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Argonaute 2 in cell-secreted microvesicles guides the function of secreted miRNAs in recipient cells.

Lv Z, Wei Y, Wang D, Zhang CY, Zen K, Li L - PLoS ONE (2014)

Bottom Line: More importantly, Ago2 in origin cell-secreted MVs (but not in recipient cells) directs the function of secreted miRNAs.Third, exogenous miR-16 delivered by MVs within the origin cells significantly reduced the Bcl2 protein level in recipient cells, and miR-16 and Bcl2 mRNA were physically associated with exogenous HA-tagged Ago2 (HA-Ago2).Finally, the effect of MV-delivered miR-16 on the production of the Bcl2 protein in recipient cells was not abolished by knocking down Ago2 in the recipient cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
MicroRNAs (miRNAs) secreted by cells into microvesicles (MVs) form a novel class of signal molecules that mediate intercellular communication. However, several fundamental aspects of secreted miRNAs remain unknown, particularly the mechanism that governs the function or fate of exogenous miRNAs in recipient cells. In the present study, we provide evidence indicating that Argonaute 2 (Ago2) plays a role in stabilizing miRNAs and facilitating the packaging of secreted miRNAs into MVs. More importantly, Ago2 in origin cell-secreted MVs (but not in recipient cells) directs the function of secreted miRNAs. First, Ago2 overexpression clearly increased the level of miR-16 in cells transfected with a miR-16 mimic by protecting the miRNAs from degradation in lysosomes. Second, Ago2 overexpression increased the level of miR-16 in cell-secreted MVs, suggesting that Ago2 may facilitate the packaging of secreted miRNAs into MVs. Third, exogenous miR-16 delivered by MVs within the origin cells significantly reduced the Bcl2 protein level in recipient cells, and miR-16 and Bcl2 mRNA were physically associated with exogenous HA-tagged Ago2 (HA-Ago2). Finally, the effect of MV-delivered miR-16 on the production of the Bcl2 protein in recipient cells was not abolished by knocking down Ago2 in the recipient cells.

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The function of secreted miR-16 in recipient cells does not require endogenous Ago2 from the recipient cells.A, Knockdown of Ago2 in 293T cells via Ago2 siRNA. 293T cells were incubated with MVs secreted by HeLa cells transfected with miR-16 or co-transfected with the miR-16 mimic and HA-Ago2 plasmid. B, Level of Bcl2 protein in 293T cells detected using Western blot analysis. *, P<0.05; **, P<0.01.
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pone-0103599-g004: The function of secreted miR-16 in recipient cells does not require endogenous Ago2 from the recipient cells.A, Knockdown of Ago2 in 293T cells via Ago2 siRNA. 293T cells were incubated with MVs secreted by HeLa cells transfected with miR-16 or co-transfected with the miR-16 mimic and HA-Ago2 plasmid. B, Level of Bcl2 protein in 293T cells detected using Western blot analysis. *, P<0.05; **, P<0.01.

Mentions: Given that exogenous HA-Ago2 can guide the function of its associated miRNA in recipient cells, we then asked whether the function of secreted miR-16 in recipient cells requires endogenous Ago2 from the recipient cells. In this experiment, we knocked down Ago2 in 293T cells via Ago2 siRNA and then incubated the cells with MVs derived from HeLa cells that had been co-transfected with the miR-16 mimic and HA-Ago2 plasmid. As shown in Figure 4A, the endogenous Ago2 level was reduced by more than half by the Ago2 siRNA treatment. However, the reduction of Bcl2 in 293T cells by MV-encapsulated miR-16 was not affected (Figure 4B). It is important that incubation with MVs secreted by miR-16 and HA-Ago2 co-transfected HeLa cells with higher capacity to inhibit the expression of Bcl2 in recipient 293T cells compared to incubation with MVs secreted by miR-16 transfected cells. This result supported our assumption that the function of secreted miR-16 in recipient cells did not require endogenous Ago2 from the recipient cells.


Argonaute 2 in cell-secreted microvesicles guides the function of secreted miRNAs in recipient cells.

Lv Z, Wei Y, Wang D, Zhang CY, Zen K, Li L - PLoS ONE (2014)

The function of secreted miR-16 in recipient cells does not require endogenous Ago2 from the recipient cells.A, Knockdown of Ago2 in 293T cells via Ago2 siRNA. 293T cells were incubated with MVs secreted by HeLa cells transfected with miR-16 or co-transfected with the miR-16 mimic and HA-Ago2 plasmid. B, Level of Bcl2 protein in 293T cells detected using Western blot analysis. *, P<0.05; **, P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4114802&req=5

pone-0103599-g004: The function of secreted miR-16 in recipient cells does not require endogenous Ago2 from the recipient cells.A, Knockdown of Ago2 in 293T cells via Ago2 siRNA. 293T cells were incubated with MVs secreted by HeLa cells transfected with miR-16 or co-transfected with the miR-16 mimic and HA-Ago2 plasmid. B, Level of Bcl2 protein in 293T cells detected using Western blot analysis. *, P<0.05; **, P<0.01.
Mentions: Given that exogenous HA-Ago2 can guide the function of its associated miRNA in recipient cells, we then asked whether the function of secreted miR-16 in recipient cells requires endogenous Ago2 from the recipient cells. In this experiment, we knocked down Ago2 in 293T cells via Ago2 siRNA and then incubated the cells with MVs derived from HeLa cells that had been co-transfected with the miR-16 mimic and HA-Ago2 plasmid. As shown in Figure 4A, the endogenous Ago2 level was reduced by more than half by the Ago2 siRNA treatment. However, the reduction of Bcl2 in 293T cells by MV-encapsulated miR-16 was not affected (Figure 4B). It is important that incubation with MVs secreted by miR-16 and HA-Ago2 co-transfected HeLa cells with higher capacity to inhibit the expression of Bcl2 in recipient 293T cells compared to incubation with MVs secreted by miR-16 transfected cells. This result supported our assumption that the function of secreted miR-16 in recipient cells did not require endogenous Ago2 from the recipient cells.

Bottom Line: More importantly, Ago2 in origin cell-secreted MVs (but not in recipient cells) directs the function of secreted miRNAs.Third, exogenous miR-16 delivered by MVs within the origin cells significantly reduced the Bcl2 protein level in recipient cells, and miR-16 and Bcl2 mRNA were physically associated with exogenous HA-tagged Ago2 (HA-Ago2).Finally, the effect of MV-delivered miR-16 on the production of the Bcl2 protein in recipient cells was not abolished by knocking down Ago2 in the recipient cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
MicroRNAs (miRNAs) secreted by cells into microvesicles (MVs) form a novel class of signal molecules that mediate intercellular communication. However, several fundamental aspects of secreted miRNAs remain unknown, particularly the mechanism that governs the function or fate of exogenous miRNAs in recipient cells. In the present study, we provide evidence indicating that Argonaute 2 (Ago2) plays a role in stabilizing miRNAs and facilitating the packaging of secreted miRNAs into MVs. More importantly, Ago2 in origin cell-secreted MVs (but not in recipient cells) directs the function of secreted miRNAs. First, Ago2 overexpression clearly increased the level of miR-16 in cells transfected with a miR-16 mimic by protecting the miRNAs from degradation in lysosomes. Second, Ago2 overexpression increased the level of miR-16 in cell-secreted MVs, suggesting that Ago2 may facilitate the packaging of secreted miRNAs into MVs. Third, exogenous miR-16 delivered by MVs within the origin cells significantly reduced the Bcl2 protein level in recipient cells, and miR-16 and Bcl2 mRNA were physically associated with exogenous HA-tagged Ago2 (HA-Ago2). Finally, the effect of MV-delivered miR-16 on the production of the Bcl2 protein in recipient cells was not abolished by knocking down Ago2 in the recipient cells.

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