentrations of Myc-6. Twenty-four hours after treatment, the expression level of MALAT1 was analyzed by real-time PCR. p < 0.05 was considered statistically significant. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. The columns represent means ± SD. (E) Knockdown of MALAT1 suppresses MG63 cell growth. MG63 cells were transfected with control siRNA or with siRNA against MALAT1. The cell viability was assessed by WST-8 assay. ∗∗p < 0.001.
Mentions: To elucidate the precise molecular mechanism(s) behind Myc-6-mediated induction of apoptosis and suppression of tumor growth, we sought to identify a putative Myc-6-target gene(s) by using DNA microarray-based analysis. After the extensive screening, we have finally obtained 14 genes whose expression levels were significantly lower in Myc-6-treated MG63 cells relative to those in untreated cells (Table 1). The close inspection of their 5′-upstream regions (up to 1 kb) revealed that MALAT1, which encodes a long noncoding RNA [21], contains a theoretical Myc-6-target sequence including E-box-like motif (at positions −258 to −251) (Fig. 4A). To verify whether Myc-6 could specifically bind to this sequence, we have performed gel retardation assay. FITC-labeled double-strand oligonucleotide corresponding to the above-mentioned MALAT1 sequence or the unrelated sequence (Mismatch, Supplementary Table 2) was generated, incubated with or without Myc-6 and the reaction mixtures were separated by polyacrylamide gel electrophoresis. Unlike the non-specific oligonucleotide, a definite mobility shift of the specific oligonucleotide was observed in the presence of Myc-6 compared to that in the absence of Myc-6 (Fig. 4B). Furthermore, this mobility shift was clearly inhibited by the excess amount of specific oligonucleotide but not by non-specific one (Fig. 4C), indicating that Myc-6 specifically binds to its theoretical target site located within 5′-upstream region of MALAT1.

Bottom Line: Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors.In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo.Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

View Article: PubMed Central - PubMed

Affiliation: Division of Orthopedic Surgery, Nihon University School of Medicine, 30-1 Oyaguchi Kami-Cho, Itabashi, Tokyo 173-8610, Japan.

ABSTRACT
Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors. In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

No MeSH data available.


Related in: MedlinePlus