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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

Specificity of miRNA targeting by 22-mer full-length antisense or 8-mer seed-directed oligonucleotides in cultured AGS cells. AGS cells were transfected with either the 22-mer full-length anti-miR-372 LNA/DNA antisense (AS372) or its scrambled sequence (SC372), or with the 8-mer LNA oligonucleotide targeting the nu 2–9 of miR-372 including the seed (TL372), or its negative control sequence (TLCo), at a final concentration of 10 nmol/l in the presence of Lipofectamine 2000. Cells were grown for 5 days. (a) RT-qPCR analysis of miR-372 levels; the bars represent the miR-372 levels normalized with snor25 and U6 and compared to mock treated cells (mean ± SD, n = 4). (b) Northern blot analysis of miR-372 (medium panel) and U6 (upper panel) in nondenaturing conditions; the lower panel schematizes miR-372 paired or not with the specific antimiR for each lane. (c) Nondenaturing northern blot analyses of miR-372, miR-373, miR-17-5p, and miR-93 in AGS cells transfected with either TL372, TL373, TL17, TL21, or TLCo at 10 nmol/l.
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fig1: Specificity of miRNA targeting by 22-mer full-length antisense or 8-mer seed-directed oligonucleotides in cultured AGS cells. AGS cells were transfected with either the 22-mer full-length anti-miR-372 LNA/DNA antisense (AS372) or its scrambled sequence (SC372), or with the 8-mer LNA oligonucleotide targeting the nu 2–9 of miR-372 including the seed (TL372), or its negative control sequence (TLCo), at a final concentration of 10 nmol/l in the presence of Lipofectamine 2000. Cells were grown for 5 days. (a) RT-qPCR analysis of miR-372 levels; the bars represent the miR-372 levels normalized with snor25 and U6 and compared to mock treated cells (mean ± SD, n = 4). (b) Northern blot analysis of miR-372 (medium panel) and U6 (upper panel) in nondenaturing conditions; the lower panel schematizes miR-372 paired or not with the specific antimiR for each lane. (c) Nondenaturing northern blot analyses of miR-372, miR-373, miR-17-5p, and miR-93 in AGS cells transfected with either TL372, TL373, TL17, TL21, or TLCo at 10 nmol/l.

Mentions: We previously reported the efficiency of full-length antisense oligonucleotides to miR-372 and miR-373 (AS372, AS373) to inhibit the functions of these miRNAs in AGS cells.11 To assess whether miR-372 seed-targeting LNAs (TL372) are as efficient as AS372 in antagonizing miR-372, 8-mer TL372, 22-mer AS372 or their respective controls TLCo and SC372 were transfected into growing AGS cells at final concentration of 10 nmol/l and miR-372 expression was evaluated by RT-qPCR and nondenaturating northern blot analyses. Upon AS372 transfection, the miR-372 level is drastically reduced as determined by RT-qPCR, and becomes undetectable by northern blot analyses, compared to untransfected cells or SC372-transfected ones (Figure 1a,b, respectively), in agreement with our previous data.11 Similar results were obtained by determining miR-373 content upon AS373 transfection (data not shown). However, the RT-qPCR data suggest that TL372 does not reduce the miR-372 content as efficiently as AS372 (Figure 1a). Northern blot analysis of miR-372 reveals a slower-migrating band in TL372-transfected cells, as compared to TLCo-, SC-transfected or untransfected cells (Figure 1b), likely being the miR-372/TL372 heteroduplex: indeed, anti-miRNA LNAs may sequester their targeted miRNA without degrading it.6 There is still room left in the miR-372/TL372 heteroduplex for the anti-miR-372 probe or the RT-qPCR primers to pair onto the miR-372 sequence and to make it detectable both by northern blot or RT-qPCR analyses (lower part of Figure 1b). On the contrary, in AS372-transfected cells, miR-372 remains sequestered on its full length by the specific 22-mer antisense oligonucleotide, and hence unable to bind either the probe or the primers: this makes it undetectable either on the northern blot or by RT-qPCR. Thus, using anti-miRNA 8-mer LNAs, quantification of residual miRNA levels by RT-qPCR may be misleading.


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)

Specificity of miRNA targeting by 22-mer full-length antisense or 8-mer seed-directed oligonucleotides in cultured AGS cells. AGS cells were transfected with either the 22-mer full-length anti-miR-372 LNA/DNA antisense (AS372) or its scrambled sequence (SC372), or with the 8-mer LNA oligonucleotide targeting the nu 2–9 of miR-372 including the seed (TL372), or its negative control sequence (TLCo), at a final concentration of 10 nmol/l in the presence of Lipofectamine 2000. Cells were grown for 5 days. (a) RT-qPCR analysis of miR-372 levels; the bars represent the miR-372 levels normalized with snor25 and U6 and compared to mock treated cells (mean ± SD, n = 4). (b) Northern blot analysis of miR-372 (medium panel) and U6 (upper panel) in nondenaturing conditions; the lower panel schematizes miR-372 paired or not with the specific antimiR for each lane. (c) Nondenaturing northern blot analyses of miR-372, miR-373, miR-17-5p, and miR-93 in AGS cells transfected with either TL372, TL373, TL17, TL21, or TLCo at 10 nmol/l.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4561653&req=5

fig1: Specificity of miRNA targeting by 22-mer full-length antisense or 8-mer seed-directed oligonucleotides in cultured AGS cells. AGS cells were transfected with either the 22-mer full-length anti-miR-372 LNA/DNA antisense (AS372) or its scrambled sequence (SC372), or with the 8-mer LNA oligonucleotide targeting the nu 2–9 of miR-372 including the seed (TL372), or its negative control sequence (TLCo), at a final concentration of 10 nmol/l in the presence of Lipofectamine 2000. Cells were grown for 5 days. (a) RT-qPCR analysis of miR-372 levels; the bars represent the miR-372 levels normalized with snor25 and U6 and compared to mock treated cells (mean ± SD, n = 4). (b) Northern blot analysis of miR-372 (medium panel) and U6 (upper panel) in nondenaturing conditions; the lower panel schematizes miR-372 paired or not with the specific antimiR for each lane. (c) Nondenaturing northern blot analyses of miR-372, miR-373, miR-17-5p, and miR-93 in AGS cells transfected with either TL372, TL373, TL17, TL21, or TLCo at 10 nmol/l.
Mentions: We previously reported the efficiency of full-length antisense oligonucleotides to miR-372 and miR-373 (AS372, AS373) to inhibit the functions of these miRNAs in AGS cells.11 To assess whether miR-372 seed-targeting LNAs (TL372) are as efficient as AS372 in antagonizing miR-372, 8-mer TL372, 22-mer AS372 or their respective controls TLCo and SC372 were transfected into growing AGS cells at final concentration of 10 nmol/l and miR-372 expression was evaluated by RT-qPCR and nondenaturating northern blot analyses. Upon AS372 transfection, the miR-372 level is drastically reduced as determined by RT-qPCR, and becomes undetectable by northern blot analyses, compared to untransfected cells or SC372-transfected ones (Figure 1a,b, respectively), in agreement with our previous data.11 Similar results were obtained by determining miR-373 content upon AS373 transfection (data not shown). However, the RT-qPCR data suggest that TL372 does not reduce the miR-372 content as efficiently as AS372 (Figure 1a). Northern blot analysis of miR-372 reveals a slower-migrating band in TL372-transfected cells, as compared to TLCo-, SC-transfected or untransfected cells (Figure 1b), likely being the miR-372/TL372 heteroduplex: indeed, anti-miRNA LNAs may sequester their targeted miRNA without degrading it.6 There is still room left in the miR-372/TL372 heteroduplex for the anti-miR-372 probe or the RT-qPCR primers to pair onto the miR-372 sequence and to make it detectable both by northern blot or RT-qPCR analyses (lower part of Figure 1b). On the contrary, in AS372-transfected cells, miR-372 remains sequestered on its full length by the specific 22-mer antisense oligonucleotide, and hence unable to bind either the probe or the primers: this makes it undetectable either on the northern blot or by RT-qPCR. Thus, using anti-miRNA 8-mer LNAs, quantification of residual miRNA levels by RT-qPCR may be misleading.

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