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Inhibition of Gastric Tumor Cell Growth Using Seed-targeting LNA as Specific, Long-lasting MicroRNA Inhibitors.

Staedel C, Varon C, Nguyen PH, Vialet B, Chambonnier L, Rousseau B, Soubeyran I, Evrard S, Couillaud F, Darfeuille F - Mol Ther Nucleic Acids (2015)

Bottom Line: They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations.Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation.Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: 1] University of Bordeaux, ARNA Laboratory, Bordeaux, France [2] INSERM, U869, Bordeaux, France.

ABSTRACT
MicroRNAs regulate eukaryotic gene expression upon pairing onto target mRNAs. This targeting is influenced by the complementarity between the microRNA "seed" sequence at its 5' end and the seed-matching sequences in the mRNA. Here, we assess the efficiency and specificity of 8-mer locked nucleic acid (LNA)-modified oligonucleotides raised against the seeds of miR-372 and miR-373, two embryonic stem cell-specific microRNAs prominently expressed in the human gastric adenocarcinoma AGS cell line. Provided that the pairing is perfect over all the eight nucleotides of the seed and starts at nucleotide 2 or 1 at the microRNA 5' end, these short LNAs inhibit miR-372/373 functions and derepress their common target, the cell cycle regulator LATS2. They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations. Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation. Their therapeutic potential is confirmed in fast-growing, miR-372-positive, primary human gastric adenocarcinoma xenografts in mice. Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies.

No MeSH data available.


Related in: MedlinePlus

8-mer LNAs stably sequester miR-372 in AGS xenografts and derepress LATS2. (a) Nondenaturing northern blot analysis of miR-372 (A,B) and miR-21 (C,D) in AGS-PM372 tumors treated with TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse (A,C) or with increasing doses (B,D). (b) Representative images of LATS2 immunostaining in AGS -PM372 tumors treated with either TLCo or TL372+373 at 5 nanomoles/mouse. Scale bars, 25 μm.
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fig4: 8-mer LNAs stably sequester miR-372 in AGS xenografts and derepress LATS2. (a) Nondenaturing northern blot analysis of miR-372 (A,B) and miR-21 (C,D) in AGS-PM372 tumors treated with TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse (A,C) or with increasing doses (B,D). (b) Representative images of LATS2 immunostaining in AGS -PM372 tumors treated with either TLCo or TL372+373 at 5 nanomoles/mouse. Scale bars, 25 μm.

Mentions: Two weeks post-treatment, the mice were euthanized. The residual tumors were removed and processed for further RNA analyses and histology. The northern blot analysis of miR-372 clearly shows a slower-migrating miR-372/TL372 heteroduplex in TL372+373-treated AGS xenografts, compared to TLCo or untreated tumors (Figure 4a, A). A similar band shift is observed on the northern blot analyzed for miR-373 expression in TL372+373-treated tumors and corresponds to the miR-373/TL373 heteroduplex (Supplementary Figure S4). The heteroduplex is formed in a TL372+373 dose-dependent manner at doses over 5 nanomoles/mouse (Figure 4a, B). On the contrary, the northern blot analysis of miR-21 does not show any band shift in the TL372+373-treated cells (Figure 4a, C and D), further stressing the specificity of miRNA targeting by 8-mer LNAs. Along with TL372+373-mediated miR-372 and miR-373 sequestrations and tumor growth inhibition, LATS2 is upregulated upon TL372+373 treatment, as compared to TLCo treatment, and accumulates in the carcinoma cell nucleus and cytoplasm (Figure 4b).


Inhibition of Gastric Tumor Cell Growth Using Seed-targeting LNA as Specific, Long-lasting MicroRNA Inhibitors.

Staedel C, Varon C, Nguyen PH, Vialet B, Chambonnier L, Rousseau B, Soubeyran I, Evrard S, Couillaud F, Darfeuille F - Mol Ther Nucleic Acids (2015)

8-mer LNAs stably sequester miR-372 in AGS xenografts and derepress LATS2. (a) Nondenaturing northern blot analysis of miR-372 (A,B) and miR-21 (C,D) in AGS-PM372 tumors treated with TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse (A,C) or with increasing doses (B,D). (b) Representative images of LATS2 immunostaining in AGS -PM372 tumors treated with either TLCo or TL372+373 at 5 nanomoles/mouse. Scale bars, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: 8-mer LNAs stably sequester miR-372 in AGS xenografts and derepress LATS2. (a) Nondenaturing northern blot analysis of miR-372 (A,B) and miR-21 (C,D) in AGS-PM372 tumors treated with TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse (A,C) or with increasing doses (B,D). (b) Representative images of LATS2 immunostaining in AGS -PM372 tumors treated with either TLCo or TL372+373 at 5 nanomoles/mouse. Scale bars, 25 μm.
Mentions: Two weeks post-treatment, the mice were euthanized. The residual tumors were removed and processed for further RNA analyses and histology. The northern blot analysis of miR-372 clearly shows a slower-migrating miR-372/TL372 heteroduplex in TL372+373-treated AGS xenografts, compared to TLCo or untreated tumors (Figure 4a, A). A similar band shift is observed on the northern blot analyzed for miR-373 expression in TL372+373-treated tumors and corresponds to the miR-373/TL373 heteroduplex (Supplementary Figure S4). The heteroduplex is formed in a TL372+373 dose-dependent manner at doses over 5 nanomoles/mouse (Figure 4a, B). On the contrary, the northern blot analysis of miR-21 does not show any band shift in the TL372+373-treated cells (Figure 4a, C and D), further stressing the specificity of miRNA targeting by 8-mer LNAs. Along with TL372+373-mediated miR-372 and miR-373 sequestrations and tumor growth inhibition, LATS2 is upregulated upon TL372+373 treatment, as compared to TLCo treatment, and accumulates in the carcinoma cell nucleus and cytoplasm (Figure 4b).

Bottom Line: They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations.Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation.Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: 1] University of Bordeaux, ARNA Laboratory, Bordeaux, France [2] INSERM, U869, Bordeaux, France.

ABSTRACT
MicroRNAs regulate eukaryotic gene expression upon pairing onto target mRNAs. This targeting is influenced by the complementarity between the microRNA "seed" sequence at its 5' end and the seed-matching sequences in the mRNA. Here, we assess the efficiency and specificity of 8-mer locked nucleic acid (LNA)-modified oligonucleotides raised against the seeds of miR-372 and miR-373, two embryonic stem cell-specific microRNAs prominently expressed in the human gastric adenocarcinoma AGS cell line. Provided that the pairing is perfect over all the eight nucleotides of the seed and starts at nucleotide 2 or 1 at the microRNA 5' end, these short LNAs inhibit miR-372/373 functions and derepress their common target, the cell cycle regulator LATS2. They decrease cell proliferation in vitro upon either transfection at nanomolar concentrations or unassisted delivery at micromolar concentrations. Subcutaneously delivered LNAs reduce tumor growth of AGS xenografts in mice, upon formation of a stable, specific heteroduplex with the targeted miR-372 and -373 and LATS2 upregulation. Their therapeutic potential is confirmed in fast-growing, miR-372-positive, primary human gastric adenocarcinoma xenografts in mice. Thus, microRNA silencing by 8-mer seed-targeting LNAs appears a valuable approach for both loss-of-function studies aimed at elucidating microRNA functions and for microRNA-based therapeutic strategies.

No MeSH data available.


Related in: MedlinePlus