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MiR-7 triggers cell cycle arrest at the G1/S transition by targeting multiple genes including Skp2 and Psme3.

Sanchez N, Gallagher M, Lao N, Gallagher C, Clarke C, Doolan P, Aherne S, Blanco A, Meleady P, Clynes M, Barron N - PLoS ONE (2013)

Bottom Line: In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase.Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1.Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.

ABSTRACT
MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.

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Impact of miR-7 on cell cycle and apoptosis.For cell cycle analysis, cells were stained with Guava Cell Cycle reagent at 3 days after treatment with pm-neg (A) or pm-7 (B). Apoptosis was evaluated with the Nexin assay reagent at day 3 (C) and day 5 (D) after transfection. The data were captured using a Guava Flow cytometer. FCS files from cell cycle assay were extracted and analysed using FCS Express Plus. Standard deviations represent four biological replicates. Significance was evaluated with a Student's t-test. ***: p-value<0.001.
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pone-0065671-g002: Impact of miR-7 on cell cycle and apoptosis.For cell cycle analysis, cells were stained with Guava Cell Cycle reagent at 3 days after treatment with pm-neg (A) or pm-7 (B). Apoptosis was evaluated with the Nexin assay reagent at day 3 (C) and day 5 (D) after transfection. The data were captured using a Guava Flow cytometer. FCS files from cell cycle assay were extracted and analysed using FCS Express Plus. Standard deviations represent four biological replicates. Significance was evaluated with a Student's t-test. ***: p-value<0.001.

Mentions: Previously, we demonstrated that up-regulation of miR-7 levels induced transient cell growth arrest in CHO cells while maintaining high cell viability [19]. Subsequent to transfection with a miR-7 mimic, cells displayed impaired growth over the following 4 days. The cells subsequently re-entered the cell cycle and proliferated normally (Fig. 1). To verify the role of miR-7 in the regulation of cell cycle, we analysed cells 72 hrs after transfection. High levels of miR-7 triggered cell accumulation in the G1 phase thus reducing the proportion of cells in S and G2 (Fig. 2A&B). There was no detectable sub-G1 population suggesting that the cells did not undergo apoptosis either in the control or in miR-7 transfected cells (Fig. 2A&B). To confirm this we measured apoptosis levels specifically and found that there were no significant changes 72 hrs after transfection (Fig. 2C). 120 hrs after transfection there was a small but significant increase in apoptosis in the pm-7 treated cells representing less than 5% of the population (Fig. 2D). It is worth noting that at this time point the cells have started to proliferate again as the effects of the transient transfection abate (Fig. 1). By way of comparison we investigated the cell cycle distribution of CHO clones over-expressing a miR-7 decoy transcript, effectively depleting endogenous levels of mature miR-7, and found an increase in the percentage of cells in S and G2/M compared to PM-7-treated cells (Fig. S1). We also measured the expression of endogenous pre-mir-7 in cells transfected with a miR-7 decoy sponge to check for any feedback loops in response to artificially deregulating the levels of mature miR-7. No change in endogenous expression was observed (data not shown). Thus the high cell viability and the lack of sub-G1 population observed in pm-7 treated cells suggest that high levels of miR-7 do not initiate apoptosis. We investigated the possibility of cellular senescence occurring subsequent to G1 arrest by analysis of SA-β-galactosidase activity. Neither treatment with the DNA intercalating agent, BrDU which causes senescence in replicating cells [20] (and did so in our positive control using HCC1419 breast cancer cells), or transfection with exogenous miR-7 induced SA-β–Gal expression in CHO cells (Fig. 3).


MiR-7 triggers cell cycle arrest at the G1/S transition by targeting multiple genes including Skp2 and Psme3.

Sanchez N, Gallagher M, Lao N, Gallagher C, Clarke C, Doolan P, Aherne S, Blanco A, Meleady P, Clynes M, Barron N - PLoS ONE (2013)

Impact of miR-7 on cell cycle and apoptosis.For cell cycle analysis, cells were stained with Guava Cell Cycle reagent at 3 days after treatment with pm-neg (A) or pm-7 (B). Apoptosis was evaluated with the Nexin assay reagent at day 3 (C) and day 5 (D) after transfection. The data were captured using a Guava Flow cytometer. FCS files from cell cycle assay were extracted and analysed using FCS Express Plus. Standard deviations represent four biological replicates. Significance was evaluated with a Student's t-test. ***: p-value<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675065&req=5

pone-0065671-g002: Impact of miR-7 on cell cycle and apoptosis.For cell cycle analysis, cells were stained with Guava Cell Cycle reagent at 3 days after treatment with pm-neg (A) or pm-7 (B). Apoptosis was evaluated with the Nexin assay reagent at day 3 (C) and day 5 (D) after transfection. The data were captured using a Guava Flow cytometer. FCS files from cell cycle assay were extracted and analysed using FCS Express Plus. Standard deviations represent four biological replicates. Significance was evaluated with a Student's t-test. ***: p-value<0.001.
Mentions: Previously, we demonstrated that up-regulation of miR-7 levels induced transient cell growth arrest in CHO cells while maintaining high cell viability [19]. Subsequent to transfection with a miR-7 mimic, cells displayed impaired growth over the following 4 days. The cells subsequently re-entered the cell cycle and proliferated normally (Fig. 1). To verify the role of miR-7 in the regulation of cell cycle, we analysed cells 72 hrs after transfection. High levels of miR-7 triggered cell accumulation in the G1 phase thus reducing the proportion of cells in S and G2 (Fig. 2A&B). There was no detectable sub-G1 population suggesting that the cells did not undergo apoptosis either in the control or in miR-7 transfected cells (Fig. 2A&B). To confirm this we measured apoptosis levels specifically and found that there were no significant changes 72 hrs after transfection (Fig. 2C). 120 hrs after transfection there was a small but significant increase in apoptosis in the pm-7 treated cells representing less than 5% of the population (Fig. 2D). It is worth noting that at this time point the cells have started to proliferate again as the effects of the transient transfection abate (Fig. 1). By way of comparison we investigated the cell cycle distribution of CHO clones over-expressing a miR-7 decoy transcript, effectively depleting endogenous levels of mature miR-7, and found an increase in the percentage of cells in S and G2/M compared to PM-7-treated cells (Fig. S1). We also measured the expression of endogenous pre-mir-7 in cells transfected with a miR-7 decoy sponge to check for any feedback loops in response to artificially deregulating the levels of mature miR-7. No change in endogenous expression was observed (data not shown). Thus the high cell viability and the lack of sub-G1 population observed in pm-7 treated cells suggest that high levels of miR-7 do not initiate apoptosis. We investigated the possibility of cellular senescence occurring subsequent to G1 arrest by analysis of SA-β-galactosidase activity. Neither treatment with the DNA intercalating agent, BrDU which causes senescence in replicating cells [20] (and did so in our positive control using HCC1419 breast cancer cells), or transfection with exogenous miR-7 induced SA-β–Gal expression in CHO cells (Fig. 3).

Bottom Line: In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase.Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1.Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.

ABSTRACT
MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.

Show MeSH
Related in: MedlinePlus