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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 miR-372 in AGS cells grown in mice and delay tumor growth. (a) Growth kinetics of AGS tumors either untreated (mock) or treated with TLCo (5 nmol/mouse) or an equimolar TL372+TL373 mix (TL) at 0.5, 5, or 50 nmol/mouse. Tumors were measured the 3rd (T3), the 9th (T9), and the 14th (T14) day following the first injection and their size was expressed in mm3 (mean ± SD, n = 5). (b) Quantification of in vivo luciferase activity at T9 on AGS-PM372 or AGS-mut372 xenografts, either untreated (mock) or treated with TL or TLCo at 5 nanomoles/mouse: bars represent the mean ± SD (n = 5) of radiance (photons/s/cm2/steridian) of the region of interest relative to the background radiance (out of the region of interest). (c) Representative in vivo bioluminescence images of AGS-mut372 xenografts in mice either untreated (A) or TL-treated (B), and AGS-PM372 xenografts either untreated (C), TLCo- (D) or TL-treated (E) as in (B).
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fig3: 8-mer LNAs inhibit miR-372 in AGS cells grown in mice and delay tumor growth. (a) Growth kinetics of AGS tumors either untreated (mock) or treated with TLCo (5 nmol/mouse) or an equimolar TL372+TL373 mix (TL) at 0.5, 5, or 50 nmol/mouse. Tumors were measured the 3rd (T3), the 9th (T9), and the 14th (T14) day following the first injection and their size was expressed in mm3 (mean ± SD, n = 5). (b) Quantification of in vivo luciferase activity at T9 on AGS-PM372 or AGS-mut372 xenografts, either untreated (mock) or treated with TL or TLCo at 5 nanomoles/mouse: bars represent the mean ± SD (n = 5) of radiance (photons/s/cm2/steridian) of the region of interest relative to the background radiance (out of the region of interest). (c) Representative in vivo bioluminescence images of AGS-mut372 xenografts in mice either untreated (A) or TL-treated (B), and AGS-PM372 xenografts either untreated (C), TLCo- (D) or TL-treated (E) as in (B).

Mentions: We then determined whether the 8-mer LNAs could be used to target miR-372 and -373 in solid tumors in vivo and subsequently reduce their growth rate. Therefore, we subcutaneously engrafted the stable reporter cell lines AGS-luc-PM372 or AGS-luc-Mut372 into female NSG mice (n = 5, one tumor per mouse). The xenografts grew at a similar rate independently on the reporter they harbor (AGS-luc-PM372 and AGS-luc-mut372 tumor size: 119.6 mm3 ± 46.2 and 108.6 mm3 ± 61.2, respectively, after 2 weeks, P value = 0.22; 157.6 mm3 ± 42.3 and 150.5 ± 54.7 respectively after 4 weeks, P value = 0.43). Ten days after AGS cell grafting, increasing concentrations of an equimolar TL372+373 mix or TLCo were subcutaneously injected at the tumor periphery for 3 consecutive days. Both the size of the tumor and in vivo bioluminescence images were periodically recorded, starting at the day of the first injection (Figure 3). A noticeable delay was observed in the growth kinetics of the tumors treated with TL372+373 at 5 or 50 nmoles/mouse as compared to untreated or TLCo-treated ones, leading to a significant reduction of tumor size after 2 weeks following the first injection (Figure 3a). Bioluminescence images recorded at day 9 following the first injection show the luciferase activity appearing in the AGS-PM372 tumors upon treatment with TL372+373 but not with TLCo (Figure 3b and Figure 3c, E versus D), whereas in mock conditions luciferase activity of AGS-PM372 tumors remains undetectable as compared to that of AGS-mut372 tumors (Figure 3b and Figure 3c, C versus A). These data indicate that the injected TL372+373 may have reached the tumor cell cytoplasm to inhibit the function of the endogenous miR-372 and miR-373 levels. The TL372+373-mediated upregulation of the luciferase activity in the AGS-PM372 tumors likely results from the combined effects on de-repression of the luciferase reporter sensing miR-372, on the one side, and reduction of the tumor size, on the other side. These combined effects could explain why the luciferase activity recorded in TL372+373-treated mice bearing the AGS-Mut372 xenografts is lower than that of the untreated ones (Figure 3b and Figure 3c, B versus A).


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 miR-372 in AGS cells grown in mice and delay tumor growth. (a) Growth kinetics of AGS tumors either untreated (mock) or treated with TLCo (5 nmol/mouse) or an equimolar TL372+TL373 mix (TL) at 0.5, 5, or 50 nmol/mouse. Tumors were measured the 3rd (T3), the 9th (T9), and the 14th (T14) day following the first injection and their size was expressed in mm3 (mean ± SD, n = 5). (b) Quantification of in vivo luciferase activity at T9 on AGS-PM372 or AGS-mut372 xenografts, either untreated (mock) or treated with TL or TLCo at 5 nanomoles/mouse: bars represent the mean ± SD (n = 5) of radiance (photons/s/cm2/steridian) of the region of interest relative to the background radiance (out of the region of interest). (c) Representative in vivo bioluminescence images of AGS-mut372 xenografts in mice either untreated (A) or TL-treated (B), and AGS-PM372 xenografts either untreated (C), TLCo- (D) or TL-treated (E) as in (B).
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Related In: Results  -  Collection

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fig3: 8-mer LNAs inhibit miR-372 in AGS cells grown in mice and delay tumor growth. (a) Growth kinetics of AGS tumors either untreated (mock) or treated with TLCo (5 nmol/mouse) or an equimolar TL372+TL373 mix (TL) at 0.5, 5, or 50 nmol/mouse. Tumors were measured the 3rd (T3), the 9th (T9), and the 14th (T14) day following the first injection and their size was expressed in mm3 (mean ± SD, n = 5). (b) Quantification of in vivo luciferase activity at T9 on AGS-PM372 or AGS-mut372 xenografts, either untreated (mock) or treated with TL or TLCo at 5 nanomoles/mouse: bars represent the mean ± SD (n = 5) of radiance (photons/s/cm2/steridian) of the region of interest relative to the background radiance (out of the region of interest). (c) Representative in vivo bioluminescence images of AGS-mut372 xenografts in mice either untreated (A) or TL-treated (B), and AGS-PM372 xenografts either untreated (C), TLCo- (D) or TL-treated (E) as in (B).
Mentions: We then determined whether the 8-mer LNAs could be used to target miR-372 and -373 in solid tumors in vivo and subsequently reduce their growth rate. Therefore, we subcutaneously engrafted the stable reporter cell lines AGS-luc-PM372 or AGS-luc-Mut372 into female NSG mice (n = 5, one tumor per mouse). The xenografts grew at a similar rate independently on the reporter they harbor (AGS-luc-PM372 and AGS-luc-mut372 tumor size: 119.6 mm3 ± 46.2 and 108.6 mm3 ± 61.2, respectively, after 2 weeks, P value = 0.22; 157.6 mm3 ± 42.3 and 150.5 ± 54.7 respectively after 4 weeks, P value = 0.43). Ten days after AGS cell grafting, increasing concentrations of an equimolar TL372+373 mix or TLCo were subcutaneously injected at the tumor periphery for 3 consecutive days. Both the size of the tumor and in vivo bioluminescence images were periodically recorded, starting at the day of the first injection (Figure 3). A noticeable delay was observed in the growth kinetics of the tumors treated with TL372+373 at 5 or 50 nmoles/mouse as compared to untreated or TLCo-treated ones, leading to a significant reduction of tumor size after 2 weeks following the first injection (Figure 3a). Bioluminescence images recorded at day 9 following the first injection show the luciferase activity appearing in the AGS-PM372 tumors upon treatment with TL372+373 but not with TLCo (Figure 3b and Figure 3c, E versus D), whereas in mock conditions luciferase activity of AGS-PM372 tumors remains undetectable as compared to that of AGS-mut372 tumors (Figure 3b and Figure 3c, C versus A). These data indicate that the injected TL372+373 may have reached the tumor cell cytoplasm to inhibit the function of the endogenous miR-372 and miR-373 levels. The TL372+373-mediated upregulation of the luciferase activity in the AGS-PM372 tumors likely results from the combined effects on de-repression of the luciferase reporter sensing miR-372, on the one side, and reduction of the tumor size, on the other side. These combined effects could explain why the luciferase activity recorded in TL372+373-treated mice bearing the AGS-Mut372 xenografts is lower than that of the untreated ones (Figure 3b and Figure 3c, B versus A).

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