MicroRNA‐216a induces endothelial senescence and inflammation via Smad3/IκBα pathway

Abstract Vascular endothelial senescence contributes to atherosclerosis and coronary artery disease (CAD), but the mechanisms are yet to be clarified. We identified that microRNA‐216a (miR‐216a) significantly increased in senescent endothelial cells. The replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established to explore the role of miR‐216a in endothelial ageing and dysfunction. Luciferase assay indicated that Smad3 was a direct target of miR‐216a. Stable expression of miR‐216a induced a premature senescence‐like phenotype in HUVECs with an impairment in proliferation and migration and led to an increased adhesion to monocytes by inhibiting Smad3 expression and thereafter modulating the degradation of NF‐κB inhibitor alpha (IκBα) and activation of adhesion molecules. Conversely, inhibition of endogenous miR‐216a in senescent HUVECs rescued Smad3 and IκBα expression and inhibited monocytes attachment. Plasma miR‐216a was significantly higher in old CAD patients (>50 years) and associated with increased 31% risk for CAD (odds ratio 1.31, 95% confidence interval 1.03‐1.66; P = .03) compared with the matched healthy controls (>50 years). Taken together, our data suggested that miR‐216a promotes endothelial senescence and inflammation as an endogenous inhibitor of Smad3/IκBα pathway, which might serve as a novel target for ageing‐related atherosclerotic diseases.

mediators in vascular ageing and inflammatory process in animal models and human studies. 4,5 To date, several miRNAs have been identified to regulate the process of endothelial senescence and inflammation, such as miR-217, 6 miR-34a, 7 miR-146a 8,9 and miR-181b. 10 Nevertheless, the effect of miRNAs that regulate endothelial cell ageing and inflammation has not been fully clarified.
Recently, through a microarray approach, a particular microRNA (miR-216a) has been first identified to show a significant differentially expression when compared the miRNA profile obtained in senescent human endothelial cells versus young cells. 6 To date, whether miR-216a, located on chromosome 2p16.1, plays a key role in endothelial cell ageing remains unclear.
Computational miRNA target analysis from miRNA databases suggests that Smad3 is a potential target gene of miR-216a. Transforming growth factor beta 1 (TGFb1) is a pleiotropic growth factor and potentially modulates the function of cells that contribute to atherosclerotic formation, such as vascular smooth muscle cells, endothelial cells, T cells and macrophages. Smad3/IjBa signalling is the main downstream mediator and responsible for the anti-inflammatory effects of TGFb1. 11 The enhancement of Smad3 phosphorylation and nuclear expression can increase NF-jB inhibitor alpha (IjBa) degradation and nuclear factor-kappa B (NF-jB) activation, which thus promotes the inflammatory reaction and atherosclerosis. 12 In this study, we applied senescent endothelial cell model and for miR-216 stable line and its negative control were serially passaged, and PDLs were calculated. The PDLs-versus-time growth curves were built to assess the replicative potential over time, respectively, for naturally cultured HUVECs and for miR-216a lentivirus transfected HUVECs.
To explore the regulatory role of miR-216a on target gene expression, miR-216a mimics or miR-216a inhibitors (GenePharma, Suzhou, China) were transfected at a final concentration of 50 nM with the use of lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). After incubation for 2 days, cells were harvested.
2.2 | Senescence-associated b-galactosidase staining Cells senescence was assessed by the senescence-associated bgalactosidase (SA-b-gal) activity via in situ staining. Images of the staining were analysed in more than five random microscopic fields per sample and percentages of SA-b-gal positive-staining cells were calculated by counting five fields. The details were described in Data S1.

| Assays for endothelial proliferation, migration, angiogenic and cell adhesion activity
The experiments for endothelial functions including cell proliferation, migration, angiogenic and cell adhesion activity were, respectively, performed in PDL8 (at 3 days after transduction of Lv-miR-216a) and PDL20 (at about 15 days after transduction of Lv-miR-216a).
The details were described in Data S1. Briefly, the MTS assay was performed to assess the potential effect of miR-216a on endothelial proliferation ability. The scratch test was performed to assess the effect of miR-216a on endothelial migration and wound healing ability. Wound healing rate was calculated: % wound healing = [(Area of original wound-Area of wound after healing)/Area of original wound] 9 100%. Each wound was analysed in five different areas.
The tube formation assay was performed to study the effect of miR-216a on angiogenic activity. Photographs of tube morphology were captured in more than five random microscopic fields, and the cumulative mean tube lengths per field of view were quantified. Cell adhesion assay was performed to assess the effect of miR-216a on endothelial adhesive capacity to monocytes. The non-adherent THP-1 cells (China Infrastructure of Cell Line Resources, Beijing, China) were removed by PBS, and the adherent cells were counted in five random fields.

| mRNA expression analysis
The mRNA expression of ageing-related genes p53 and p21, intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), and TGFb1 pathway genes TGFb1, Smad2, Smad3 and IjBa were analysed by real-time PCR, respectively. Total RNA of HUVECs was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA); then, cDNAs were synthesized using PrimeScript Reverse Transcriptase assay (Takara, Dalian, China) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Green qPCR mix (YEASEN, Shanghai, China) on the ABI 7500 System (Applied Biosystems, Foster City, CA, USA). GAPDH levels were used as internal controls, and fold changes were calculated by relative quantification (2 ÀDDCt ). The primers applied for real-time PCR were shown in Table S1.

| miRNA isolation and expression analysis
Enriched miRNAs were extracted from HUVECs using miRNeasy Mini Kit (QIAGEN, Hilden, Germany). miRNAs in patients' plasma were extracted by miRNeasy Plasma Kit (QIAGEN, Hilden, Germany) with introducing a spiked C.elegans miR-39 (cel-39). Real-time PCR was conducted to examine miR-216a using miScript II reverse transcription kit and miScript SYBR Green PCR kit (QIAGEN, Hilden, Germany) with ABI 7500 System according to the manufacturer's protocol. The U6 small nuclear RNA was used as internal reference for HUVECs; cel-39 was applied as external normalized reference for human plasma samples.

| Telomerase enzyme activity and telomere length measurement
To assess the telomerase activity during endothelial senescence, real-time PCR was performed. 13 Relative mean telomere length in the endothelial cells was determined by a quantitative real-time PCR-based method that compares telomere repeat copy number (T) to single-copy gene copy number b-globin (S) (T/S ratio). 14 The details were shown in Data S1. Resources, Beijing, China). The details were described in Data S1.

| Western blot analysis
HUVECs were harvested, proteins extracts were isolated, and the effects of miR-216a on Smad2, Smad3, p65 and IjBa protein levels were determined by Western blot assay. Bands were visualized with FluorChem R, M and E Systems (ProteinSimple, CA, USA) and quantified with AlphaView Software. The details were shown in Data S1.

| Subjects for CAD case-control study
Patients in this study were unrelated and consecutively admitted to FuWai Hospital from May 2016 to May 2017. All subjects provided their written informed consents with the declaration to be of Han nationality. The study was approved by the ethics committee of FuWai Hospital. Inclusion criteria of CAD were defined as having ≥50% narrowing of the lumina of at least 1 of the major coronary arteries by coronary angiography. 15 The controls were selected from the subjects admitted to FuWai hospital for excluding coronary heart disease, with ≤20% stenosis of major coronary artery and without any vascular disease. All subjects were excluded with a history of other heart diseases including cardiomyopathy, valvular heart disease and congenital heart disease.
The present study comprised 176 CAD patients and 342 agematched control subjects, and the clinical features were recorded.
Blood samples were collected in the morning after an overnight fast.
Plasma biochemical indexes were examined.

| Statistical analysis
The one-sample Kolmogorov-Smirnov test was used to assess the normality of quantitative variables. Quantitative variables were presented as means AE SD or medians and interquartile ranges (IQRs), and categorical variables as percentages. The v 2 test was used to compare qualitative variables. Group differences of quantitative variables were compared by Student's t test or one-way ANOVA as appropriate, and the Mann-Whitney nonparametric test was used to compare variables with non-normal distributions. Logistic regression models were performed to evaluate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between plasma miR-216a levels and CAD. Data were analysed by SPSS Statistics 20.0 (SPSS Inc, Chicago, USA), and P < .05 was considered to be significant. HUVECs ( Figure 1A). The expression levels of senescence-related genes p53 and p21 were, respectively, up-regulated by 69% (P = .003) and 61% (P = .001) in PDL44 HUVECs. In addition, telomere, the specialized DNA-protein structure located at the end of YANG ET AL.
| 2741 chromosomes, is believed to be a marker for biological clock that determines cellular behaviour at the cellular level, with shorter telomere indicating cellular ageing. As expected, telomere length in PDL44 HUVECs was shorter by 42% (P = .01) ( Figure 1B). PDL8 and PDL44 cells were identified as young and ageing endothelial cells, respectively.
miR-216a, located at chromosome 2p16.1, was highly conservative in different species. The expression of miR-216a was almost absent in young PDL8 and markedly up-regulated by 59% in senescent PDL44 cells (P = .03) during endothelial ageing ( Figure 1C). The miR-216 family consists of two members, miR-216a and miR-216b.
Compared with PDL8 cells, miR-216b was not significantly increased in PDL44 cells ( Figure S1).
To explore the expression of plasma miR-216a under atherosclerotic stimuli during endothelial senescence, the young PDL8, middle PDL20 and senescent PDL44 lines were treated with ox-LDL. The results showed that miR-216a expression remained an increasing trend in response to ox-LDL stimuli during the endothelial ageing process. However, only in old PDL44 cells, ox-LDL stimuli significantly promoted miR-216a expression by 65% (P = .04) compared to the ox-LDL-untreated group ( Figure S2).

| Overexpression of miR-216a induces a premature senescent-like phenotype in endothelial cells
To investigate whether miR-216a promoted the process of endothelial senescence, miR-216a stable line was constructed by transfection with pre-miR-216a recombinant lentiviruses (Lv-miR-216a) in HUVECs and serially passaged. The data showed that miR-216a sig- much slower growth rate than naturally passaged cells, and reached a significant growth arrest at about PDL20 which was similar as the senescent status of naturally passaged PDL44 ( Figure S3). We therefore used PDL20 stable line to explore the effects of miR-216a.
We further detected the expression of senescence-related cyclins including p53 and p21, telomere length and telomerase activity. Stable overexpression of miR-216a significantly up-regulated p53 and p21 mRNA levels and down-regulated telomerase activity in PDL8 and PDL20 stable lines, respectively. Telomere length remarkably shortened with the increase of PDLs, although no significant difference was observed between negative control and the stable miR-216a lentiviral infection cells ( Figure 2B).

| miR-216a promotes monocytes adhesion to endothelial cells monolayers
Because the PDL20 with miR-216a stable transfection (Lv-miR-216a) showed an enlarged shape and slow growth rate which was similar as the senescent status of naturally passaged PDL44, we therefore used PDL20 stable line to explore the effects of miR216a on endothelial functions. The results showed that miR-216a increased the adhesion ability of THP-1 cells to HUVECs by 106% (P = .002) and 95% (P = .001) ( Figure 3A) in PDL8 and PDL20 stable line of Lv-miR216a, respectively. The expressions of adhesion molecules ICAM1 and VCAM1 were also remarkably increased by 34% and 52% in PDL8 Lv-miR216a cells and were up-regulated by 30% and 1.79-fold in PDL20 Lv-miR216a cells, respectively ( Figure 3B).
On the other hand, for the experiments of miR-216a inhibitor, the naturally senescent PDL44 line was used and transiently transfected by antagomiR-216a to explore the changes of endothelial functions after the inhibition of endogenous miR-216a. Once miR-216a function was inhibited, the endothelial adhesion ability of to THP-1 cells was decreased by 19% (P = .01) ( Figure 3C).

| miR-216a inhibits proliferation and migration during endothelial cells growth
The MTS assay, scratch test and tube formation assay were performed to assess the effects of miR-216a on endothelial cells proliferation, migration and angiogenesis function, respectively. miR-216a overexpression was not found to change the cells proliferation activity and wound healing ability at the beginning of infection with miR-216a in PDL8 cells. After infection at about 15 days, stable overexpression of miR-216a suppressed cells proliferation by 15% (P < .001) and migration ability by 16% (P < .001) in senescent PDL20 stable line, respectively ( Figure 4A and C).
Similarly, antagomiR-216a was transiently transfected into naturally senescent PDL44 line to investigate the effect of miR-216a on proliferation and migration. Once miR-216a function was inhibited, the endothelial proliferative ability was increased by 12% (P = .02) and migration ability was increased by 24% (P = .03), respectively ( Figure 4B and D). Additionally, miR-216a had no significant effect on angiogenic activity of endothelial cells in both young PDL8 and senescent PDL20 stable line of Lv-miR-216a ( Figure S4). TGFb1, Smad3 and IjBa were markedly decreased in senescent PDL44 as compared to young PDL8 HUVECs; moreover, this decrease was negatively correlated with natural miR-216a level.

| Smad3 is a direct target of miR-216a
There was no correlation between Smad2 mRNA expression and miR-216a level ( Figure 6A).
In PDL8 cells, overexpression of miR-216a resulted in marked degradation of Smad3 as well as IjBa, but no significant change on Smad2, p65 protein levels and their phosphorylation ( Figure 6B). Next, to confirm the negative regulation of miR-216a on IjBa, Smad3 siR-NAs and miR-216a inhibitor were transfected to PDL8 and PDL44 cells, respectively. As expected, IjBa expression was diminished by silencing Smad3, and consequently, the mRNA expression levels of downstream adhesion molecules ICAM1 and VCAM1 were up-regulated ( Figure 6C). On the contrary, once endogenous miR-216a was inhibited, Smad3 and IjBa expression were up-regulated and ICAM1 and VCAM1 expression were down-regulated ( Figure 6D). All these results supported the role of miR-216a as a natural inhibitor of Smad3 in promoting endothelial inflammation via Smad3/IjBa pathway.

| Plasma miR-216a level is elevated in old patients with coronary artery diseases
The regulatory effects of miR-216a on endothelial senescence and dysfunction suggest an important role of miR-216a in the pathogenesis of age-associated vascular diseases. To assess the association between miR-216a and risk of CAD, particularly in those ageing individuals, plasma miR-216a levels were examined in 176 CAD patients and 342 age, gender-matched healthy controls. The clinical characteristics of patients and controls were described in

| DISCUSSION
Vascular endothelial cells senescence is one of major risk factors for atherosclerosis and cardiovascular diseases. Emerging evidence indicate that miRNAs represent a common molecular mechanism that underlies the cell senescence. In the current study, we provided evidence for the first time that the age-progressive increase in miR- has been reported to express in vascular wall, including endothelial cells, vascular smooth muscle cells, monocytes/macrophages, regulatory T cells and myofibroblasts. 16 Expression of TGFb1 and receptors is increased in human atherosclerotic plaque lesion, especially in fibroproliferative regions, compared with non-atherosclerotic regions. 17,18 TGFb1 can stimulate endothelial migration, proliferation and angiogenesis at low concentrations, but inhibit these functions at higher concentrations, which associated with increased extracellular matrix. 19 In this study, the TGFb1 expression in senescent PDL44 HUVECs was significantly decreased compared with that in young PDL8 cell line. inhibition of miR-216a can rescue the impairment of autophagy during endothelial ageing. 21 However, the causal link between miR-216a and endothelial senescence was not studied in these reports. Here, we first clarified that miR-216a induced endothelial senescence and promoted endothelial inflammation via Smad3/IjBa pathway, which thus extended the knowledge on miR-216a.
In the current study, our results found that the presence of increased SA-b-gal activity was later than the elevation of p53 expression in PDL8 line after miR-216a transfection for 3 days. p53 is well-recognized to play a key role in cell-cycle control, DNA repair and cellular stress responses, and recent studies has reported that p53 activation of p53 can regulate apoptosis, cellular senescence and organismal ageing. 22 SA-b-gal is an extensively utilized marker for cell senescence, and the b-galactosidase activity can be detected at pH 6.0 in replicative or induced senescence cells but undetectable from proliferating cells. 23 Levels of p53 often gradually increase during the process of replicative senescence, whereas the presence of SA-b-gal marker can distinguish senescent cells during cell passages independent of DNA synthesis. 23 Thus, a time-lag may exist between the time-point of increased p53 levels and the presence of SA-b-gal activity. For example, it has been reported that exogenous IGF-binding protein 5 (IGFBP-5) markedly increased p53 expression after 90 minutes in HUVECs, while SA-b-gal activity was found to be elevated after prolonged treatment IGFBP-5 for 6 days. 24 miR-216a is expressed in several vascular cells, including monocytes/macrophages, 20 vascular smooth muscle cells and senescent endothelial cells. 21 Circulating endothelial progenitor cells (EPCs) can modulate the repair and regeneration of the endothelium associated with vascular ageing, atherosclerosis and CAD. It has been reported that elderly healthy subjects had lower circulating EPCs with impaired function and increased senescence when compared to young people. 25 Circulating EPCs obtained from CAD patients, compared with from age-matched healthy subjects, showed shorter telomere length and lower telomerase activity. 26 These emerging evidence supported that the decreased number and increased senescence of circulating EPCs in older CAD patients contribute to vascular endothelial dysfunction and ageing. Further comparison of miR-216a expression in plasma and circulating angiogenic cells with senescent phenotype between older and younger CAD patients, such as EPCs, would help clarify the underlying mechanisms of miR-216a in vascular repair and ageing related to atherosclerotic diseases.
In summary, we provided evidence for the first time that the ageprogressive increase in miR-216a triggers endothelial senescence and promotes the endothelial inflammation by targeting Smad3 and leading to NF-jB signalling activation. Moreover, in aged CAD patients, miR-216a plasma level was significantly higher than in healthy controls. This extends the knowledge of mechanisms involved in both the functions of miRNAs and their application serving as new potential biomarkers and therapeutic targets for endothelial dysfunction and related atherosclerotic diseases in aged individuals.

ACKNOWLEDG EMENTS
We thank the technicians and staffs at the State Key Laboratory of Cardiovascular Disease for data collection and management.

CONFLI CTS OF INTEREST
We have no conflicts of interest to disclose.