Original Article

MicroRNA-29a-3p Reduces TNFa-Induced
Endothelial Dysfunction by Targeting
Tumor Necrosis Factor Receptor 1
Xinrui Deng,1,4 Xia Chu,1,4 Peng Wang,1 Xiaohui Ma,1 Chunbo Wei,1 Changhao Sun,1,2 Jianjun Yang,3 and Ying Li1,2
1Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, No. 157 Baojian Road, Nangang District, Harbin 150081, China; 2ResearchInstitute of Food, Nutrition and Health, Sino-Russian Medical Research Center, Harbin Medical University, No. 157 Baojian Road, Nangang District, Harbin 150081, China;
3Department of Nutrition and Food Hygiene, School of Public Health and Management, Ningxia Medical University, No. 1160 Shengli Road, Xingqing District, Yinchuan

750004, China

miR-29a-3p has been shown to be associated with cardiovascular diseases; however, the effect of miR-29a-3p on endothelial
dysfunction is unclear. This study aimed to reveal the effects
and mechanisms of miR-29a-3p on endothelial dysfunction.
The levels of vascular cell adhesion molecule 1 (VCAM-1),
intercellular adhesion molecule 1 (ICAM-1), and E-selectin
were determined by real-time PCR and immunoﬂuorescencestaining to reveal the degree of tumor necrosis factor alpha(TNFa)-induced endothelial dysfunction. A luciferase activity
assay and cell transfection with a miR-29a-3p mimic or an inhibitor were used to reveal the underlying mechanisms of miR29a-3p action. Furthermore, the effects of miR-29a-3p on
endothelial dysfunction were assessed in C57BL/6 mice injectedwith TNFa and/or a miR-29a-3p agomir. The results showedthat the expression of TNFa-induced adhesion molecules invascular endothelial cells (EA.hy926 cells, human aortic endothelial cells [HAECs], and primary human umbilical vein endothelial cells [pHUVECs]) and smooth muscle cells (humanumbilical vein smooth muscle cells [HUVSMCs]) was signiﬁcantly decreased following transfection with miR-29a-3p.
This effect was reversed by cotransfection with a miR-29a-3p
inhibitor. As a key target of miR-29a-3p, tumor necrosis factor
receptor 1 mediated the effect of miR-29a-3p. Moreover, miR29a-3p decreased the plasma levels of TNFa-induced VCAM-1(32.62%), ICAM-1 (38.22%), and E-selectin (39.32%) in vivo.
These data indicate that miR-29a-3p plays a protective role in
TNFa-induced endothelial dysfunction, suggesting that miR29a-3p is a novel target for the prevention and treatment of
atherosclerosis.

vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion
molecule 1 (ICAM-1), and E-selectin.8–11 However, the detailed
mechanisms leading to endothelial dysfunction remain incompletely
understood.
MicroRNAs (miRNAs, miRs) are short (21–23 nt) noncoding RNAs
that regulate target genes by degrading mRNAs or by inhibiting
posttranscriptional gene expression by binding to partially complementary mRNA recognition sequences.12,13 miRNAs have been
implicated in the control of a number of biological functions,
including metabolism, differentiation, development, proliferation,
apoptosis, and oncogenesis.14 Recently, the role of miRNAs in thepathogenesis and physiological processes of cardiovascular diseases
has been frequently investigated,15–19 and some miRNAs, including
miR-29, have attracted signiﬁcant attention.20–24
Previous studies have reported that miR-29 can play pivotal roles in
the regulation of ﬁbrosis in a variety of organs, such as hepaticﬁbrosis,25,26 cardiac ﬁbrosis,20 renal ﬁbrosis,27 and pulmonaryﬁbrosis.28,29 In recent years, researchers have been concerned about
the role of miR-29 in cardiovascular diseases.21,23,24,30,31 Sassi
et al.23 reported that miR-29 promotes pathological hypertrophy
and overall cardiac insufﬁciency of cardiac myocytes rather thanﬁbrotic heart disease. These studies imply that miR-29 may be
involved in the progression of cardiovascular diseases, althoughdifferent studies suggest that the role of miR-29 in cardiac function
or atherosclerosis is debatable. However, the role of miR-29 in

Received 15 February 2019; accepted 12 October 2019;https://doi.org/10.1016/j.omtn.2019.10.014.

INTRODUCTION

4

Atherosclerosis is a chronic multifactorial vascular disease that is
considered to be the leading cause of morbidity and mortality among
cardiovascular diseases worldwide.1–3 There is growing evidence that
endothelial dysfunction may be the cause of important early events inatherosclerosis that promote the development and progression ofplaques.4–7 Endothelial dysfunction is commonly detected by theelevation of soluble markers, such as adhesion molecules, including

These authors contributed equally to this work.

Correspondence: Ying Li, Department of Nutrition and Food Hygiene, Public
Health College, Harbin Medical University, No. 157 Baojian Road, Nangang District, Harbin 150081, China.
E-mail: liying_helen@163.com
Correspondence: Jianjun Yang, Department of Nutrition and Food Hygiene,
School of Public Health and Management, Ningxia Medical University, No. 1160
Shengli Road, Xingqing District, Yinchuan 750004, China.
E-mail: yangjianjun_1970@163.com

Molecular Therapy: Nucleic Acids Vol. 18 December 2019 ª 2019 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

903

vascular endothelial function and its underlying mechanisms havenot been determined.
In this study, we investigated the effect of miR-29a-3p on tumor necrosis factor alpha (TNFa)-induced endothelial dysfunction and its
underlying mechanisms in vitro and in vivo. Here, we report for theﬁrst time that miR-29a-3p can ameliorate TNFa-induced endothelial
dysfunction by targeting tumor necrosis factor receptor 1 (TNF-R1),
which means that miR-29a may be a potential novel target for the
early prevention of atherosclerosis.

RESULTS
miR-29a-3p Modulates VCAM-1, ICAM-1, and E-selectin
Expression in EA.hy926 Cells, HAECs, pHUVECs, and HUVSMCs

To analyze the potential effect of miR-29a-3p on endothelial dysfunction, we assessed the levels of adhesion molecules, including
VCAM-1, ICAM-1 and E-selectin, in EA.hy926 cells, human aortic
endothelial cells (HAECs), primary human umbilical vein endothelial
cells (pHUVECs), and human umbilical vein smooth muscle cells(HUVSMCs). As expected, the transfection of an exogenous miR29a-3p mimic and a miR-29a-3p inhibitor signiﬁcantly increased
and decreased intracellular miR-29a-3p expression, respectively, suggesting successful transfection (Figures S1A–S1H).
Compared with the normal group, in the TNFa group, VCAM-1,
ICAM-1, and E-selectin mRNA levels dramatically increased,
whereas the transfection of EA.hy926 cells with miR-29a-3p mimic
noticeably prevented VCAM-1, ICAM-1, and E-selectin production.
In addition, transfection with a miR-29a-3p inhibitor enhanced theincreases in ICAM-1 mRNA levels that were induced by TNFa. To
test whether the changes were inﬂuenced by miR-29a-3p, the cells
were cotransfected with the miR-29a-3p mimic and its inhibitor,
and the effect of miR-29a-3p in the TNFa model was signiﬁcantly
diminished (Figures 1A–1C). These results suggested that miR-29a3p modulated TNFa-induced expression of VCAM-1, ICAM-1,
and E-selectin in EA.hy926 cells.
Furthermore, we examined the effect of miR-29a-3p on VCAM-1,
ICAM-1, and E-selectin protein expression using immunoﬂuorescence staining. A similar trend was observed in the protein levels of
VCAM-1, ICAM-1, and E-selectin in EA.hy926 cells that was similar
to what was observed in the mRNA level. The upregulation of miR29a-3p dramatically decreased TNFa-induced VCAM-1 protein
expression, and the inhibition effect was partly reversed by co-application with the miR-29a-3p inhibitor, while transfection with themiR-29a-3p inhibitor alone partly enhanced the effect of TNFa on
VCAM-1, ICAM-1, and E-selectin expression (Figure 1D). Furthermore, TNFa-induced ICAM-1 and E-selectin protein expression,
which was visualized with R-phycoerythrin (PE), was also affectedby miR-29a-3p, and that was similar to what was observed for
VCAM-1 (Figures 1E and 1F).
To further determine whether miR-29a-3p modulates TNFa-induced
expression of adhesion molecules in other endothelial cells, VCAM-1,

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ICAM-1, and E-selectin expression was examined at both the mRNAand protein levels in HAECs (Figure 2) and pHUVECs (Figure 3).
VCAM-1, ICAM-1, and E-selectin expression was increased in thepresence of TNFa, and treatment with the miR-29a-3p mimic reduced
their levels in HAECs and pHUVECs. Cotransfection with the miR29a-3p inhibitor partly restored the expression of VCAM-1,
ICAM-1, and E-selectin. In addition, transfection with the miR29a-3p inhibitor alone increased TNFa-induced mRNA levels of
VCAM-1, ICAM-1, and E-selectin in HAECs and pHUVECs. The results suggest that miR-29a-3p ameliorates TNFa-induced VCAM-1,
ICAM-1, and E-selectin expression in three kinds of endothelial celllines (EA.hy926 cells, HAECs, and pHUVECs).
Next, the effects of miR-29a-3p on TNFa-induced expression of adhesion molecule (VCAM-1, ICAM-1, and E-selectin) were assessed in
HUVSMCs. TNFa strikingly upregulated VCAM-1, ICAM-1, andE-selectin expression at both the mRNA and protein levels in
HUVSMCs. In the presence of TNFa, treatment with the miR-29a3p mimic dramatically inhibited the increase in VCAM-1, ICAM-1,
and E-selectin expression that was mediated by TNFa; however, coapplication with the miR-29a-3p inhibitor restored the ICAM-1
mRNA level. Compared with the TNFa group, the miR-29a-3p inhibitor increased VCAM-1 and E-selectin expression at the mRNA level(Figures 4A–4C). The accumulation of VCAM-1, ICAM-1, and E-selectin proteins, which was measured by immunoﬂuorescence staining,
dramatically decreased in the miR-29a-3p mimic group and increasedin the miR-29a-3p inhibitor group compared with the TNFa group;
however, cotreatment with the miR-29a-3p inhibitor partially abrogated the effect of the miR-29a-3p mimic (Figures 4D–4F).
miR-29a-3p Specifically Suppresses the Levels of TNF-R1
Protein Expression

A bioinformatics analysis was performed by an online algorithm,
TargetScan 6.2, to predict the potential targets of miR-29a-3p.
Considering that the important inﬂuence of TNF-R1 on endothelial
dysfunction is induced by pro-atherosclerotic cytokines, such as
TNFa, we designed our experiments to test the connection between
miR-29a-3p and TNF-R1. TargetScan predicted that the binding
sequence in the 30 UTR of TNFRSF1A, which encodes TNF-R1, wasa very good match for the miR-29a-3p seed (Figure 5A). In addition,a luciferase assay revealed that miR-29a-3p overexpression inhibited
luciferase activity in HEK293 cells transfected with a reporter plasmidcarrying the wild-type (WT) 30 UTR of TNFRSF1A; the luciferase activity of the mutated (Mut) TNF-R1 30 UTR vector was unaffected bysimultaneous transfection with the miR-29a-3p mimic (Figure 5B).
Furthermore, the luciferase activity of the TNF-R1 30 UTR WT or
Mut was uninﬂuenced by miR-29a-3p inhibitor (Figure 5B).
To further conﬁrm our luciferase results, similar transfection procedures were conducted in EA.hy926 cells, HAECs, pHUVECs, andHUVSMCs. The overexpression of miR-29a-3p decreased the
TNF-R1 protein levels compared to those in the NC group, and cotreatment with the miR-29a-3p inhibitor partly attenuated the miR29a-3p-mediated downregulation of TNF-R1 expression, whilewww.moleculartherapy.org

Figure 1. miR-29a-3p Modulates TNFa-Induced VCAM-1, ICAM-1 and E-selectin Expression in EA.hy926 Cells(A–F) The mRNA levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) were detected, and the protein expression levels of VCAM-1 (D), ICAM-1 (E), and E-selectin (F) wereanalyzed by immunofluorescence staining in differentially transfected EA.hy926 cells. The nuclei were counterstained with DAPI (blue). Each experiment was performed atleast three times. EA.hy926 cells were transfected with miR-29a-3p or/and miR-29a-3p inhibitor for 48 h and then treated with or without TNFa for 3 h (mRNA detection) or for
6 h (protein detection). A scrambled sequence was used as a negative control (NC). Error bars are defined as the SD. Scale bars, 50 mm. *p < 0.05 and **p < 0.01 indicate
statistically significant differences. miR-29a-3p, cells transfected with miR-29a-3p mimic; miR-29a-3p inhibitor, cells transfected with miR-29a-3p inhibitor; miR-29a-3p+inhibitor, cells transfected with miR-29a-3p mimic and its inhibitor.

treatment with the miR-29a-3p inhibitor alone increased the protein
levels of TNF-R1 (Figures 5C–5F). Collectively, the above data suggest that TNF-R1 is a genuine target of miR-29a-3p.
miR-29a-3p Affect TNFa-Induced VCAM-1, ICAM-1, and Eselectin Expression by Targeting TNF-R1

In summary, the miR-29a-3p mimic speciﬁcally suppressed TNF-R1
expression and modulated TNFa-induced VCAM-1, ICAM-1, andE-selectin expression. To further verify that miR-29a-3p affects
TNFa-induced VCAM-1, ICAM-1, and E-selectin expression bytargeting TNF-R1, pHUVECs were cotransfected with the miR29a-3p mimic and a TNF-R1-overexpression plasmid, or they
were cotransfected with the miR-29a-3p inhibitor and a TNF-R1small interfering RNA (siRNA). Transfection with the TNF-R1overexpression plasmid or the TNF-R1-siRNA signiﬁcantly
increased or decreased intracellular TNF-R1 protein expression,

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Figure 2. miR-29a-3p Modulates TNFa-Induced VCAM-1, ICAM-1, and E-selectin Expression in HAECs(A–F) The mRNA levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) were detected, and the protein expression levels of VCAM-1 (D), ICAM-1 (E), and E-selectin (F) wereanalyzed by immunofluorescence staining in differentially transfected HAECs. The nuclei were counterstained with DAPI (blue). Each experiment was performed at least threetimes. HAECs were transfected with miR-29a-3p or/and miR-29a-3p inhibitor for 48 h and then treated with or without TNFa for 3 h (mRNA detection) or for 6 h (protein
detection). A scrambled sequence was used as a negative control (NC). Error bars are defined as the SD. Scale bars, 50 mm. **p < 0.01 indicates statistically significant
differences. miR-29a-3p, cells transfected with miR-29a-3p mimic; miR-29a-3p inhibitor, cells transfected with miR-29a-3p inhibitor; miR-29a-3p+inhibitor, cells transfectedwith miR-29a-3p mimic and its inhibitor.

respectively, suggesting successful transfection (Figure S2). As expected, overexpression of TNF-R1 caused a robust increase in themRNA levels of VCAM-1 compared to the levels observed in the
TNFa group; however, cotransfection with the miR-29a-3p mimic
and the TNF-R1-overexpression plasmid partly eliminated this increase. We found that treatment with the miR-29a-3p mimic
decreased TNFa-induced mRNA levels of VCAM-1 (Figure 4A),
and we observed a similar result here, whereas TNF-R1 overexpres-906

Molecular Therapy: Nucleic Acids Vol. 18 December 2019sion markedly abrogated the decrease caused by treatment with themiR-29a-3p mimic (Figure 6A). Moreover, the mRNA levels of
ICAM-1 and E-selectin presented the same changes (Figures 6B
and 6C). Furthermore, the downregulation of TNF-R1 by siRNA
effectively negated TNFa-induced VCAM-1, ICAM-1, and E-selectin expression, while cotransfection with the miR-29a-3p inhibitor
did not signiﬁcantly impact their mRNA levels (Figures 6D–6F).
The results collectively indicate that miR-29a-3p reduces thewww.moleculartherapy.org

Figure 3. miR-29a-3p Modulates TNFa-Induced VCAM-1, ICAM-1, and E-selectin Expression in pHUVECs(A–F) The mRNA levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) were detected, and the protein expression levels of VCAM-1 (D), ICAM-1 (E), and E-selectin (F) wereanalyzed by immunofluorescence staining in differentially transfected pHUVECs. The nuclei were counterstained with DAPI (blue). Each experiment was performed at least
three times. pHUVECs were transfected with miR-29a-3p or/and miR-29a-3p inhibitor for 48 h and then treated with or without TNFa for 3 h (mRNA detection) or for 6 h(protein detection). A scrambled sequence was used as a negative control (NC). Error bars are defined as the SD. Scale bars, 50 mm. **p < 0.01 indicates statisticallysignificant differences. miR-29a-3p, cells transfected with miR-29a-3p mimic; miR-29a-3p inhibitor, cells transfected with miR-29a-3p inhibitor; miR-29a-3p+inhibitor, cells
transfected with miR-29a-3p mimic and its inhibitor.

expression of TNFa-induced adhesion molecules (VCAM-1,
ICAM-1, and E-selectin) by targeting TNF-R1.
miR-29a-3p Affects TNF-R1 Expression in the Aortic
Endothelium and VCAM-1, ICAM-1, and E-selectin Expression
In Vivo

To further conﬁrm the results in vitro, expression measurementsof adhesion molecules (VCAM-1, ICAM-1, and E-selectin) andTNF-R1 in vivo were collected. The mice received a tail vein
injection of saline or a miR-29a-3p agomir and an intraperitoneal
injection of saline or TNFa (Figure 7A). TNF-R1 expression infrozen sections of aortas was determined by immunohistochemistry staining, and the location of TNF-R1 antigen was indicatedby a dark brown diaminobenzidine (DAB) reaction product (Figure 7B). The results indicated that although TNF-R1 expression
was decreased in the TNFa group compared with the control

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Figure 4. miR-29a-3p Modulates TNFa-Induced VCAM-1, ICAM-1, and E-selectin Expression in HUVSMCs(A–F) The mRNA levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) were detected, and the protein expression levels of VCAM-1 (D), ICAM-1 (E), and E-selectin (F) wereanalyzed by immunofluorescence staining in differentially transfected HUVSMCs. The nuclei were counterstained with DAPI (blue). Each experiment was performed at least
three times. HUVSMCs were transfected with miR-29a-3p or/and miR-29a-3p inhibitor for 48 h and then treated with or without TNFa for 3 h (mRNA detection) or for 6 h(protein detection). A scrambled sequence was used as a negative control (NC). Error bars are defined as the SD. Scale bars, 50 mm. **p < 0.01 indicates statisticallysignificant differences. miR-29a-3p, cells transfected with miR-29a-3p mimic; miR-29a-3p inhibitor, cells transfected with miR-29a-3p inhibitor; miR-29a-3p+inhibitor, cells
transfected with miR-29a-3p mimic and its inhibitor.

group, miR-29a-3p overexpression almost entirely eliminated
TNF-R1 expression in the aortic endothelium of mice treatedwith TNFa.
Furthermore, the levels of adhesion molecules in the plasma andin the aortic endothelium were detected by ELISA and immunohistochemistry staining, respectively. The slides treated with PBS
instead of the primary antibody were negative (Figure S3). The

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results showed that the TNFa treatment increased the levels of
VCAM-1, ICAM-1, and E-selectin, and the intervention of miR29a-3p decreased the elevation of these TNFa-induced adhesion
molecules in blood circulation (Figures 8A–8C). Furthermore,similar results were found in the aortic endothelium. TNFa
signiﬁcantly induced the expression, and miR-29a-3p reduced
the increases in adhesion molecules mediated by TNFa (Figures
8D–8F).

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Figure 5. TNF-R1 Expression Is Repressed by miR29a-3p(A) Schematic representation of the 30 UTR of TNFRSF1A
shows a putative miR-29a-3p binding site. The seedlocation for the WT 30 UTR of TNFRSF1A is indicated inred, whereas the Mut 30 UTR is indicated in blue. (B) The
effect of miR-29a-3p on the 30 UTR of TNFRSF1A wasdetermined by luciferase activity assays. HEK293 cells
were cotransfected with TNFSF1A-30 UTR wild-type (WT)or mutant (Mut) plasmid, and miR-29a-3p or its inhibitor by
Lipofectamine 2000. After 24 h, luciferase activities were
measured. (C–F) TNF-R1 protein expression wasmeasured in EA.hy926 cells (C), HAECs (D), pHUVECs (E),and HUVSMCs (F) that had been differentially transfected.
A scrambled sequence was used as a negative control(NC). After 48 h of transfection, TNF-R1 protein expression was assessed by western blot. Each test was performed at least three times. Error bars are defined as theSD. *p < 0.05 and **p < 0.01 indicate statistically significant differences. miR-29a-3p, cells transfected with miR29a-3p mimic.

DISCUSSION
In this study, we demonstrated for the ﬁrst time that miR-29a-3p
shows a remarkable inhibitory effect on the expression of TNFa-induced adhesion molecules (VCAM-1, ICAM-1, and E-selectin) in
vascular endothelial cells (EA.hy926 cells, HAECs, and pHUVECs)
and vascular smooth muscle cells (HUVSMCs); these in vitro results
were validated with an animal model. The underlying mechanism
revealed that TNF-R1, the direct target of miR-29a-3p, mediatedthe ameliorative role of miR-29a-3p in TNFa-induced endothelial
dysfunction.
As a pleiotropic proinﬂammatory cytokine, TNFa could be used
to establish a model of endothelial dysfunction in vitro andin vivo.32–35 Likewise, in our experiments, we found that TNFa
markedly induced the expression of endothelial dysfunction
biomarkers (VCAM-1, ICAM-1, and E-selectin),36–38 directly
providing evidence of the important role of TNFa in endothelial
dysfunction.39–41 In addition, unlike previous reports that used
one or two kinds of cells to test their hypotheses, in our study,
three kinds of endothelial cells (EA.hy926 cells, pHUVECs, and
HAECs) and one kind of vascular smooth muscle cell
(HUVSMCs), which also express a variety of cellular adhesion
molecules,42 were used to verify the ameliorative effect of miR29a-3p on TNFa-induced VCAM-1, ICAM-1, and E-selectin
expression. Indeed, elevated levels of adhesion molecules
VCAM-1, ICAM-1, and E-selectin, as markers of endothelial
dysfunction, play large roles in the progression of atheroscle-rosis;11,43–45 thus, the changes in these adhesion molecule levels were measured to assessthe degree of endothelial dysfunction in thisstudy. Therefore, the reduction of adhesion
molecules caused by the transfection with amiR-29a-3p mimic suggested that miR-29a-3p plays a crucialrole in regulating endothelial dysfunction.
Many studies have described the aberrant expression of miR-29 inheart diseases, such as cardiac ﬁbrosis,20 congestive heart failure,30
and ischemia-reperfusion injury.31,46,47 miR-29 has been found to
be involved in thoracic aortic development processes,48 cardiovascular aging,49 and the pathogenesis of some aortic diseases.50–53 In
particular, Cushing et al.54 reported that miR-29 was required for
postnatal growth and development because of its role in vascularsmooth muscle cell differentiation and vessel wall formation; further
supporting this role, the authors found a reduction in heart weight inmiR-29 null mice. These results imply that miR-29 may play a role in
the progression of some cardiovascular diseases. However, little is
known regarding the function of miR-29 in the endothelium. Therefore, in this study, we ﬁrst proposed the protective effects of miR-29 inpreventing endothelial dysfunction. This result supports the idea ofmiR-29 functioning in the modulation of cardiovascular diseases.
TNF-R1 is encoded by TNFRSF1A gene and is one of the main TNF
receptors; it is responsible for most TNF biological properties, such as
antiviral activity and activation of the transcription factor nuclear factor kB (NF-kB), which it does in a wide variety of cell types.55,56 As is
known, the activation of NF-kB is essential for the transcriptional
regulation of adhesion molecules VCAM-1, ICAM-1, and E-selectin.57–60 Furthermore, Zhang et al.61 reported that inhibition of
TNF-R1 controlled aortic atherosclerosis by reducing the expression

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Figure 6. miR-29a-3p Affects TNFa-Induced VCAM-1, ICAM-1, and E-selectin Expression by Targeting TNF-R1(A–C) The mRNA levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) were detected in pHUVECs transfected with miR-29a-3p or/and the plasmid containing human
TNFRSF1A (TNF-R1 overexpression). (D–F) The mRNA levels of VCAM-1 (D), ICAM-1 (E), and E-selectin (F) were measured in pHUVECs transfected with miR-29a-3p
inhibitor or/and TNF-R1-siRNA. After 24 h, the cells were treated with TNFa for 3 h before collection. A scrambled sequence was used as a negative control (NC). Each test
was performed three times. Error bars are defined as the SD. *p < 0.05 and **p < 0.01 indicate statistically significant differences. miR-29a-3p, cells transfected with miR-29a3p mimic.

of the adhesion molecules VCAM-1 and ICAM-1 in vivo. In thisstudy, we found that TNF-R1 was a genuine target of miR-29a-3p
and veriﬁed their relationship for the ﬁrst time. Therefore, the targeted reduction of TNF-R1 expression by the transfection of themiR-29a-3p mimic might be the reason for the protective effect ofmiR-29a-3p on TNFa-induced endothelial dysfunction. In this study,
the overexpression and inhibition of TNF-R1 were used to conﬁrm
this speculation. The data indicate that TNF-R1, as the direct targetof miR-29a-3p, mediates the ameliorative role of miR-29a-3p in
TNFa-induced endothelial dysfunction.
In conclusion, we have clearly demonstrated that miR-29a-3p
remarkably improves TNFa-induced vascular endothelial dysfunction in vitro and in vivo by targeting TNF-R1. Our ﬁndings revealthat regulation of miR-29a-3p may be a potential strategy for the early
prevention of atherosclerosis by reducing endothelial dysfunction.

MATERIALS AND METHODSNJ, USA). The antibodies used for immunohistochemistry staining
were TNF-R1 (sc-8436, Santa Cruz Biotechnology, Santa Cruz,
CA, USA), VCAM-1 (BA0406, Boster Biological Technology, China),
ICAM-1 (WL02268, Wanleibio, China), and E-selectin (abs122144a,
Absin, China).
Cell Culture, Transfection, and TNFa Treatments

The human endothelial cell line (EA.hy926 cells, ATCC, Manassas,
VA, USA), HAECs (BNCC, China), and HEK293 cells (ATCC, Manassas, VA, USA) were maintained in DMEM (HyClone, South Logan,
UT, USA) containing 10% fetal bovine serum (FBS; PAA Laboratories, Austria). pHUVECs were obtained from AllCells (China)
and cultured incomplete medium (AllCells, China). The HUVSMCs
were purchased from ScienCell (Carlsbad, CA, USA) and cultured inbasal medium (ScienCell, Carlsbad, CA, USA) with smooth muscle
cell growth supplement (ScienCell, Carlsbad, CA, USA) and 10%
FBS (PAA Laboratories, Austria). All cells were cultured at 37 C in
5% CO2 and 95% air atmosphere.

Reagents

Human TNFa (recombinant human TNFa protein, P01375) andmouse TNFa (recombinant mouse TNFa protein, P06804) were purchased from R&D (Minneapolis, MN, USA). The antibodies used forwestern blotting were as follows: anti-TNF-R1 (C25C1) was from Cell
Signaling Technology (Beverly, MA, USA), and anti-b-actin was fromSanta Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies used
for immunoﬂuorescence staining were VCAM-1 (551146), ICAM-1(555511), and E-selectin (551145) (BD Biosciences, Franklin Lakes,

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To determine the effects of miR-29a-3p on the expression of the adhesion molecules VCAM-1, ICAM-1, and E-selectin, all cells were transfected with exogenous hsa-miR-29a-3p (miRBase: MIMAT0000086).
Speciﬁcally, cells were transfected with 50 nM miR-29a-3p mimic(RiboBio, China) or/and 100 nM miR-29a-3p inhibitor (RiboBio,
China) with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA,
USA) according to the manufacturers’ protocols. siRNAs against
TNF-R1 (TNF-R1-siRNA) were purchased from Santa Cruz

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Figure 7. miR-29a-3p Agomir Affects TNF-R1 Expression in Arteries In Vivo
(A) Schematic representation of experimental mouse design is shown. Briefly, 8-week-old male mice received a tail vein injection of saline or miR-29a-3p agomir and werethen administered an intraperitoneal injection of saline or TNFa. (B) Immunohistochemistry staining was performed for TNF-R1 in the Mouse aorta at harvest. Error bars aredefined as the SD. Scale bars, 50 mm. **p < 0.01 indicates statistically significant differences.

Biotechnology (Santa Cruz, CA, USA). The eukaryotic-expressing
vector plasmid pDoubleEX-EGFP-TNFRSF1A containing the human
TNFRSF1A (GenBank: NM-001065) gene was constructed by Changsha Yingrun Biotechnology (Yingrun Biotechnology, China) and puriﬁed by a Plasmid Midi Kit (QIAGEN, Germany). The pHUVECs
were transfected with 100 nM miR-29a-3p inhibitor or/and 100 nM
TNF-R1-siRNA. In addition, the pHUVECs were transfected with
50 nM miR-29a-3p mimic or/and the expression vector plasmidpDoubleEX-EGFP-TNFRSF1A (TNF-R1 overexpression, 1.5 mg/well).
A scrambled sequence was used as a negative control (NC).
Forty-eight hours after the transfection, the cells were treated with orwithout 10 ng/mL recombinant human TNFa protein (R&D Systems,
Minneapolis, MN, USA) for 3 h (to test mRNA levels) or 6 h (to test
protein levels), and then they were used for the subsequent experiments. Each in vitro test was performed at least three times.

Protein Isolation and Western Blot

Western blotting was performed as previously described62 with thefollowing primary antibodies: TNF-R1 (Cell Signaling Technology,
Danvers, MA, USA) and b-actin (Santa Cruz Biotechnology, Dallas,
TX, USA). Anti-rabbit alkaline-phosphatase-conjugated antibody(Promega, Madison, WI, USA) was used as a secondary antibody.
RNA Isolation and Quantitative Real-Time PCR

For mRNA assessment, total RNA was isolated from the treated
EA.hy926 cells, HAECs, pHUVECs, and HUVSMCs with different
amounts of TRIzol reagent (Invitrogen) according to its manufacturer’s instructions. For expression experiments, b-actin was used
as an internal control. Primers (VCAM-1, ICAM-1, E-selectin, andb-actin) were synthesized by Invitrogen, and their primer sequences
are listed in Table S1. Real-time PCR was performed with a SYBRGreen PCR Master Mix by a 7500 Fast Real-Time PCR System(Applied Biosystems, Foster City, CA, USA).62

Target Prediction and Luciferase Activity Assay

The target genes of miR-29a-3p were predicted with TargetScan 6.2(http://www.targetscan.org/). The pmiRRB-REPORTTM Dual-Luciferase reporter vectors carrying the WT or Mut 30 UTR of TNFRSF1A
were constructed (RiboBio, China). HEK293 cells were cotransfectedwith 200 ng of recombinant plasmid, 50 nM miR-29a-3p mimic, or
100 nM miR-29a-3p inhibitor. After 24 h of transfection, the luciferase activities were measured with a Dual-Luciferase reporter assay
kit (Promega, Madison, WI, USA) on a luminometer (GloMaxTM
20/20, Promega).

Immunofluorescence Analysis of Cell Surface Expression of
VCAM-1, ICAM-1, and E-selectin

Cells were grown to conﬂuence in 24-well plates. After the indicatedtreatments, the cells were washed with PBS and ﬁxed with 4% paraformaldehyde (PFA). Then the cells were incubated with a ﬂuorescein
isothiocyanate (FITC)-conjugated VCAM-1 antibody, a PE-conjugated ICAM-1 antibody, or a PE-conjugated E-selectin antibody(BD Biosciences, Franklin Lakes, NJ, USA) for 48 h. After that, thecell nuclei were counterstained with DAPI (Beyotime Biotechnology,

Molecular Therapy: Nucleic Acids Vol. 18 December 2019911

Molecular Therapy: Nucleic Acids

Figure 8. miR-29a-3p Agomir Affects TNFa-Induced VCAM-1, ICAM-1, and E-selectin Expression In Vivo
(A–F) The levels of VCAM-1 (A), ICAM-1 (B), and E-selectin (C) in plasma and the expressions of VCAM-1 (D), ICAM-1 (E), and E-selectin (F) in aorta endothelium of mice indifferent treatment groups were measured by ELISA and immunohistochemistry staining, respectively. The mice received a tail vein injection of saline or miR-29a-3p agomir
and were then administered an intraperitoneal injection of saline or TNFa. Error bars are defined as the SD. Scale bars, 50 mm. *p < 0.05 and **p < 0.01 indicate statisticallysignificant differences.

China) for 15 min. The ﬂuorescent signal was detected with a confocal
microscope (Nikon, Japan). Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA) was used to calculate the immunoﬂuorescence light intensity. The average light intensity was obtained bysummation of light intensity divided by the area of interest.
Animal Experimental Protocol and Adhesion Molecule Analysis

All protocols in this study were approved by the Medical Ethics Committee of Harbin Medical University (China) and were performed inaccordance with the NIH regulations for the care and use of animals
in research. Twenty C57BL/6 mice (males, 7 weeks old) were used inthis experiment (Beijing Vital River Laboratory Animal Technology,
China). The mice were maintained on a 12-h light/12-h dark cycle,
given water and food ad libitum, and housed in an environmentallycontrolled room at 22 ± 2 C with 50% ± 5% humidity. After an initialacclimation period, the mice were randomly divided into three groups(control, TNFa, and miR-29a-3p agomir + TNFa), with six mice per

912

Molecular Therapy: Nucleic Acids Vol. 18 December 2019group. Based on mmu-miR-29a-3p (miRBase: MIMAT0000535), amiR-29a-3p agomir (micrONTMmmu-miR-29a-3p agomir) wasconstructed by RiboBio (China). On days 0, 3, and 6, the micereceived a tail vein injection (intravenous [i.v.]) of saline or miR29a-3p agomir (micrONTMmmu-miR-29a-3p agomir, RiboBio,
China) at a dose of 20 nmol per mouse, which was administered every
3 days for a total of three injections. On days 6, 7, 8, and 9, the mice
were administered an intraperitoneal (i.p.) injection of recombinantmouse TNFa protein (R&D Systems, Minneapolis, MN, USA) at adose of 30 mg/kg body weight (BW), which they received daily for 3
consecutive days. Saline was used as a negative control over thesame period. After the last TNFa injection, all mice were fasted for
12 h and sacriﬁced under anesthesia with barbital sodium.
Measurements of Adhesion Molecules in Plasma

Mouse plasma was collected by centrifugation of blood in EDTA K2
tubes. Later, the plasma was frozen at 80 C for ELISA analysis.

www.moleculartherapy.org

Plasma VCAM-1, ICAM-1, and E-selectin levels were measured
with ELISA kits (Cusabio, China) following the manufacturer’s
instructions.
Immunohistochemical Localization of TNF-R1, VCAM-1, ICAM1, and E-selectin

The mouse aorta and heart were perfused with 10 mL of PBS at harvest and dissected from the surrounding tissues. Then, the aortae were
submersed in 4% PFA overnight for further measurements. After incubation in PFA, the aortae were washed with PBS and then put in
15% and 30% sucrose in turn until the tissues had successively sunkto the bottom of tubes. Finally, the upper portion of the heart andthe proximal aorta were obtained and embedded in an optimal cutting temperature (OCT) compound (Thermo Fisher Scientiﬁc, Waltham, MA, USA) and frozen rapidly. The aorta sections were cut at
a 6-mm thickness by a cryostat microtome (Thermo Scientiﬁc, Cryoster NX70) and stored at 80 C until ﬁnal analysis.
The presence of TNF-R1, VCAM-1, ICAM-1, and E-selectin in frozen
sections was assessed by immunohistochemical staining. The frozen
aortic tissue sections were soaked in acetone for ﬁxation, 4% Triton
X-100 for permeabilization, and 3% H2O2 for removal of endogenous
peroxidase, and then they were incubated with blocking reagents (goatserum, Wanleibio, China) overnight to reduce background irritation.
Subsequently, the sections were incubated with different primary antibodies for 48 h. Immunohistochemistry for TNF-R1 was performed
with a mouse anti-TNF-R1 primary antibody diluted 1:50 (Santa
Cruz Biotechnology, CA, USA) and a SABC-POD Mouse IgG kit (Boster Biological Technology, China). To accomplish the immunohistochemistry staining of VCAM-1, ICAM-1, and E-selectin, a rabbit
anti-VCAM-1 primary antibody (Boster Biological Technology,
China) or a rabbit anti-ICAM-1 primary antibody (Wanleibio, China)
or a rabbit anti-E-selectin primary antibody (Absin, China) was used
after being diluted with PBS at 1:250, 1:100, and 1:200, respectively.
Then, a SABC-POD rabbit IgG kit (Boster Biological Technology,
China) was used as a secondary antibody. Next, the signals were visualized by a DAB chromogen system (ZSGB-BIO, China). Cell nuclei
were counterstained with Mayer’s hematoxylin (Solarbio, China) for
20 min. PBS replaced the primary antibody in negative control slides.
Image-Pro Plus 6.0 software was used to calculate the integrated opticalintensity. The average optical intensity was obtained by summation of
the integrated optical intensity divided by the area of interest.
Statistical Analysis

Values expressed as the mean ± SD were obtained from three separate
experiments. The signiﬁcance of differences was determined by oneway ANOVA. Continuous variables were compared by t tests between
two groups. SPSS 10.0 software (SPSS, Chicago, IL, USA) was used forall statistical analyses. p % 0.05 was considered to indicate a signiﬁcant difference.

AUTHOR CONTRIBUTIONS
Y.L., X.C., and X.D. designed the research. X.D. performed the majorparts of the experiments. P.W., X.M., and C.W. performed parts of theresearch. X.D. analyzed the data, tested statistics, coordinated the ﬁgures, and wrote the article. Y.L. and X.C. revised the article.

CONFLICTS OF INTEREST
The authors declare no competing interests.

ACKNOWLEDGMENTS
This research was supported by grants from the Open Research Fundfor Top Disciplines of Public Health and Preventive Medicine at
Ningxia Medical University (30181302), the National Science Foundation of China (81673153), and the National Key R&D Programof China (2017YFC1307401 to Changhao Sun).

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Supplemental Information

MicroRNA-29a-3p Reduces TNFa-Induced
Endothelial Dysfunction by Targeting
Tumor Necrosis Factor Receptor 1
Xinrui Deng, Xia Chu, Peng Wang, Xiaohui Ma, Chunbo Wei, Changhao Sun, Jianjun
Yang, and Ying Li

Supplemental information

Figure S1. MiR-29a-3p increases miR-29a-3p expression in different cells and its
inhibitor did the opposite. The transfection efficiencies of EA.hy926 cells (A),HAECs (C), pHUVECs (E) and HUVSMCs (G) with miR-29a-3p mimic are shown.
The transfection efficiencies of EA.hy926 cells (B), HAECs (D), pHUVECs (F) andHUVSMCs (H) with miR-29a-3p inhibitor are shown. Each experiment was performed

at least three times. * (P<0.05) and ** (P<0.01) indicate statistically significant
differences. Error bars are defined as the s.d. miR-29a-3p, cells transfected with miR29a-3p mimic.

Figure S2. TNF-R1-siRNA decreases TNF-R1 expression in pHUVECs and
TNF-R1-overexpression plasmid did the opposite. The transfection efficiencies of
pHUVECs with TNF-R1-overexpression plasmid or TNF-R1-siRNA are shown. The
experiment was performed at least three times. ** (P<0.01) indicates statisticallysignificant differences. Error bars are defined as the s.d.

Figure S3. The immunohistochemistry staining negative control of TNF-R1,
VCAM-1, ICAM-1 and E-selectin. PBS replaced the primary antibody in the adjacentslides and other treatments were same with images in test. Scale bar ═ 50 μm.

Table S1. Primer sequences used for real-time PCRGene
VCAM-1

Accession No.
NM_001078.3

ICAM-1

NM_000201.2

E-selectin

NM_000450.2β-actin

NM-001101

Primer sequence
F:5’-TTCCCTAGAGATCCAGAAATCGAG-3’
R:5’-CTTGCAGCTTACAGTGACAGAGC-3’
F:5’-CTCCAATGTGCCAGGCTTG-3’
R:5’-CAGTGGGAAAGTGCCATCCT-3’
F:5’-TGAAGCTCCCACTGAGTCCAA-3’
R:5’-GGTGCTAATGTCAGGAGGGAGA-3’
F:5’-ACCATTGGCAATGAGCG-3’
R:5’-GTGCCAGGGCAGTGATCT-3’