3.9 miR-26a inhibits Smad3 activation by targeting Smad4
We next sought to determine how miR-26a regulates Smad3 activation.
MiRanda (www.microRNA.org) analysis indicated that Smad4 contains a
binding site for miR-26a (Fig. 9a). We confirmed that Smad4 was a direct
target of miR-26a by luciferase reporter assay (Fig. 9b). Consistently,
Smad4 expression was significantly downregulated with miR-26a mimic
overexpression, whereas miR-26a inhibitor treatment increased the
protein level of Smad4 in AngII-induced VSMCs (Fig. 9c, d).
To explore the effect of Smad4 on Smad3 activation, we examined
siRNA-mediated knockdown of reduced level of Smad4 in VSMCs. We selected
siRNA with the best silencing effect on Smad4 to transfect AngII-treated
VSMCs (Supplemental Fig. S2c, d). Smad4 expression was highly relevant
for the expression and nuclear translocation of p-Smad3. Inhibition of
Smad4 could reduce p-Smad3 protein expression and decreased the nuclear
translocation of p-Smad3 (Fig. 9e-h). Therefore, Smad4 promoted the
activation of Smad3. In summary, miR-26a regulated Smad3 activation by
targeting Smad4.
Combined with previous results (Fig. 7-9), a positive feedback loop
between miR-26a and Smad3/4 may be involved in hypertensive VR (Fig.
10).
Discussion
In this study, we evaluated the role and mechanisms of miR-26a in
controlling hypertensive VR (Fig. 10). MiR-26a was significantly
downregulated in the thoracic aorta and plasma of SHRs with VR.
Overexpression of miR-26a effectively reduced ECM deposition and VSMC
hyperproliferation both in vivo and in vitro. Also, AngII potentiated
Smad3 binding to the miR-26a promoter, inhibiting miR-26a transcription,
which in turn promoted Smad3 activation by upregulating Smad4, thereby
further downregulating miR-26a. These findings reveal a possible new
positive feedback loop between miR-26a and Smad3/4, which may be related
to hypertensive VR. Moreover, miR-26a mitigated ECM accumulation and
excessive VSMC proliferation in AngII-induced VSMCs by targeting CTGF
and the EZH2/p21 pathway, respectively. Our study suggests that miR-26a
is a protective molecule in hypertensive VR and can be considered a
promising target for treating hypertensive VR.
The crucial finding of this study is that miR-26a plays a protective
role in hypertensive VR. Previous studies have shown that miR-26a is
closely related to cardiovascular diseases. Li et al. found low
expression of miR-26a in plasma of patients with acute myocardial
infarction as compared with healthy people (Li et al. , 2015).
Zhang et al. showed that miR-26a plays a role in the process of
myocardial fibrosis after acute myocardial infarction by inhibiting
phosphatase and tensin homolog expression, enhancing matrix
metalloproteinase 9 level and promoting the PI3K/AKT pathway (Zhanget al. , 2018). Chiang et al. discovered that miR-26a
ameliorates cardiac dysfunction and fibrosis in myocardial infarction
(Chiang et al. , 2020). In this study, we first observed that
miR-26a expression was downregulated in the thoracic aorta and plasma of
SHRs. We next used rAAV-miR-26a to overexpress miR-26a in the thoracic
aorta and plasma of SHRs. The overexpression of miR-26a reduced media
thickening of the thoracic aorta and diminished the severity of VR
induced by hypertension. However, due to the limitations of this
experiment, we have not been able to obtain data on the extent of SHR
aortic remodeling before miR-26a treatment. Therefore, our conclusion
was based on the hypothesis that there was no difference in VR of SHRs
in each group before intervention. Meanwhile, we found that
overexpression of miR-26a appears to have an effect on blood pressure,
it is unknown whether miR-26a directly reduces VR or if it is secondary
to reduced blood pressure, which can continue to study in future.
One of the characteristics of VR is pathological remodeling of ECM,
which is often accompanied by increased ECM release and collagen
deposition (Ricard-Blum et al. , 2019). ECM is a structural
scaffold of the blood vessel wall, which controls cellular functions in
the pathological environment. When stimulated or injured, the deposition
of ECM protein, especially Col I, alters the collagen/elastin ratio and
hemodynamics, leading to increased vascular stiffness and affecting
blood vessel function (Lee et al. , 2015). We found the CVF of the
thoracic-aorta media layer reduced in miR-26a-overexpressed SHRs. In
vitro studies showed that miR-26a mimic treatment inhibited but miR-26a
inhibitor treatment promoted Col I and III expression in AngII-induced
VSMCs. CTGF belongs to matricellular protein expressed by various cells
in response to stimuli and is an important molecule prominently
implicated in increased deposition of ECM (Li et al. , 2016;
Petrosino et al. , 2019). Our results showed that that CTGF is a
downstream target gene of miR-26a and the level of miR-26a was inversely
associated with CTGF expression both in vivo and in vitro, so miR-26a
may diminish ECM deposition by targeting CTGF. Additionally, the Col I
is a downstream target gene of miR-26a (Wei et al. , 2013).
MiR-26a may directly inhibit collagen synthesis to reduce ECM
deposition, which needs further study.
Another pathological manifestation of hypertensive VR is excessive VSMC
proliferation, which is determined by the cell cycle (Deniset et
al. , 2018; Lu et al. , 2018; He et al. , 2019). EZH2
accelerates the cell cycle, whereas p21 arrests the cell cycle (Karimianet al. , 2016; Dai et al. , 2018). EZH2/p21 has been found a
critical signaling pathway to regulate cell proliferation (Lu et
al. , 2011; Li et al. , 2019). We found that transfection with
rAAV-miR-26a suppressed PCNA and EZH2 expression, but p21 was
upregulated in the thoracic aorta of SHRs. Similarly, in vitro
experiments showed that miR-26a inhibited the progression of the cell
cycle accompanied by downregulated protein level of EZH2, whereas p21
level was correspondingly increased in AngII-induced VSMCs; miR-26a
inhibitor treatment had the opposite result. Furthermore, EZH2 is a
target gene of miR-26a, so miR-26a participates in VSMC proliferation by
targeting the EZH2/p21 pathway. Cyclin D2 can promote the G1/S
transition (Gul et al. , 2018; Pei et al. , 2018), and
previous studies with luciferase reporter assay showed that cyclin D2 is
also a target gene of miR-26a (Zhou et al. , 2016). Consistently,
we found that miR-26a suppressed cyclin D2 expression in AngII-induced
VSMCs. Thus, miR-26a may be involved in excessive VSMC proliferation by
directly regulating cyclin D2 apart from targeting the EZH2/p21 pathway.
Our study confirmed that AngII regulates miR-26a level dose- and
time-dependently. However, how AngII regulates miR-26a expression is
unclear. Some studies demonstrated that miRs are subject to
sophisticated regulation, and superposition of subtle changes in the
expression of several molecules may be responsible for miRs exerting
their effects (Krol et al. , 2010; Liang et al. , 2014).
Previous studies determined that miR-26a inhibits TGFβ-dependent Smad
signaling (Leeper et al. , 2011). We focused on TGFβ‐independent
Smad signal regulatory mechanisms of miR-26a. In this study, we
validated that Smad3 could directly suppress miR-26a expression, and
AngII could promote this effect; AngII regulates miR-26a by activating
Smad3. Previous studies reported a functional crosstalk between miR-26a
and other molecules via a feedback loop (Jiang et al. , 2018; Liuet al. , 2018). We found that miR-26a downregulation enhanced
Smad4 expression via targeted regulation and then induced Smad3
activation and p-Smad3 nuclear translocation. Smad3 activation caused
further downregulation of miR-26a, thereby forming a Smads/miR-26a
positive feedback loop. Once the loop is activated, it will run
repeatedly, eventually leading to increased ECM accumulation, excessive
VSMC proliferation and VR.
To sum up, miR-26a plays a protective role in hypertensive VR. The
effect of miR-26a on VR was mediated by activation of a Smads/miR-26a
positive feedback loop. In addition, miR-26a inhibited ECM deposition by
targeting CTGF and attenuated VSMC proliferation by regulating the
EZH2/p21 pathway. We clarify the functions and mechanisms of miR-26a in
hypertensive VR and suggest that miR-26a may be a novel therapeutic
target of hypertensive VR.