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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 inhibit the growth of a miR-372-positive human gastric tumor. (a) TL372+373 (TL) dose-dependent effects on tumor growth: the horizontal bars inside the boxes indicate the median and the capped bars the minimal and the maximal data values (n = 5) of the relative tumor size increase during the 14 days following the first day of injection. (b) Representative images of LATS2 immunostaining in GC10 xenografts treated with either TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse. Scale bars, 25 μm.
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fig5: 8-mer LNAs inhibit the growth of a miR-372-positive human gastric tumor. (a) TL372+373 (TL) dose-dependent effects on tumor growth: the horizontal bars inside the boxes indicate the median and the capped bars the minimal and the maximal data values (n = 5) of the relative tumor size increase during the 14 days following the first day of injection. (b) Representative images of LATS2 immunostaining in GC10 xenografts treated with either TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse. Scale bars, 25 μm.

Mentions: To assess whether the human gastric AGS cell line is relevant of other human gastric carcinoma, we selected two tumors in our collection of resected tumors from gastric cancer patients, to be xenografted in mice and treated with an equimolar TL372+373 mix. GC10 is a highly aggressive, fast growing gastric carcinoma of intestinal type; it expressed both miR-200b, which attests for its epithelial origin, and miR-372 in every epithelial cell, as shown by in situ hybridization (Supplementary Figure S5 left panels). GC06 is a gastric carcinoma of diffuse type that grows slower than GC10; it homogenously expressed miR-200b in every cell, but miR-372 only in some cell clusters (Supplementary Figure S5, right panels). Figure 5a shows that untreated GC10 tumors gain 200% volume in 2 weeks in average, whereas GC06 tumors gain only 60% volume during the same time, suggesting a causal relationship between the high miR-372 expression and rapid tumor growth. Indeed, TL372+373 treatment markedly inhibits GC10 growth at doses as low as 5 nanomoles per mouse. It is inefficient on GC06 (Figure 5a). TL372+373-treated GC10 xenografts exhibit an upregulated LATS2 expression as compared to TLCo-treated ones (Figure 5b).


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 inhibit the growth of a miR-372-positive human gastric tumor. (a) TL372+373 (TL) dose-dependent effects on tumor growth: the horizontal bars inside the boxes indicate the median and the capped bars the minimal and the maximal data values (n = 5) of the relative tumor size increase during the 14 days following the first day of injection. (b) Representative images of LATS2 immunostaining in GC10 xenografts treated with either TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse. Scale bars, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4561653&req=5

fig5: 8-mer LNAs inhibit the growth of a miR-372-positive human gastric tumor. (a) TL372+373 (TL) dose-dependent effects on tumor growth: the horizontal bars inside the boxes indicate the median and the capped bars the minimal and the maximal data values (n = 5) of the relative tumor size increase during the 14 days following the first day of injection. (b) Representative images of LATS2 immunostaining in GC10 xenografts treated with either TLCo or an equimolar TL372+TL373 mix at 5 nanomoles/mouse. Scale bars, 25 μm.
Mentions: To assess whether the human gastric AGS cell line is relevant of other human gastric carcinoma, we selected two tumors in our collection of resected tumors from gastric cancer patients, to be xenografted in mice and treated with an equimolar TL372+373 mix. GC10 is a highly aggressive, fast growing gastric carcinoma of intestinal type; it expressed both miR-200b, which attests for its epithelial origin, and miR-372 in every epithelial cell, as shown by in situ hybridization (Supplementary Figure S5 left panels). GC06 is a gastric carcinoma of diffuse type that grows slower than GC10; it homogenously expressed miR-200b in every cell, but miR-372 only in some cell clusters (Supplementary Figure S5, right panels). Figure 5a shows that untreated GC10 tumors gain 200% volume in 2 weeks in average, whereas GC06 tumors gain only 60% volume during the same time, suggesting a causal relationship between the high miR-372 expression and rapid tumor growth. Indeed, TL372+373 treatment markedly inhibits GC10 growth at doses as low as 5 nanomoles per mouse. It is inefficient on GC06 (Figure 5a). TL372+373-treated GC10 xenografts exhibit an upregulated LATS2 expression as compared to TLCo-treated ones (Figure 5b).

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