Biochemical and Biophysical Research Communications
MicroRNA-34a regulation of endothelial senescence
Research highlights
► MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. ► MiR-34a expression increases during endothelial cell senescence and in older mice. ► SIRT1 is a miR-34a target gene in endothelial cells. ► SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells.
Introduction
Senescence of endothelial cells may play an important role in cardiovascular diseases such as atherosclerosis and thrombosis [1], [2], [3], [4]. Changes in many transcription factors are associated with aging [5], [6], [7]. Sirtuin 1 (SIRT1), the mammalian homolog of Sir2, regulates cell cycle, cellular senescence, and metabolism by deacetylating p53, Forkhead box subfamily O (Foxo), Ku70 and peroxisome proliferator-activated receptor gamma (PPAR-gamma) [8], [9], [10], [11], [12], [13], [14]. Over-expression of Sir2 can extend the life span of yeast, worms, and flies [15], [16], [17]. In human fibroblasts, increased expression of SIRT1 can delay cellular senescence and extend cellular life span [18], [19]. SIRT1 also has a role in vascular homeostasis by controlling endothelial cell senescence and angiogenesis [20], [21]. However, the mechanisms regulating senescence of endothelial cells mostly remain unknown.
MicroRNA (miRNA) are small non-coding RNAs that inhibits gene expression by binding to complementary sequences of 3′UTR in their target mRNAs [22], [23], [24], [25]. MiRNAs modulate a variety of biological functions such as cell development, cell differentiation, and apoptosis [26], [27], [28]. MiRNAs regulate endothelial biology, since knockdown of Dicer or Drosha in human endothelial cells decrease angiogenesis in vitro [29], [30]. Several miRNAs involved in endothelial cell functions have been identified. MiR-126, miR-92, and miR-221/222 control the growth of new blood vessels [31], [32], [33], [34], [35]. A recent report showed that miR-217 regulates senescence in endothelial cells [36], but the role of other miRNAs that regulate senescence in endothelial cells are not fully clarified.
MiR-34a has been shown to regulate genes involved in cell cycle regulation and apoptosis in a p53 dependent or independent manner in cancer cells [37], [38], [39], [40], [41], [42], [43], [44]. However, the function of miR-34a in endothelial cells is not known. We now show that miR-34a is abundantly expressed in primary endothelial cells. We investigated the role of miR-34a in endothelial cells and found that miR-34a regulates SIRT1 expression and controls cell senescence. Aging endothelial cells expressed high levels of miR-34a and low levels of SIRT1 protein. Over-expression of miR-34a decreased SIRT1 protein level and increased acetylated p53 level in endothelial cells. Our data suggest that miR-34a regulates endothelial senescence in part through SIRT1.
Section snippets
Cell culture and transfection
Pooled HUVECs (population doubling level; PDL 7–45), HAEC, and HMVEC were cultured in endothelial basal medium (EBM2) supplemented with growth factors (Lonza). Reagents were obtained from Sigma (St. Louis, MO). For primary endothelial cell culture, serial passage was performed when the cells got 80% confluence. The number of PDL was calculated as described previously [45]. We used cells in PDL < 10 unless we mentioned in the manuscript. Pre-miRNAs and AS-miRNAs were obtained from Applied
MiR-34a is expressed in endothelial cells
We previously performed microarray for miRNAs in human umbilical cord endothelial cell (HUVEC) [33]. Since we found that miR-34a is moderately expressed in HUVEC, we analyzed miR-34a levels in several types of primary endothelial cells, including HUVEC, human aortic endothelial cell (HAEC) and human dermal microvascular endothelial cell (HMVEC) by qRT-PCR. These primary endothelial cells (within passage 5) expressed miR-34a at levels higher than HCT116 colon cancer cells and HeLa epithelial
Discussion
The major findings of this study are that endothelial cells express miR-34a, expression of miR-34a increases during senescence, and miR-34a regulates levels of SIRT1. Since SIRT1 is linked to cellular aging, our study suggests that a specific miRNA may regulate aging pathways.
Acknowledgments
The author is grateful to Dr. Charles Lowenstein for helpful discussion and critical reading of the manuscript. This work was supported by an SDG grant from American Heart Association (0835446N).
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