Limits...
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 against miR-372 and miR-373 de-repress LATS2 and inhibit the growth of cultured AGS cells. (a) Relative luciferase activity (mean ± SD, n = 3) of the LATS2 reporter system pGL3-LATS2wt containing the wt LATS2 3'UTR, or of the mutated LATS2 reporter system pGL3-LATS2mut, compared to that of the control vector pGL3. The vectors have been cotransfected with pRL-SV40 and 10 nmol/l of the indicated oligonucleotides using lipofectamin, and their expression was analyzed 48 hours later. (b) Western blot analysis of LATS2 (upper panel) and α-tubulin (lower panel) protein expression in untreated MKN74 cells (positive control) or in AGS cells treated with an equimolar mix of either 22-mer anti-miR-372+miR-373 antisenses (AS) or their scrambled sequence (SC), or with 8-mer LNA against miR-372 and miR-373 (TL) or their negative control (TLCo). (c) Relative growth rate (mean ± SD, n = 6) of tiny LNA-transfected cultured AGS cells between day 2 and day 5 post-treatment, compared to TLCo-treated cells. (d) LATS2 immunofluorescent staining in AGS cells in the same conditions than in (b); scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: 8-mer LNAs against miR-372 and miR-373 de-repress LATS2 and inhibit the growth of cultured AGS cells. (a) Relative luciferase activity (mean ± SD, n = 3) of the LATS2 reporter system pGL3-LATS2wt containing the wt LATS2 3'UTR, or of the mutated LATS2 reporter system pGL3-LATS2mut, compared to that of the control vector pGL3. The vectors have been cotransfected with pRL-SV40 and 10 nmol/l of the indicated oligonucleotides using lipofectamin, and their expression was analyzed 48 hours later. (b) Western blot analysis of LATS2 (upper panel) and α-tubulin (lower panel) protein expression in untreated MKN74 cells (positive control) or in AGS cells treated with an equimolar mix of either 22-mer anti-miR-372+miR-373 antisenses (AS) or their scrambled sequence (SC), or with 8-mer LNA against miR-372 and miR-373 (TL) or their negative control (TLCo). (c) Relative growth rate (mean ± SD, n = 6) of tiny LNA-transfected cultured AGS cells between day 2 and day 5 post-treatment, compared to TLCo-treated cells. (d) LATS2 immunofluorescent staining in AGS cells in the same conditions than in (b); scale bars, 10 μm.

Mentions: LATS2 is one of the major targets of both miR-372 and miR-373, and contains two miR-372 and -373 pairing sites in its 3′UTR11. It has been validated in other cancer cells.15,17 To compare the efficiency of the 8-mer LNA and full-length AS against miR-372 and miR-373 to derepress LATS2, the luciferase reporter plasmid containing the 3′UTR of LATS2, named pGL3-3'UTR-LATS2wt, was transiently transfected into AGS cells along with 8-mer LNAs or 22-mer AS. The resulting luciferase activity was compared to that of either the control pGL3 vector or the pGL3-3'UTR-LATS2mut vector harboring mutated miR-372 and -373 pairing sites. In the absence of oligonucleotides, the luciferase activity generated by pGL3-3′UTR-LATS2wt, which senses the endogenous miR-372 and miR-373 levels, represented 30% of that of the control pGL3 reporter and 37% of that of the pGL3-3′UTR-LATS2mut vector (Figure 2a). In the presence of an equimolar mix of TL372+TL373, but not TLCo or SC372, the luciferase activity of the pGL3-3'UTR-LATS2wt increased in the same extend that in the presence of an equimolar mix of AS372+AS373 (Figure 2a). The TL372+373-derepressed luciferase activity reached the level of that generated by the pGL3-3′UTR-LATS2mut. This indicates that the 8-mer LNAs are as efficient as the AS372+373 to inhibit miR-372 and -373 functions and derepress LATS2. In consequence, LATS2 protein accumulates in cells treated by either 8-mer or full-length antisenses against miR-372 and miR-373, but not in cells treated by the respective control oligonucleotides, in which LATS2 expression pattern remained as faint as in untreated cells (Figure 2b,d). The derepressed LATS2 mainly accumulates in the cell nucleus upon treatment with either 22-mer AS or 8-mer LNAs (Figure 2d). TL372+373-transfected AGS cells exhibit a noticeably reduced proliferation rate, which is slowed down by 50 or 40% compared to those of untransfected or TLCo-transfected cells, respectively (Figure 2c). TLCo-transfected cells grow slightly slower as compared to untransfected cells: this effect could be attributed either to the transfection procedure using Lipofactamin or to some noxious properties of the LNA/phosphorothiate chemistry of oligonucleotides, which may have reached unspecific targets as well through their strong binding capacities, leading to slight off-target harmfulness. Of note, TL21, which totally sequesters miR-21, another oncogenic miRNA that is also highly expressed in AGS cells (Supplementary Figure S2),11 has no more effect on AGS proliferation rate that TLCo, suggesting that this cell line may not be particularly dependent on miR-21 for its growth. All together, these results show that 8-mer LNAs, inhibiting miR-372 and -373 functions and derepressing LATS2, inhibit AGS cell growth. Their effects are similar as those of the 22-mer AS, as reported in our previous data.11


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 against miR-372 and miR-373 de-repress LATS2 and inhibit the growth of cultured AGS cells. (a) Relative luciferase activity (mean ± SD, n = 3) of the LATS2 reporter system pGL3-LATS2wt containing the wt LATS2 3'UTR, or of the mutated LATS2 reporter system pGL3-LATS2mut, compared to that of the control vector pGL3. The vectors have been cotransfected with pRL-SV40 and 10 nmol/l of the indicated oligonucleotides using lipofectamin, and their expression was analyzed 48 hours later. (b) Western blot analysis of LATS2 (upper panel) and α-tubulin (lower panel) protein expression in untreated MKN74 cells (positive control) or in AGS cells treated with an equimolar mix of either 22-mer anti-miR-372+miR-373 antisenses (AS) or their scrambled sequence (SC), or with 8-mer LNA against miR-372 and miR-373 (TL) or their negative control (TLCo). (c) Relative growth rate (mean ± SD, n = 6) of tiny LNA-transfected cultured AGS cells between day 2 and day 5 post-treatment, compared to TLCo-treated cells. (d) LATS2 immunofluorescent staining in AGS cells in the same conditions than in (b); scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: 8-mer LNAs against miR-372 and miR-373 de-repress LATS2 and inhibit the growth of cultured AGS cells. (a) Relative luciferase activity (mean ± SD, n = 3) of the LATS2 reporter system pGL3-LATS2wt containing the wt LATS2 3'UTR, or of the mutated LATS2 reporter system pGL3-LATS2mut, compared to that of the control vector pGL3. The vectors have been cotransfected with pRL-SV40 and 10 nmol/l of the indicated oligonucleotides using lipofectamin, and their expression was analyzed 48 hours later. (b) Western blot analysis of LATS2 (upper panel) and α-tubulin (lower panel) protein expression in untreated MKN74 cells (positive control) or in AGS cells treated with an equimolar mix of either 22-mer anti-miR-372+miR-373 antisenses (AS) or their scrambled sequence (SC), or with 8-mer LNA against miR-372 and miR-373 (TL) or their negative control (TLCo). (c) Relative growth rate (mean ± SD, n = 6) of tiny LNA-transfected cultured AGS cells between day 2 and day 5 post-treatment, compared to TLCo-treated cells. (d) LATS2 immunofluorescent staining in AGS cells in the same conditions than in (b); scale bars, 10 μm.
Mentions: LATS2 is one of the major targets of both miR-372 and miR-373, and contains two miR-372 and -373 pairing sites in its 3′UTR11. It has been validated in other cancer cells.15,17 To compare the efficiency of the 8-mer LNA and full-length AS against miR-372 and miR-373 to derepress LATS2, the luciferase reporter plasmid containing the 3′UTR of LATS2, named pGL3-3'UTR-LATS2wt, was transiently transfected into AGS cells along with 8-mer LNAs or 22-mer AS. The resulting luciferase activity was compared to that of either the control pGL3 vector or the pGL3-3'UTR-LATS2mut vector harboring mutated miR-372 and -373 pairing sites. In the absence of oligonucleotides, the luciferase activity generated by pGL3-3′UTR-LATS2wt, which senses the endogenous miR-372 and miR-373 levels, represented 30% of that of the control pGL3 reporter and 37% of that of the pGL3-3′UTR-LATS2mut vector (Figure 2a). In the presence of an equimolar mix of TL372+TL373, but not TLCo or SC372, the luciferase activity of the pGL3-3'UTR-LATS2wt increased in the same extend that in the presence of an equimolar mix of AS372+AS373 (Figure 2a). The TL372+373-derepressed luciferase activity reached the level of that generated by the pGL3-3′UTR-LATS2mut. This indicates that the 8-mer LNAs are as efficient as the AS372+373 to inhibit miR-372 and -373 functions and derepress LATS2. In consequence, LATS2 protein accumulates in cells treated by either 8-mer or full-length antisenses against miR-372 and miR-373, but not in cells treated by the respective control oligonucleotides, in which LATS2 expression pattern remained as faint as in untreated cells (Figure 2b,d). The derepressed LATS2 mainly accumulates in the cell nucleus upon treatment with either 22-mer AS or 8-mer LNAs (Figure 2d). TL372+373-transfected AGS cells exhibit a noticeably reduced proliferation rate, which is slowed down by 50 or 40% compared to those of untransfected or TLCo-transfected cells, respectively (Figure 2c). TLCo-transfected cells grow slightly slower as compared to untransfected cells: this effect could be attributed either to the transfection procedure using Lipofactamin or to some noxious properties of the LNA/phosphorothiate chemistry of oligonucleotides, which may have reached unspecific targets as well through their strong binding capacities, leading to slight off-target harmfulness. Of note, TL21, which totally sequesters miR-21, another oncogenic miRNA that is also highly expressed in AGS cells (Supplementary Figure S2),11 has no more effect on AGS proliferation rate that TLCo, suggesting that this cell line may not be particularly dependent on miR-21 for its growth. All together, these results show that 8-mer LNAs, inhibiting miR-372 and -373 functions and derepressing LATS2, inhibit AGS cell growth. Their effects are similar as those of the 22-mer AS, as reported in our previous data.11

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