Ginsenoside-Rg1, a component of ginseng, can induce angiogenesis by reducing levels of miR-23a. miR-23a normally targets and reduces expression of the MET tyrosine kinase receptor, which promotes angiogenesis. However, ginsenoside-Rg1 decreases miR-23a levels, relieving repression of MET and allowing its protein levels and pro-angiogenic activity to increase. Experiments confirmed that ginsenoside-Rg1 induces angiogenesis through this mechanism of downregulating miR-23a and subsequently upregulating MET.

1. Toxicology and Applied Pharmacology
Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine
kinase receptor expression through miR-23a.
Keywords:
Ginsenoside-Rg1
MET
Hepatocyte growth factor receptor
Angiogenesis
miR-23a
microRNA
a b s t r a c t
Therapeutic angiogenesis has been implicated in ischemic-
diseases and wound healing. Ginsenoside-Rg1 (Rg1),
one of the most abundant active components of ginseng, has been
demonstrated as an angiogenesisstimulating
compound in different models. There is increasing evidence
implicating microRNAs (miRNAs), a
group of non-coding RNAs, as important regulators of
angiogenesis, but the role of microRNAs in Rg1-induced
angiogenesis has not been fully explored. In this report, we found
that stimulating endothelial cells with Rg1
could reduce miR-23a expression. In silico experiments predicted
hepatocyte growth factor receptor (MET), a
well-established mediator of angiogenesis, as the target of miR-
23a. Transfection of the miR-23a precursor or
inhibitor oligonucleotides validated the inverse relationship of miR-

2. 23a andMET expression. Luciferase reporter
assays further confirmed the interaction between miR-23a and
theMET mRNA 3′-UTR. Intriguingly, ginsenoside-
Rg1 was found to increase MET protein expression in a time-
dependent manner.We further demonstrated that
ginsenoside-Rg1-induced angiogenic activities were indeed
mediated through the down-regulation of miR-23a
and subsequent up-regulation ofMET protein expression, as
confirmed by gain- and loss-of-function angiogenic
experiments. In summary, our results demonstrated that
ginsenoside-Rg1 could induce angiogenesis by the
inverse regulation of MET tyrosine kinase receptor expression
through miR-23a. This study has broadened our
understanding of the non-genomic effects of ginsenoside-Rg1, and
provided molecular evidence that warrant
further development of natural compound as novel angiogenesis-
promoting therapy.
Introduction
Angiogenesis is the formation of new blood vessels from preexisting
blood vessels. It is involved in both physiological and pathological
conditions such as embryo development (Heinke et al., 2012),
wound healing (Li et al., 2005), atherosclerosis (Bochkov et al., 2006),
and tumor growth (Carmeliet and Jain, 2000). During angiogenesis,
complex cell–cell interactions and various ligand-receptor activations
are involved, but endothelial cells play the central role in this process
(Augustin et al., 1994). Once activated by angiogenic factors, endothelial

3. cells release proteolytic enzymes and migrate to distant sites, where
they assemble into new blood vessels. Among the angiogenesis regulatory
factors, vascular endothelial growth factor (VEGF) is the best
studied and plays a prime role in angiogenesis (Ferrara et al., 2003);
however, a number of growth factors, such as epidermal growth factor
(Ongusaha et al., 2004), insulin-like growth factor (Tomita et al., 2003;
Delafontaine et al., 2004) and hepatocyte growth factor (Tomita et al.,
2003), are also involved in supporting angiogenesis.
Hepatocyte growth factor/scatter factor (HGF/SF), a plasminogenlike,
multi-domain protein, is important for cell proliferation, survival
and motility. Upon ligand binding of HGF to the transmembrane tyrosine
kinase receptor, hepatocyte growth factor receptor (also known
as mesenchymal–epithelial transition factor, MET) dimerizes and recruits
different cytoplasmic adaptor proteins (Gherardi et al., 2012). It
has been well documented that HGF can stimulate endothelial cell proliferation
and induce angiogenesis both in vitro and in vivo (Van Belle
et al., 1998), and the downstream signaling pathways play important
roles in HGF/MET-mediated angiogenesis.
MicroRNAs (miRNAs) are a group of small RNAs of approximately 18–
24 nts. Although miRNAs are non-coding RNAs, they are important in regulating
over 30% of gene expression at the post-transcriptional level
(Filipowicz et al., 2008; Carthew and Sontheimer, 2009; Macfarlane and
Murphy, 2010). Mature miRNAs in the cytoplasm recognize the 3 -′
untranslated region (3 -UTR) of target mRNAs, and their partial complementary′
binding to the 3 -UTR may lead to translational repression of the′
mRNA.Ginseng is the most extensively used Chinese medicine worldwide,
and ginsenosides are steroid-like triterpene saponins that are the pharmacologically
active components of ginseng. Over 30 ginsenosides have
been identified and are classified into three groups: protopanaxadiol,
protopanaxatriol and oleic acid derivatives (Shibata et al., 1963).
Ginsenoside-Rg1 is one of the most abundant protopanaxatriols, and it
has been shown to affect various biological activities, such as blood
pressure regulation (Chen et al., 2012), anti-inflammation (Du et al.,
2011) and neuro-protection (Chen et al., 2006).
Our previous studies have demonstrated that ginsenoside-Rg1 can
promote angiogenesis in vitro (Yue et al., 2005) and in vivo (Leung
et al., 2006b) through activation of the glucocorticoid receptor (Leung
et al., 2006a). Furthermore, miRNA array expression profiling has
shown that Rg1 can modulate the expression of a subset of miRNAs to
induce angiogenesis (Chan et al., 2009, 2013), but the functional role
of those miRNAs has not been fully explored. miR-23a is one of the
microRNAs that is regulated by Rg1. In this report, we investigate the
functional role of miR-23a in ginsenoside-Rg1-induced angiogenesis.