A drug-RNA interaction predictor software (rnanut.net/drip) was used to examine the potential interaction between Rb2 and miR-216a on the basis of its sequence and structural features. The MST assay is a biophysical technology for testing interactions between protein and protein or nucleic acid, as well as interactions between nucleic acid and small molecules (15). In the current study, the MST experiment was applied to assess the interaction between Rb2 and miR-216a. The wild-type or mutant-type probe of miR-216a was labeled with 5-carboxyfluorescein (5′FAM), and the sequences were 5′-UAAUCUCAGCUGGCAACUGUGA-3′ for wild-type probe, and 5′-UAUAGAGUGCUGGCAACUGUGA-3′ for mutant-type probe. Rb2 (purity 98.26%; MedChemExpress) was dissolved in RNase-free water, and then serial doubling dilutions of Rb2 were created from 1 mM to 30 nM for the MST experiment. The 5′FAM-labeled probe of miR-216a (500 nM) and Rb2 dilutions were mixed in a 1:1 ratio and incubated for 5 min at room temperature in the dark. The mixtures were then filled into the capillaries, and the florescence was detected at 400 nm of excitation light wavelength using the Monolith NT.115 MTS instrument (NanoTemper Technologies GmbH). The binding ability between miR-216a and Rb2 was assessed using the dissociation constant (K_d).

The primary human umbilical vein endothelial cells (HUVECs) were isolated from the human umbilical cord veins, as described previously (9,16,17). The umbilical cord samples were donated by 3 healthy pregnant women (aged, 25–30 years) at Beijing Haidian Material & Child Health Hospital (Beijing, China) and collected from January to February, 2020. Informed consent was obtained from the donors. The study was approved by the ethics committee of Fuwai Hospital. The HUVECs were cultured using the endothelial cell medium (ScienCell Research Laboratories, Inc.) supplemented with 1% endothelial cell growth supplement (ScienCell Research Laboratories, Inc.), 5% FBS (ScienCell Research Laboratories, Inc.) and 1% penicillin-streptomycin solution in an incubator with 5% CO₂ at 37°C.

A replicative senescent model of endothelial cells was established by passaging, and the number of population-doubling level (PDL) was calculated using the equation: PD=log₂ (Ch/Cs), in which Ch is the number of viable cells at harvest and Cs is the number of cells seeded (6). The PDLs vs. time growth curves were obtained to assess the cellular replication potentiality over time. PDL8 was identified as young HUVECs at a culture of 5–8 days, and PDL44 as senescent cells at a culture of ~75 days. In this study, given the potential difference in the differentiate rate of HUVECs, in order to calculate the PDLs accurately when establishing the replicative senescence model, the HUVECs derived from different donors were not mixed. However, the main experiments were replicated in HUVECs from three independent donors to assess the findings.

Human aortic endothelial cells (HAECs) from a pool of three individuals (lot nos. 11,035, 3,535 and 11,385; cat. no. 6100; ScienCell Research Laboratories, Inc.) were cultured to evaluate the effects of Rb2. HAECs were cultured in the same condition as HUVECs.

The PDL8 and PDL44 HUVECs were cultured and grown to 90% confluency, and were then treated with Rb2 at various concentrations of 0.1, 1, 10 and 100 µM for 24 h at 37°C, in order to investigate the effect of Rb2 on the expression of endogenous miR-216a.

miRNA expression analysis was performed via reverse transcription-quantitative (RT-q)PCR. In brief, the total RNA was extracted from cultured cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was reverse transcribed with All-in-One™ miRNA First-Strand cDNA Synthesis kit (GeneCopoeia, Inc) at 37°C for 15 min and 85°C for 5 min. Next, the expression of miR-216a was measured by All-in-One™ miRNA qPCR kit (GeneCopoeia, Inc) using an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction conditions were as follows: initial denaturation at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 1 min and extension at 72°C for 10 min. U6 was used as internal reference and the relative gene expression of miR-216a was analyzed using the relative quantification (2^−ΔΔCq) method (18). The primer sequences of miR-216a and U6 (GeneCopoeia, Inc.) were as follows: miR-216a-5p 5′-TCTCAGCTGGCAACTGTGAAA-3′ and U6 5′-GGTCGGGCAGGAAAGAGGGC-3′.

Lentiviruses expressing miR-216a and the negative control (NC) vector were constructed by Shanghai Genechem Co., Ltd. According to the manufacturer's instructions, the coding sequence of pre-miR-216a was inserted into GV369 vector (Ubi-EGFP-MCS-IRES-puromycin) and the construct was verified via DNA sequencing. Next, to collect the lentiviral particles, 293T cells were transfected with 20 µg lentiviral vectors mixed with 1 ml transfection reagent (Genechem Co., Ltd) at 37°C for 48 h, and cell culture supernatants were harvested 48 h after transfection, centrifuged at 4,000 × g at 4°C for 10 min and passed through the 0.45 µM filter to remove cellular debris. Finally, lentiviral particles were resolved in PBS and aliquoted for storage at −80°C as viral stocks.

To establish the stable cell line of miR-216a, HUVECs were infected with pre-miR-216a recombinant lentiviruses (Lv-miR-216a) or NC vectors (Lv-NC) (Shanghai Genechem Co., Ltd.). PDL4 HUVECs were seeded in 24-well cell culture plate (Corning, Inc.) at a density of 5×10⁴ cells/well and added with 5 µl lentiviral vector diluent of 1×10⁸ transducing unit (TU/ml) in each well for a 24-h infection at 37°C, and then the medium was replaced with fresh growth medium. The multiplicity of infection was 10. After 3 days, cells were treated with 400 ng/ml puromycin (Sigma-Aldrich; Merck KGaA) for selection, and the lentivirus infection efficiency (>95%) was evaluated via the enhanced green fluorescent protein florescence analysis. Then, infected HUVECs were passaged every 3 days and treated with puromycin for selection. PDLs were calculated during the passage. PDL25 cells with stable expression of miR-216a (at ~40 days following Lv-miR-216a infection) were assessed as the senescent cell line and used for subsequent experiments.

To examine whether Rb2 could affect the target gene expression of miR-216a, PDL8 HUVECs were transfected with miR-216a mimics (Shanghai GenePharma Co., Ltd.) at a final concentration of 50 nM using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After incubation for 6 h at 37°C, cells were treated with Rb2 (10 µM) at 37°C for 48 h and harvested for expression analysis.

To evaluate the effect of Rb2 on the Smad3/IκBα pathway, PDL8 HUVECs were transfected with Smad3 siRNA (si-Smad3; Shanghai GenePharma Co., Ltd.) at a final concentration of 40 nM using Lipofectamine 3000 reagent. After incubation for 6 h at 37°C, cells were treated with Rb2 (10 µM) at 37°C for 48 h and then were harvested for expression analysis. The sequences were as follows: miR-216a mimics sense, 5′-UAAUCUCAGCUGGCAACUGUGA-3′ and anti-sense, 5′-ACAGUUGCCAGCUGAGAUUAUU-3′; and Smad3 siRNA sense, 5′-AGGACGAGGUCUGCGUGAAUCCCUA-3′ and anti-sense, 5′-UAGGGAUUCACGCAGACCUCGUCCU-3′.

Endothelial senescence was assessed via in situ staining for SA-β-gal positive cells using Senescence β-Galactosidase Staining kit (Beyotime Institute of Biotechnology). β-galactosidase catalyzes the hydrolysis of X-gal, producing a blue color that can be easily visualized under a microscope (19). According to the manufacturer's protocol, cells were treated with fresh β-galactosidase staining solution (pH 6.0) for 16 h at 37°C without CO₂. The SA-β-gal-positive cells were identified as blue stained cells and images were analyzed using Leica DMI4000 B inverted light microscopes (magnification, ×10; Leica Microsystems GmbH). The percentage of SA-β-gal positive-staining cells was calculated by counting five random microscopic fields per sample. The experiment was repeated five times for each group.

The endothelial proliferative and adhesive abilities were assessed in the senescent PDL25 HUVECs (at ~40 days after transfection of Lv-miR-216a). The cell proliferative ability was assessed using the Cell Counting Kit-8 (CCK-8) method (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, HUVECs were seeded into 96-well plate at a density of 5×10³ cells/ml and cultured in a 5% CO₂ incubator at 37°C for 24 h, and were then treated with Rb2 (10 µM) for 24 h at 37°C. Next, the cells were incubated with 10 µl CCK-8 solution for an additional 1, 2 and 3 h at 37°C. The absorbance was detected at a wavelength of 450 nm on a LB960 microplate reader (Titertek-Berthold).

The human acute monocytic leukemia cell line (THP-1; China Infrastructure of Cell Line Resources, Beijing, China) was used to assess the endothelial adhesive ability to monocytes. THP-1 cells were labeled with CellTracker™ CM-Dil (red fluorescence; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min at 37°C, washed twice with PBS and then were re-suspended in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA) at a density of 1×10⁶ cells/ml. The CM-Dil labeled THP-1 cells were incubated with PDL25 HUVECs at 37°C for 30 min in an incubator containing 5% CO₂. The non-adherent THP-1 cells were removed by PBS, and the adherent cells were fixed with 4% paraformaldehyde (Beijing Leagene Biotechnology Co., Ltd.) at room temperature for 15 min. The nuclei of cells were stained with DAPI (OriGene Technologies, Inc.) at room temperature for 20 min, and the number of adhesive THP-1 cells to endothelial cells was observed and imaged in five random fields at wavelengths of 359 and 549 nm using a confocal laser scanning microscope (magnification, ×20) SP8 (Leica Microsystems GmbH). The experiments were repeated five times.

The mRNA expression levels of p53, p21, vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule (ICAM1) and Smad3 were analyzed via RT-qPCR (primer sequences are listed in Table SI). Briefly, total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was reverse transcribed using PrimeScript™ RT Master Mix (Takara Bio, Inc.) under the following thermocycling conditions: 37°C for 15 min, 85°C for 5 sec. RT-qPCR was performed using SYBR Green qPCR mix (Shanghai Yeasen Biotechnology Co., Ltd.) on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction conditions were conducted as following: Initial denaturation for 10 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing and elongation at 60°C for 1 min, and final extension at 72°C for 1 min. GAPDH expression was used as internal control, and the expression of target gene was calculated using relative quantification (2^−ΔΔCq) method (18).

Protein expression levels were determined via western blot analysis. In brief, HUVECs were harvested and proteins extracts were isolated with ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitor (Roche Diagnostics GmbH). The total concentration of protein was determined using a Pierce BCA Protein Assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). Each sample protein was loaded at 20 µg and separated by 10% SDS-PAGE, and then transferred to nitrocellulose membranes (EMD Millipore). Subsequently, the membrane was blocked with 10% non-fat milk at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C. The primary antibodies were purchased from Cell Signaling Technology, Inc., including the anti-p53 (1:1,000; cat. no. 2527T), anti-p21^Waf1/Cip1 (1:1,000; cat. no. 2947T), anti-ICAM1 (1:1,000; cat. no. 67836T), anti-VCAM1 (1:1,000; cat. no. 39036S), anti-Smad3 (1:1,000; cat. no. 9523T) and anti-IκBα (1:1,000; cat. no. 4812S). The mouse monoclonal anti-GAPDH antibody (1:2,000; cat. no. TA309157; OriGene Technologies, Inc.) was applied as an internal control. Then, membranes were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) or anti-mouse IgG secondary antibody (1:2,000; ZB2305; OriGene Technologies, Inc.) for 1.5 h at room temperature. Bands were visualized with FluorChem R, M and E Systems (ProteinSimple) and analyzed using ImageJ software (v1.5.1; National Institutes of Health).

Data are presented as the mean ± SD. For the comparison between two groups, group differences were analyzed compared using an unpaired Student t-test. For the comparisons ≥3 groups, group differences were analyzed using one-way ANOVA with Tukey's post hoc test. Each experiment was repeated ≥3 times independently. Data were analyzed using SPSS 19.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

The bioinformatics analysis with drug-RNA interaction predictor software (rnanut.net/drip) suggested that Rb2 may have a high affinity with miR-216a. In the subsequent MST experiment for assessing the interaction between Rb2 and miR-216a, the results demonstrated that Rb2 bound to the wild-type probe of miR-216a and the K_d value was 17.6 µM (Fig. 1A). By contrast, if the seed sequence of miR-216a was mutated, Rb2 was not detected to bind the mutant-type probe (Fig. 1B), indicating that Rb2 can bind to miR-216a specifically.

A premature senescent model of endothelial cells induced by miR-216a was established. The PDLs-vs.-time growth curves indicated that, in the PDL25 line at ~40 days after transfection with Lv-miR-216a, the cells showed senescent features, characterized by enlarged size and flattened morphology (Fig. 2A), as well as accumulation of SA-β-gal within cytoplasm (blue staining; Fig. 2B), which was similar as the senescent status of naturally passaged PDL44 (at a culture of ~75 days). The percentage of SA-β-gal-positive cells was markedly increased by 130% in the PDL25 line transfected with Lv-miR-216a compared with the NC (P=0.01; Fig. 2C). Moreover, the mRNA expression levels of the senescent-related cell cycle inhibitors p21 and p53 were upregulated by 34% (P=0.05) and 38% (P=0.02; Fig. 2D), respectively; further protein expression analysis also showed that p21 (P=0.05) and p53 (P<0.01) were significantly upregulated (Fig. 2E).

To verify whether pre-miR-216a recombinant lentiviruses were successfully transfected in HUVECs, miR-216a expression was examined using RT-qPCR. Compared with NC (Lv-NC), the expression level of miR-216a in the Lv-miR-216a cell line was increased by 6900% (P<0.01; Fig. S2).

Serial concentrations of Rb2 were applied to examine the effects of Rb2 on endogenous miR-216a expression. In young PDL8 HUVECs, Rb2 treatment with concentrations of 1, 10 or 100 µM significantly decreased miR-216a expression by 40% (P=0.01), 30% (P=0.02) and 36% (P=0.02), respectively (Fig. 3A). Moreover, in senescent PDL44 HUVECs, Rb2 did not appear to affect miR-216a expression at low doses, whereas a relative high dose (100 µM) of Rb2 exerted a significant inhibitory effect on miR-216a expression by 44% (P=0.01; Fig. S3), supporting the previous finding that endogenous miR-216a was highly expressed in senescent endothelial cells (9). However, the concentration of 100 µM Rb2 has previously been shown to induce cytotoxic effects on endothelial cells (20). In HAECs, Rb2 (10 µM) also decreased the expression level of endogenous miR-216a by 34% (P=0.02; Fig. S4). Given that the K_d value was 17.6 µM in the MST experiment assessing the special binding ability between Rb2 and miR-216a, the concentration of 10 µM of Rb2 was selected as the optimal dose and used in further experiments.

Next, the potential role of Rb2 in endothelial senescence and dysfunctions induced by miR-216a was assessed. In the premature senescent model of PDL25 HUVECs with Lv-miR-216a transfection, the percentage of SA-β-gal-positive cells were increased by 174% (P<0.01; Fig. 3B) compared with the Lv-NC; whereas the senescent features and accumulation of SA-β-gal within cytoplasm induced by miR-216a were reversed following Rb2 treatment, and the percentage of SA-β-gal-positive cells decreased by 76% in the Lv-miR-216a+Rb2 group compared with the Lv-miR-216a group (P<0.01; Fig. 3B). The elevated mRNA and protein expression levels of p53 (P=0.05) and p21 (P=0.04) induced by miR-216a were not significantly affected by Rb2 treatment (Fig. 3C and D).

In senescent PDL25 HUVECs with overexpression of miR-216a, the adhesive ability of endothelial cells (DAPI-staining) to THP-1 cells (CM-Dil-staining) was increased by 180% compared with the NC (P<0.01), whereas this promoting effect of miR-216a was significantly inhibited by Rb2 treatment (P<0.01; Fig. 4A and B). Consistently, the mRNA expression level of adhesion cytokine VCAM1 was upregulated by 78% by Lv-miR-216a (P=0.02), while this effect was inhibited by Rb2 (P=0.04; Fig. 4C), and these effects were also observed via further protein expression analysis (both P<0.05; Fig. 4D). Additionally, the proliferative ability of endothelial cells was examined in senescent PDL25 cells, and the inhibitory effect of miR-216a was not affected by Rb2 (Fig. S5).

Our previous study has shown that miR-216a can directly inhibit the Smad3/IκBα signaling pathway, and therefore, promote the endothelial inflammatory process (6). Thus, the current study transfected miR-216a mimics in PDL8 HUVECs to observe the effect of miR-216a on the protein expression levels of Smad3 and IκBα. To verify whether miR-216a mimics was successfully transfected, miR-216a expression was examined using RT-qPCR. Compared with the NC group, the expression level of miR-216a was increased by 422-fold (P<0.01; (Fig. S6). The results consistently demonstrated that miR-216a decreased the protein expression levels of Smad3 and IκBα (P<0.01) in PDL8 HUVECs transfected with miR-216a mimics, and the role of miR-216a was significantly reversed by Rb2 treatment (P<0.01; Fig. 5A).

To further assess whether Rb2 reversed the role of endogenous miR-216a in the Smad3/IκBa pathway, Smad3 siRNA was transfected to PDL8 HUVECs. As expected, the mRNA and protein expression levels of Smad3 were greatly decreased by silencing Smad3 (P<0.01; Fig. 5B), and consequently the mRNA and protein expression levels of downstream adhesion molecules ICAM1 and VCAM1 were upregulated (both P<0.05; Fig. 5C). By contrast, when exposed to Rb2, the expression levels of ICAM1 and VCAM1 were reversed, although the findings were not significant (Fig. 5C). These results suggested that Rb2 attenuated the inflammatory process by inhibiting the expression level of Smad3 targeted by miR-216a.