The Effects of Some MicroRNAs in Vascular Neointimal Formation May Depend on Cell Cycle Phase
To the Editor:
We would like to respond to the letter by Silvestri et al1 regarding the potential role of cell cycle phase in micro (mi)RNA-mediated effects on vascular smooth muscle cells (VSMCs) and vascular neointimal formation based on our recent study.2 In our study, we demonstrated that miR-21 knockdown and serum deprivation induced a disproportionate apoptosis, indicating a synergistic effect with an unclear mechanism. In addition, miR-21 knockdown had only a mild effect on the expression level of a candidate target, PTEN (phosphatase and tensin homology deleted from chromosome 10).
Recently, Vasudevan et al showed that serum deprivation with consequent arrest of the cell cycle may have profound effects on the mechanism of action of some miRNAs.3,4 Unlike cell growth under normal conditions, miRNA may enhance mRNA translation in cells with serum deprivation and consequent growth arrest.
Based on the novel information, Silvestri et al provided us with some questions about the results and the potential explanations. First, what was the percentage of VSMCs arrested in the G0/G1 phase of the cell cycle by serum deprivation? If most of the cells were growth-arrested, then miR-21 may be stimulating rather than inhibiting translation of its mRNA targets. Second, heterogeneous VSMCs with the possibility of divergent actions exerted on them by miR-21, and this may explain the different results on apoptosis observed in serum-deprived VSMCs as compared with cells cultured with 10% serum. The heterogeneity of the cultured cells may also be the justification for the unpredicted small effect on PTEN express changes after miR-21 knockdown. Finally, as indicated by the title of the letter by Silvestri, does the effect of miRNA in vascular neointimal formation depend on cell cycle phase?
We appreciated the complimentary appraisal of our study from Silvestri et al. Based on our experimental data, we would like to discuss these comments. Although we did not measure the percentage of VSMCs arrested in the G0/G1 phase of the cell cycle by serum deprivation, 48 hours of serum free was able to push most of the cells arrested in the G0/G1 phase. Of course, during the treatment of miR-21 inhibitor, some cells were not in the G0/G1 phase, and thus the cells were heterogeneous at the beginning of miR-21 inhibition. We also compared the effects of miR-21 inhibition on apoptosis and its mRNA target PTEN expression. We think that the effects of miR-21 inhibition on apoptosis and PTEN in VSMCs cannot be explained by cell cycle phase for the following reasons. First, after serum deprivation, miR-21 expression level was quickly increased, and the increased miR-21 had a protective role against serum deprivation-mediated cell apoptosis. The protective role against apoptosis was also found in 10% of serum-cultured VSMCs, as demonstrated by miR-21 inhibition. Because miR-21 inhibition and serum deprivation had different targets in cell apoptosis, they had a synergistic effect. Second, in cultured VSMCs with or without serum deprivation, we both found miR-21 inhibition increased PTEN expression. In addition, miR-21 inhibition could not decrease PTEN expression in VSMCs even after 3 days of serum deprivation, during which the cells arrested in the G0/G1 phase. Third, we think the reason for the minor effect of miR-21 on PTEN expression could be that PTEN is only 1 of its multiple target genes or is not its major target. At least no opposite effects were found in miR-21–mediated effects on apoptosis and PTEN expression in VSMCs with different phases of the cell cycle. Therefore, opposite effects of miRNA at different cell cycle phases may be miRNA-specific and target gene–specific.
Does cell cycle phase affect miRNAs and their effects on its gene targets? Our answer to this question is yes. First, our unpublished data have clearly shown that multiple miRNA expression was different in growth-arrested (G0/G1 phase) VSMCs and proliferative (late S/G2 phase) VSMCs. Second, the effect of miR-145 on VSMC-specific marker genes is significantly decreased in confluence growth-arrested cells, compared with that in subconfluence, proliferative cells. Until now, we have not found the opposite effects of miRNAs on VSMCs with different cell cycle phases; however, we cannot eliminate these possibilities. Identifying these potential opposite effects could be important for vascular biology research.
In diseased vessels characterized by neointima lesion formation, such as those with atherosclerosis, postangioplasty restenosis and graft vasculopathy, VSMCs are heterogeneous with different cell cycle phases. We have found that cell cycle phases affect functions of some miRNAs such as miR-145; thus, we conclude that the effects of some miRNAs in vascular neointimal formation may depend on cell cycle phase.
Sources of Funding
This work was supported by a National Institutes of Health Grant HL080133 (to C.Z.).
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