Endothelial replicative senescence delayed by the inhibition of MTORC1 signaling involves MicroRNA-107

Accepted Manuscript Title: Endothelial Replicative Senescence Delayed by the Inhibition of MTORC1 Signaling Involves MicroRNA-107 Authors: Eng-Soon Khor, Pooi-Fong Wong PII: DOI: Reference:

S1357-2725(18)30128-6 https://doi.org/10.1016/j.biocel.2018.05.016 BC 5373

To appear in:

The International Journal of Biochemistry & Cell Biology

Received date: Revised date: Accepted date:

7-2-2018 22-5-2018 29-5-2018

Please cite this article as: Khor E-Soon, Wong P-Fong, Endothelial Replicative Senescence Delayed by the Inhibition of MTORC1 Signaling Involves MicroRNA-107, International Journal of Biochemistry and Cell Biology (2018), https://doi.org/10.1016/j.biocel.2018.05.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Endothelial Replicative Senescence Delayed by the Inhibition of MTORC1 Signaling Involves MicroRNA-107

Eng-Soon Khor a, Pooi-Fong Wong a,* Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur,

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Malaysia. * Corresponding author: Email address: [email protected] (P.F. Wong).

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Other authors: Email address: [email protected] (E.S. Khor)

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There are 6 figures and 2 supplementary figures in this paper.

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4875 words (abstract, main text, figure legends)

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ABSTRACT

Accumulation of senescent endothelial cells can contribute to endothelium dysfunction.

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Suppression of MTOR signaling has been shown to delay senescence but the mechanism that underpins this effect, particularly one that involves miRNAs, remains to be further defined. This study sought to identify miRNAs involved in MTORC1-mediated inhibition of

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replicative senescence in endothelial cells. Pre-senescent HUVECs were prolonged treated with low dose rapamycin (1 nM), an MTOR inhibitor. Rapamycin treatment down-regulated the phosphorylated MTOR, RPS6 and 4EBP1 expressions, which confirmed MTORC1 suppression. Prolonged low dose rapamycin treatment has significantly reduced the percentage of senescence-associated beta galactosidase (SA-β gal) positively stained 1

senescent cells and P16INK4A expression in these cells. On the contrary, the percentage of BrdU-labelled proliferating cells has significantly increased. RPTOR, a positive regulator of MTORC1 was knockdown using RPTOR siRNA to inhibit MTORC1 activation. RPTOR knockdown was evidenced by significant suppressions of RPTOR mRNA and protein expression levels. In these cells, the expression of miR-107 was down-regulated whereas

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miR-145-5p and miR-217 were up-regulated. Target gene prediction revealed PTEN as the target of miR-107 and this was confirmed by biotin pull-down assay. Over-expression of

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miR-107 has decreased PTEN expression, increased MTORC1 activity, induced cell cycle

arrest at G0/G1 phase and up-regulated P16INK4A expression but mitigated tube formation.

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Collectively, our findings revealed that delayed endothelial replicative senescence caused by

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the inhibition of MTORC1 activation could be modulated by miR-107 via its influence on

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PTEN.

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Abbreviations: SA-β gal, senescence-associated beta galactosidase; MTOR, mammalian target of rapamycin; 4EBP1, eIF4E binding protein 1; S6K1, S6 kinase 1; miRNA, microRNA; RISC, RNAinduced silencing complex; UTR, un-translated region; HUVEC, human umbilical vein endothelial cell; ECM; endothelial cell medium; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; RPTOR, regulatory associated protein of MTOR complex 1; PTEN, phosphatase and tensin homolog; RPS6, ribosomal protein S6; CDK6, cyclin dependent kinase 6.

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Key words: MicroRNA-107; Mammalian target of rapamycin; Rapamycin; Cellular senescence;

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Human endothelial cell

1. Introduction

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Cellular senescence, which is also known as replicative senescence, is defined as a state of irreversible cell cycle arrest after prolonged population doubling of the cells. Although non-proliferative, these cells remain metabolically active with altered cellular functions (Campisi 2000). Its hallmark characteristics include enlarged and flattened cell morphology, increased senescence-associated beta galactosidase (SA-β gal) activity, increased granularity

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and vacuolization.

Cellular senescence can potentially impair the repair capacity of the endothelial lining and

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eventually contribute to age-related vascular diseases such as cardiovascular diseases and

atherosclerosis (Fenton et al. 2001). Accumulated evidence showed that endothelial cell

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senescence is induced by reactive oxygen species (ROS) through the p53-dependent DNA

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damage pathway (Adelibieke et al. 2012; Kim et al. 2009) but the involvement of other

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critical pathway such as the mammalian target of rapamycin (MTOR) pathway remains to be

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further explored.

MTOR is a serine/threonine protein kinase of the phosphatidylinositol-3-OH kinase

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(PI3K) related family. It plays a pivotal role in regulating various eukaryotic physiological functions (Wang and Proud 2011). MTOR complex 1 (MTORC1) regulates cell growth and

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proliferation, whereas, the function of MTOR complex 2 (MTORC2) is less well defined. Activation of MTORC1 inhibits translation initiation factor 4E (eIF4E) binding protein 1

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(4EBP1) and activates S6 kinase 1 (S6K1) through phosphorylation, which in turn favour critical protein synthesis (Blommaart et al. 1995; Hara et al. 1998). Inhibition of MTOR by

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rapamycin has been shown to delay senescence in vitro (Demidenko et al. 2009; Pospelova et al. 2012), in vivo (Khor et al. 2016), and prolong lifespan in mice (Harrison et al. 2009). In particular, attenuation of MTORC1 has been shown to delay cellular senescence in fibroblasts (Kolesnichenko et al. 2012).

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MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at posttranscriptional level. MiRNAs are incorporated into the effector ribonucleoprotein complex RISC (RNA-induced silencing complex) and are guided to the targeted mRNAs by binding to the 3’ un-translated region (3’ UTR). Upon binding, whether in perfect and partial

translational repression would ensue, respectively (Pillai et al. 2007).

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complementarity to the targeted mRNA 3’ UTRs, endo-nucleolytic degradation and

MiRNAs are known to regulate cellular senescence and endothelial cell functions (Ono et

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al. 2011; Wong et al. 2017; Jong et al. 2013). Nevertheless, molecular mechanisms that underpin miRNAs involvement in endothelial senescence remain to be further clarified due to

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the complexity of miRNA networks in mRNA regulation. The present study sought to

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investigate the role of miRNAs associated with MTORC1 pathway in endothelial senescence.

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Delineation of the mechanisms involved will unveil the specific role of miRNA, its potential

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interactive genes and shed more lights into the modulation of age-related vascular diseases by

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miRNAs.

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2.1 Cell culture

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2. Materials and methods

Human umbilical vein endothelial cells (HUVECs; ScienCell) were cultured at 37 °C

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with 5% CO2 in endothelial cell medium (ECM) (ScienCell, #1001) supplemented with 5% fetal bovine serum (FBS) (ScienCell, #0025), 1% endothelial cell growth supplement (ECGS) (ScienCell, #1052) and 1% penicillin/streptomycin solution (P/S) (ScienCell, #0503). 2.2 Rapamycin treatment

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HUVECs at passage 15 were seeded with 6.0 x 105 cells per T-75 flask and 3.5 x 104 cells per well in 12-well plate for short and long-term treatments, respectively. Treatments began after 24 h of seeding. To determine which concentration of rapamycin can inhibit MTOR activation, the cells were treated with final concentrations of 0, 0.1, 0.5, 1.0 and 2.0 nM rapamycin (Calbiochem, #553210) in a total volume of 10 ml of culture medium per flask for

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24 h. To determine the effect of prolonged rapamycin treatment on HUVECs proliferation, the cells were treated with final concentrations of 1.0, 2.0 and 4.0 nM rapamycin for 18 days.

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During the 18 days of treatment, the cells were trypsinised at approximately 80% confluency and re-seeded equally in 12-well plates before each subsequent treatment. After 18 days of

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treatment, rapamycin was removed and the cells were continuously sub-cultured with equal

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seeding number until passage 27-28 in the absence of rapamycin. Cells treated with 0.0002%

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2.3 SA-β gal flow cytometry assay

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dimethyl sulfoxide (DMSO) were used as negative control.

The percentage of senescent (SA-β gal positive) HUVECs that were treated with

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rapamycin for 18 days and then sub-cultured until passage 25-27 in the absence of rapamycin were determined by flow cytometry. The cells were trypsinised and 7.5 x 104 cells/well were

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re-seeded in 6-well plates. After 48 h, the cells were treated with 2 ml of 50 nM of bafilomycin A1 (Calbiochem, #196000) per well for 1 h in CO2 incubator. Next, 5-

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dodecanoylaminofluorescein Di-β-D-galactopyranoside (C12FDG) (Life Technologies, #D2893) was added into the 2 ml mixture of medium and bafilomycin at the final concentration of 33 µM and incubated for 2 h. Lastly, 400 µl of 5% FBS in 1X PBS was used to re-suspend the cell and the cell suspension was collected in polystyrene round-bottom tube (BD Falcon). Negative controls used were the C12FDG-stained A549 cells and young

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HUVECs. In addition, unstained A549 and young HUVECs were used as controls to normalise background fluorescence.

2.4 BrdU incorporation assay

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HUVECs that were treated with rapamycin for 18 days and then sub-cultured until passage 25-27 in the absence of rapamycin were used for BrdU dye incorporation assay to

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determine the percentage of proliferating cells in the culture. The cells were trypsinised and 6.0 x 105 cells/flask were re-seeded in T-75 flask. After 48 h or when 70% confluency was

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obtained, the cells were starved by incubating in 10 ml of ECM containing 0.5% FBS without

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ECGS and P/S for 8 h in a CO2 incubator. Then, ECGS-free medium was discarded and

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replaced with complete ECM containing a final concentration of 10 µM of BrdU reagent. Cells were labelled with BrdU for 24 h in a CO2 incubator. Subsequent steps were performed

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according to the manufacturer’s instructions of the BrdU flow kit (BD Pharmingen). Negative

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controls used were the anti BrdU-APC-stained A549 cells and young HUVECs. In addition, unstained A549 and young HUVECs that were collected right after 1 h of incubation with

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DNase solution were used as experimental controls to normalise background fluorescence.

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2.5 siRNA transfection of Regulatory-associated protein of MTOR complex 1 (RPTOR)

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HUVECs at passage 15 (pre-senescent) were seeded with 1.4 x 105 cells per well in 6-

well plates. Twenty-four hours after seeding, each well was rinsed twice with 1X HBSS (Biowest, #L0606) and replenished with 1.6 ml of fresh medium containing basal ECM supplemented with 1% ECGS. Next, the cells were transfected with 100 nM of validated ONTARGETplus SMARTpool RPTOR siRNA (L-004107-00-0010; Dharmacon), which contains

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of a mixture of four individual siRNAs that target RPTOR at various positions in 0.2%

DharmaFECT reagent (Dharmacon) in a final total volume of 2 ml of medium. After 6 h of transfection, the medium was supplemented with 5% FBS, and cells were incubated for an additional 18 h. Twenty-four hours post-transfection, the old medium was discarded and

RNA and protein, 24 h and 48 h post-transfection, respectively.

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2.6 Mimic miRNA transfection

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replaced with fresh complete ECM medium. The transfected cells were harvested for total

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HUVECs at passage 9-10 were seeded with 1.4 x 105 cell per well in 6-well plates. The

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seeded cells were transfected with 50 nM mimic miR-107 (RiboBio) in 0.1% DharmaFECT

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reagent as described above.

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2.7 Protein extraction and immunoblotting

Total protein extraction and immunoblotting were performed following our previous

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study (Wong et al. 2017). Details of antibodies used in the present study are provided in

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supplementary materials and methods.

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2.8 Quantitative RT-PCR (qRT-PCR)

Total RNA isolation was performed using RNeasy Mini Kit (Qiagen) for mRNA

expression study and using miRNeasy Mini Kit (Qiagen) with enrichment of small RNA species for miRNA expression study. Purity of the extracted RNAs was assessed using NanoDrop 2000 (Thermo Scientific). For mRNA expression quantification, cDNA was

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synthesised from 250 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814), followed by real time PCR amplification using TaqMan Fast Advanced Master Mix (Applied Biosystems, #4444557). For miRNA expression quantification, cDNA was synthesized from 10 ng of total RNA using miRCURY LNA TM Universal RT microRNA PCR Universal cDNA Synthesis Kit II (Exiqon), followed by

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LNATM real time PCR amplification using ExiLENT SYBR Green Master Mix (Exiqon).

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2.9 miRNA qRT-PCR array

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Amplification of miRNAs was performed on a customised miRCURY LNATM

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microRNA Array (Exiqon) with pre-coated miRNA primers. UniSP6, the RNA spike-in

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control primer (CP) was used as the negative control while UniSP3, the RNA spike-in inter-

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plate calibrator (IPC) was used as the PCR positive control for plate normalisation. All miRNAs expression were normalised to that of 5S-rRNA. Details of miRNA primers are

In vitro tube formation assay

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2.10

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provided in supplementary materials and methods.

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Frozen growth factor-reduced (GFR) matrigel (BD Biosciences) was thawed at 4 0C a day

prior to the experiment. Firstly, 40-50 µl of matrigel was loaded into 96-well plates on ice

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using pre-chilled micro-pipette tips prior to incubation in CO2 incubator at 37 0C for at least 15 min. Meanwhile, HUVECs transfected for 48 h with mimic miRNA were trypsinised and 1.0 x 104 of HUVECs were seeded onto the matrigel and incubated for 4-6 h before images were captured using the Canon PowerShot A640 Digital Camera affixed to an inverted microscope (Carl Zeiss).

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2.11

Target prediction and biotin pull-down assay

Prediction of the interaction between miR-107 and its target genes was performed using the three most commonly used web-based prediction tools, including miRanda, PITA and

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TargetScan. Biotin pull-down assay was performed following our previous study (Wong et al.

2017). The detailed protocol for biotin pull-down assay is provided in supplementary

Statistical analysis

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2.12

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materials and methods.

All data are presented as mean ± standard deviation (SD). Statistical analysis were

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performed between two groups using unpaired Student’s t-test and between more than two groups using one-way analysis of variance (ANOVA) test, where applicable, by GraphPad

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3. Results

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Prism v6.0 software. Statistically significance is achieved when p <0.05.

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3.1 Rapamycin treatment attenuates MTORC1 signaling in HUVECs

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To determine which concentration of rapamycin could inhibit MTOR, HUVECs at

passage 15 were treated with rapamycin for 24 h at several low doses (0.1-2.0 nM) followed by immunoblotting analysis. The expression of phosphorylated MTOR (p-MTOR), which is the main component of MTORC1, was significantly suppressed at 0.5 nM rapamycin, whereas insignificant inhibition was observed at 1 or 2 nM (Fig. 1A). Moreover, its

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downstream proteins, phosphorylated ribosomal protein S6 (p-RPS6) (Fig. 1B) and phosphorylated 4EBP1 (p-4EBP1) (Fig. 1C) were significantly down-regulated with 0.5 nM rapamycin and at higher doses, which confirmed MTORC1 attenuation by low dose rapamycin treatment.

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3.2 Long-term low dose rapamycin treatment decreases senescence phenotypes of HUVECs

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HUVECs that were prolonged treated with low dose rapamycin (18 days in culture) and

then sub-cultured in the absence of rapamycin until passage 25-27 displayed normal cell

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morphology whereby the cells were relatively smaller in size compared to the larger cell size and higher granularity of the vehicle-treated senescent cells (Fig. 2B). There was also a

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significant decrease in SA-β gal activity in these cells, whereby, the HUVECs culture treated

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with 1 nM rapamycin consisted of only 18.7% SA-β gal-positive cells compared to the higher

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percentage of SA-β gal-positive cells in the vehicle control cells (44.8%, Fig. 2C). These

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results show that long-term exposure to low dose rapamycin has suppressed the development of senescence phenotype in HUVECs even though they were continuously sub-cultured till

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late passages to induce replicative senescence.

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3.3 Long-term low dose exposure to rapamycin preserves the proliferative potential of

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HUVECs

The proliferative potential of long-term low dose rapamycin treated cells was investigated

by calculating population doubling and the percentage of cells in S-phase. In the first three weeks of rapamycin treatment, population doubling was initially suppressed in the presence of rapamycin. Population doubling then gradually increased after the removal of rapamycin.

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Five weeks after the cessation of treatment, population doubling of the vehicle-treated senescent HUVECs has stopped. At day 40, population doubling of senescent HUVECs pretreated with 2 nM and 4 nM rapamycin has reached a plateau while the 1 nM rapamycin-pretreated senescent HUVECs continued to proliferate whereby cell growth eventually surpassed that of the vehicle-treated cells (Fig. 2A), despite equal re-seeding of cells at every sub-

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culture. This shows that long-term low dose exposure to rapamycin has preserved the

proliferative potential of HUVECs. Concomitantly, protein expression of P16INK4A was

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significantly down-regulated in 1 nM and 2 nM rapamycin-pre-treated senescent HUVECs (Fig. 2E). This shows that long-term low dose rapamycin treatment followed by rapamycin

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removal mitigated growth arrest associated with cellular senescence. Treatment with 1 nM

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rapamycin also resulted in increased proliferation with 43.1% cells in S-phase, a significant

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increase compared to the vehicle control with 15.8% cells in S-phase (Fig. 2D). These

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observations show that prolonged treatment with low dose rapamycin has prevented the loss

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of proliferative potential in senescent HUVECs at late passage.

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3.4 Knockdown of MTORC1-associated protein (RPTOR) in HUVECs

RPTOR is a positive regulator of MTOR and an interacting protein of MTORC1

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substrates, S6K1 and 4EBP1 (Laplante and Sabatini 2009). HUVECs at passage 15 were transfected with RPTOR siRNA to specifically suppress the MTORC1 pathway. Significant

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reductions in RPTOR mRNA (Fig. 3A) and protein expressions (Fig. 3B) were obtained following transfection with 100 nM RPTOR siRNA. Growth kinetic analysis of the transfected HUVECs showed a significant decrease in the normalised cell index (CI) from 84 h post-transfection onwards. This suggests that inhibition of MTORC1 activity via RPTOR knockdown suppressed cell proliferation (Fig. 3C).

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3.5 MTORC1-mediated suppression of endothelial senescence de-regulates miRNAs expression

To identify miRNAs that potentially play a role in both MTORC1 signaling and cellular

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senescence, miRNA profiling using PCR array was performed on HUVECs that were

prolonged treated with 1 nM rapamycin and on the pre-senescent HUVECs at passage 15

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with 100 nM RPTOR siRNA knockdown. Analysis revealed that miR-107 was downregulated by half-fold while miR-145-5p and miR-217 were up-regulated by 3 – 4 folds (Fig.

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S1).

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3.6 Over-expression of miR-107 accelerates cellular senescence and suppresses in vitro tube

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formation

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Over-expression of miR-107 in young HUVECs was established by transfection with 50 nM mimic miR-107 (Fig. 4A). Cell cycle analysis showed a significant increase in the

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percentage of cell population arrested at G0|G1 phase in the mimic miR-107 transfected cells compared to the mimic negative control cells (Fig. 4B). Moreover, the percentage of cell

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population in S-phase was also significantly lower in the mimic miR-107 transfected cells compared to the mimic negative control cells. This result shows that over-expression of miR-

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107 induced G0/G1 cell cycle arrest. Over-expression of miR-107 also resulted in significant augmentation of the expression of P16INK4A (Fig. 4C). In addition, over-expression of miR107 also suppressed in vitro tube formation (Fig. 4D) as demonstrated by a significant reduction in total tubule length and number of junctions (Fig. 4E).

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3.7 Target prediction of miR-107

miRanda, PITA and TargetScan 7.1 predicted that phosphatase and tensin homolog (PTEN) is a potential target gene for miR-107. PTEN gene scored good mirSVR and PITA scores of -1.0899 (Fig. S2A) and -11.44 (Fig. S2C), respectively. In TargetScanHuman 7.1,

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PTEN gene was predicted to be conserved (PCT: 0.32) and likely to be repressed (context++

score: -0.37) by miR-107 (Fig. S2B). For further verification, miRNA-mRNA complex pull-

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down assay was performed. Accordingly, qRT-PCR analysis demonstrated a significant

enrichment of PTEN mRNA in the pull-down miRNA-mRNA complex (Fig. 5A). In

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addition, PTEN protein expression was down-regulated following the over-expression of

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miR-107 (Fig. 5B) similar to that observed in the 1 nM rapamycin-treated pre-senescent

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HUVECs (Fig. 5D). Increased expression of p-RPS6, which indicated increased MTORC1

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activity was observed following the over-expression miR-107 in HUVECs (Fig. 5C).

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4. Discussion

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Previous studies reported that MTOR drives endothelial cellular senescence (Rajapakse et al. 2011) and vascular aging in rat (Rice et al. 2005), whereby, over-activation of MTOR

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switches the cells from irreversible cell cycle arrest to cellular senescence (Blagosklonny 2014). However, inhibition of MTOR pathway could also reduce cellular senescence

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(Rajapakse et al. 2011; Pospelova et al. 2012; Demidenko et al. 2009) and here, we sought to investigate the effects of MTORC1 inhibition on HUVECs growth and identify the miRNAs that are involved. It was previously demonstrated that the MTOR inhibitor, rapamycin at 0.5 nM and 1 nM could inhibit S6 kinase phosphorylation in MCF7 breast cancer cells (Foster and Toschi 2009) and MTOR phosphorylation in glioblastoma cells (Anandharaj et al. 2011),

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respectively. Similarly, we found that rapamycin as low as 0.5 nM inhibited the phosphorylation of MTOR, RPS6 and 4EBP1 proteins in HUVECs. It is established that long-term attenuation of MTORC1 by rapamycin delays the onset of senescence (Kolesnichenko et al. 2012; Pospelova et al. 2012) as rapamycin could repress the expression of senescence-associated proteins and cytokines (Xia et al. 2017). However, it is

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reported that rapamycin at certain concentrations could also partially inhibit cell proliferation

(Leontieva and Blagosklonny 2016; Pospelova et al. 2012). In the present study, prolonged

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exposure to low dose, 1 nM rapamycin resulted in stagnant population doubling and

eventually attenuated replicative senescence (reduced SA-β gal activity and P16INK4A

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expression) in comparison to the vehicle control cells that senesced when propagated in

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parallel with equal seeding number. These low dose rapamycin-treated cells also had elevated

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BrdU dye uptake activity, indicating improved proliferative potential. Hence, our findings are

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in agreement with those of Demidenko et al. (2009) and Pospelova et al. (2012) which demonstrated that rapamycin could decelerate the loss of proliferative potential whereby, cell

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proliferation would resume upon the removal of rapamycin (Demidenko et al. 2009; Pospelova et al. 2012). Similarly, Gidfar et al. (2017) also observed that SA-β gal activity

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was reduced in 2 nM rapamycin-treated human corneal epithelial cells (Gidfar et al. 2017). It is observed that prolonged exposure to higher rapamycin doses, 2 nM and 4 nM

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rapamycin resulted in cell proliferation arrest and the development of replicative senescence, similar to the vehicle control cells propagated in parallel. This suggests that overtime

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accumulation of relatively high rapamycin dose is overall detrimental to cell health. Hence, an optimized dose of rapamycin is necessary to inhibit cell proliferation or delay senescence as larger doses of rapamycin could kill cells indiscriminately as reported earlier (Pospelova et al. 2012; Leontieva and Blagosklonny 2016).

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Prolonged rapamycin treatment has been shown to also inhibit MTORC2 (Fang et al. 2013). Therefore, we performed knockdown of RPTOR to specifically inhibit MTORC1 and identify miRNAs that were de-regulated during MTORC1 inhibition. Not only our results corroborated with those of the previous studies that demonstrated successful RPTOR knockdown in HUVECs (Zhang et al. 2014) using 100 nM RPTOR siRNA; we further

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identified miR-107, miR-145-5p and miR-217 that are potentially involved in both

endothelial senescence and MTORC1 pathway. Previously, miR-145 and miR-217 were

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found up-regulated in senescent human urothelial carcinoma cells and human endothelial

cells, respectively (Chen et al. 2015). In addition, Jin et al. (2013) also observed an up-

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regulation of miR-217 by more than 1-fold in rapamycin-treated HUVECs (Jin et al. 2013).

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However, miR-145 has also been demonstrated to enhance the proliferation of endothelial

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progenitor cells (Chen et al. 2005), but this could be due to cell type-dependent effect. These

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findings, nevertheless, are supportive of the role of both miR-145 and miR-217 in replicative senescence.

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Cellular senescence is caused by inappropriate growth promotion during cell cycle arrest which then drives the arrested cells into senescence (gero-conversion) (Leontieva and

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Blagosklonny 2014; Demidenko et al. 2010; Blagosklonny 2012; Blagosklonny 2009). This suggests that conversion from reversible arrest to senescence can be prevented by suppressing

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growth-promoting pathway such as MTOR (Leontieva and Blagosklonny 2016). Accordingly, we showed the inhibition of MTORC1 by prolonged low dose rapamycin has

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augmented cell proliferation and reduced senescence phenotypes, suggesting that geroconversion has been suppressed in these cells. However, inhibition of MTORC1 by RPTOR knockdown has instead inhibited cell proliferation, similar to the observations in earlier studies (Li et al. 2011; Wang et al. 2015). It is not surprising to observe cell proliferation inhibition instead of promotion during transient inhibition of MTORC1, which is

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accompanied by the down-regulation of miR-107 as we only achieved increased population doubling after 46 days in culture with prolonged rapamycin treatment. The role of miR-107 in the knockdown RPTOR cells that displayed inhibited proliferation as opposed to improved proliferative potential in 1 nM rapamycin-treated cells remains unclear. This irreconcilation could be due to the identification of miRNA in two different settings.

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To date, there is no report on the role of miR-107 in endothelial senescence although a

previous study has demonstrated that miR-107 is down-regulated in older individuals (Noren

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Hooten et al. 2010). It was previously reported that miR-107 is highly expressed in human

endothelial cells (Suarez et al. 2007) and its down-regulation promotes endothelial progenitor

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cells differentiation (Meng et al. 2012). Our findings revealed that miR-107 was down-

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regulated in prolonged low dose rapamycin-induced delayed senescence in HUVECs whereas

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its over-expression induced cell cycle arrest at G0/G1 phase and increased expression of

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P16INK4A, a cellular senescence marker. Previously, an earlier study reported that miR-107 down-regulates cyclin dependent kinase 6 (CDK6) (Takahashi et al. 2009), a cell cycle

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regulator. Since P16INK4A is the inhibitor of CDK6, this finding indicates that cell cycle arrest at G0/G1 phase could be mediated through the interaction of P16INK4A and CDK6.

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These findings collectively suggest that miR-107 plays a role in cellular senescence. In addition, miR-107 down-regulation is also associated with MTORC1 inhibition caused by

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RPTOR knockdown. Hence, restoration of HUVECs growth is likely contributed by the modulation of cell cycle by miR-107, underscoring its role in regulating cell growth,

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differentiation and death via critical cell survival pathway such as MTOR. Our results showed that increased MTORC1 activity, as evidenced by the up-regulation

of p-RPS6 protein expression following the over-expression of mimic miR-107 has led to the down-regulation of PTEN expression. PTEN is identified as a target of miR-107 in a recent study (Xiong et al. 2017) and in ours. PTEN is a negative regulator of the PI3K-AKT-MTOR

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signaling (Zhou et al. 2007) and it was demonstrated that MTOR pathway is constitutively up-regulated in cancerous cell due to the loss of PTEN (Seront et al. 2013). Hence, it is plausible that miR-107 could augment MTORC1 activity via its influence on PTEN. Intriguingly, PTEN protein expression was down-regulated in 1 nM rapamycin-treated HUVECs even though miR-107 was found down-regulated in these cells, suggesting of the

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involvement of a less straightforward regulatory mechanism. In this regard, Das et al. (2012) demonstrated that inhibition of MTORC1 by rapamycin or RPTOR knockdown decreases

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PTEN levels and in contrast, constitutive activation of MTOR increases the expression of

PTEN transcriptionally. They further showed that rapamycin-mediated down-regulation of

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PTEN enhances MTORC1 activity by activating the downstream protein AKT,

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demonstrating the presence of a feedback regulation in the PTEN-MTORC1 interaction (Das

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et al. 2012). Instead of up-regulation, the down-regulation of PTEN in the 1 nM rapamycin-

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treated HUVECs, hence, is in agreement to the observation by Das et al. (2012) (Fig. 6, solid arrow).

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Up-regulation of miR-107 has been shown to inhibit human glioma angiogenesis (Chen et al. 2016) and tumour angiogenesis in mice (Yamakuchi et al. 2010). Similarly, our data

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showed over-expression of miR-107 inhibited tube formation. Although a previous study has demonstrated that loss of PTEN up-regulates angiogenesis (Tian et al. 2010), we postulate its

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anti-angiogenesis effect involves other mechanisms such as VEGF pathway but this requires further clarification in the future. Moreover, loss of PTEN is associated with increased

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P16INK4A-induced cellular senescence in mouse embryonic fibroblasts (Alimonti et al. 2010) and this suggests that up-regulation of miR-107 may favour augmentation of P16INK4A expression and induction of endothelial senescence that could in turn inhibit tube formation.

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In summary, the present study has demonstrated that attenuation of MTORC1 by longterm low dose rapamycin treatment delays endothelial senescence, with concomitant decrease in miR-107 expression level. Over-expression of miR-107 increases MTORC1 activity and down-regulates PTEN expression which lead to endothelial replicative senescence and

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inhibition of tube formation.

Conflict of interest

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The authors declared no competing interest.

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Financial support

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This research was supported by a University of Malaya Research Grant (RG500-13HTM) and

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University of Malaya Postgraduate Research Grant (PG306-2016A).

Author contributions

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P. F. Wong designed the study; E. S. Khor performed the experiment, analysed the data and

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Fig. 1. Suppression of MTORC1 activation with low dose rapamycin treatment and

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siRNA knockdown of RPTOR in HUVECs. (A) (B) (C) Representative immuno-blot images of MTOR, RPS6 and 4EBP1 following various rapamycin doses treatment.

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Densitometry analysis of p-MTOR, p-RPS6 and p-4EBP1 normalised to total MTOR, RPS6

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and 4EBP1, respectively. Rapamycin is abbreviated as RAPA. Data represent the mean ± SD,

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n=3. Statistical significance as analysed by one-way ANOVA is expressed as *p <0.05, ***p

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<0.001, ****p <0.0001 versus vehicle control.

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Fig. 2. Decreased senescence phenotypes and increased proliferative potential with longterm low dose rapamycin treatment in HUVECs. (A) Cumulative population doubling

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curve illustrates the growth of prolonged low dose rapamycin-treated HUVECs followed by

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rapamycin removal for over 46 days. (B) Representative bright field images show the cell morphology of HUVECs at passage 25. Rapamycin-treated HUVECs were relatively smaller in cell size. (C) Representative flow cytometry dot plot shows cell population expressing SA-

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β gal activity. Bar graph displays the percentage of SA-β gal positively stained cell in 1 – 4 nM rapamycin-treated HUVECs. (D) Representative flow cytometry dot plot and bar graph show the population and the percentage of cells with BrdU uptake activity in HUVECs (passage 27-28) pre-treated with 1 – 4 nM rapamycin (18 days in culture). (E) Representative immuno-blot image and densitometry analysis of P16INK4A protein expression in 1 – 4 nM 22

rapamycin-treated HUVECs. Rapamycin is abbreviated as RAPA. Data represent the mean ± SD, n=3. Statistical significance as analysed by one-way ANOVA is expressed as *p <0.05, **p <0.01, ****p <0.0001 versus vehicle control.

Fig. 3. siRNA knockdown of RPTOR in HUVECs. (A) qRT-PCR analysis shows the

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down-regulation of RPTOR mRNA expression following siRNA RPTOR knockdown.

Protein bands intensity were normalised to that of GAPDH. (B) Representative immuno-blot

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image and densitometry analysis show the down-regulation of RPTOR protein expression. (C) Representative Real-Time Cell Analysis (RTCA) profile of HUVECs transfected with

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RPTOR siRNA. Results shown are mean of eight replicate wells for each group with cell

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index (CI) values normalised to the point after 24 h of cell seeding. MR indicates fresh media replenishment after the transfection medium was discarded. Data represent the mean ± SD,

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n=3. Statistical significance as analysed by one-way ANOVA and Student’s t-test are

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expressed as *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 versus vehicle control.

Fig. 4. Over-expression of miR-107 in HUVECs induced cellular senescence and

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diminished in vitro tube formation. (A) qRT-PCR analysis shows increased expression of miR-107 after transfection with mimic miR-107. RNU6 was used for normalisation. (B) The

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percentages of cell population in cell cycle phases and (C) P16INK4A protein expression in mimic miR-107 transfected cells. (D) (E) Representative phase contrast images and bar

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graphs of in vitro tube formation and total tubule length and number of junctions, respectively. Data represent the mean ± SD, n=3. Statistical significance as analysed by oneway ANOVA and Student’s t-test are expressed as *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001 versus vehicle control.

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Fig. 5. PTEN as miR-107 target. (A) qRT-PCR analysis displays the enrichment ratio of PTEN normalised to GAPDH following pull-down of miR-107-mRNA complex. Result showed enrichment of PTEN in HUVECs transfected with biotinylated hsa-miR-107. Representative immuno-blot images display PTEN expression in HUVECs transfected with mimic miR-107 (B) and treated with 1 nM rapamycin (D). Densitometry analysis of PTEN

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normalised to beta-actin. Rapamycin is abbreviated as RAPA. (C) Representative immuneblot image and densitometry analysis show p-RPS6 expression in mimic miR-107 transfected

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cells. Data represent the mean ± SD, n=3. Statistical significance as analysed by Student’s t-

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test is expressed as *p <0.05 versus vehicle control.

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Fig. 6. Schematic representation of miR-107-PTEN-MTORC1 regulation. Rapamycin-

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mediated down-regulation of MTORC1 reduces the PTEN expression. The solid arrow

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indicates the direct relationship of MTORC1 in modulating PTEN which acts upstream (Das

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