miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition

Abstract

We examined the effect of reactive oxygen species (ROS) on MicroRNAs (miRNAs) expression in endothelial cells in vitro, and in mouse skeletal muscle following acute hindlimb ischemia. Human umbilical vein endothelial cells (HUVEC) were exposed to 200 lM hydrogen peroxide (H 2 O 2 ) for 8 to 24 h; miRNAs profiling showed that miR-200c and the co-transcribed miR-141 increased more than eightfold. The other miR-200 gene family members were also induced, albeit to a lower level. Furthermore, miR-200c upregulation was not endothelium restricted, and occurred also on exposure to an oxidative stress-inducing drug: 1,3-bis(2 chloroethyl)-1nitrosourea (BCNU). miR-200c overexpression induced HUVEC growth arrest, apoptosis and senescence; these phenomena were also induced by Reactive oxygen species (ROS) play a causal role in a variety of cardiovascular diseases, including ischemia, ischemia/ reperfusion (I/R) injury, diabetic vasculopathy and atherosclerosis, and in aging. 1-3 ROS, which include H 2 O 2 , superoxide anion and hydroxyl radicals have been demonstrated to inhibit cell growth and to induce cell death and senescence. 1 MicroRNAs (miRNAs) are small non-coding RNAs, usually 21-23 nucleotides long, which regulate the stability and/or the translational efficiency of target messenger RNAs (mRNAs). 4 They appear to be closely conserved across species and to them have been ascribed diverse functions, including regulation of proliferation, differentiation, senescence and death. 5 The objective of the present work was to establish the effect of ROS on miRNAs expression, and to determine whether miRNAs modulate endothelial cells (EC) response to oxidative stress. In light of the important role that tumor suppressor proteins retinoblastoma (pRb) and p53 have in responses to ROS, we examined their contribution to miRNAs expression on oxidative stress exposure. The retinoblastoma family, which includes pRb, p130 and p107, is an integral part of the mechanism that regulates proliferation and senescence via phosphorylation-sensitive interactions, regulating either positively or negatively E2F transcription factors family. 6 H 2 O 2 causes rapid pRb dephosphorylation by the activity of protein phosphatase 2A 7,8 and successively, by the increase of p53 protein, which in turns upregulates the CDK inhibitor p21 Waf1/Cip1/Sdi1 (p21). 7 The ROS effect on miRNAs expression was also evaluated in vivo, in a mouse model of hindlimb ischemia, both in wild-type (wt) and in p66 ShcA -null (p66 ShcAÀ/À ) mice. The mammalian adaptor protein p66 ShcA regulates ROS metabolism and apoptosis. The cytoplasmic fraction of p66 ShcA is phosphorylated in serine 36 residue in response to several stimuli, including UV and H 2 O 2 . 9 Moreover, a fraction of p66 ShcA is localized in the mitochondria and functions as a redox enzyme that generates ROS; 10 accordingly p66 ShcAÀ/À mice display lower levels of intracellular ROS 9 and decreased oxidative stress levels and tissue damage following ischemia and I/R injury. 2,3 In the present work, we show tha...

MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in vascular remodelling

“…However, our findings clearly show that miR-146a is not involved in oxidative stress response in neither younger nor senescent HUVECs. These results are in accordance with previous data showing that robust expression of miR-146a is not associated with growth arrest or stress-induced stasis (Bhaumik et al 2009;Magenta et al 2011;Li et al 2009). We also confirmed that miR-200c and miR-141 were up-regulated in younger and senescent HUVECs in response to oxidative stress (Magenta et al 2011;Li et al 2009).…”

“…Human umbilical vein endothelial cells (HUVECs) have been extensively used as an in vitro endothelial model representing common functional and morphological features of the in vivo endothelial cells (Unterluggauer et al 2007). This model was recently used to identify miRs and their relative target proteins associated with replicative and/or stress-induced senescence (Ito et al 2010;Menghini et al 2009;Hackl et al 2010;Vasa-Nicotera et al 2011;Magenta et al 2011). However, these recent reports have not been conclusive on identifying specific miRs as markers of vascular ageing-associated dysfunction in vivo.…”

MiR-200c regulates ROS-induced apoptosis in murine BV-2 cells by targeting FAP-1

“…And the proteins with equal amounts of 15 μg was separated on an SDS-PAGE gel (10% (v/v) polyacrylamide) and transferred onto a polyvinylidene fluoride membrane at 300 mA for 2 h as previously reported. 10 The primary antibodies were listed as follows: GAPDH, cleavedcaspase-3, Bcl-2, Bax, Bim and FAP-1 (Cell Signaling, Danvers, MA, USA). The protein bands were quantified using a PhosphorImager and ImageQuant (Amersham Biosciences, Piscataway, NJ, USA) software analysis.…”

“…9 MiR-200c was demonstrated to be significantly upregulated following H 2 O 2 treatment. 10 However, little research has been conducted on the expression of miR-200c after SCI. In this regard, we explored the miRNA expression file after SCI in mice.…”

Oxidative stress-induced overexpression of miR-25: the mechanism underlying the degeneration of melanocytes in vitiligo

“…27 Recently, several studies have found that the expression profile of miRNAs could be regulated by oxidative stress and mediate the pathogenic effect of ROS in some diseases and animal models, which deepens the understanding of redox biology and implicates new therapeutic strategies for oxidative stress-related diseases. [28][29][30] Given this crosstalk between redox signaling and miRNAs, we hypothesized that enhanced oxidative stress in vitiligo could induce the aberrant expression of miRNAs, which promotes the degeneration of melanocytes.…”