Human umbilical vein endothelial cells (HUVECs; ATCC^® PCS-100-013) were bought from ACTT (Manassas, VA, USA) and were cultured in RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂. HUVECs in the logarithmic growth phase were digested with trypsin and then cell suspensions of 1–5×10⁴ cells/ml were made. A total of 100 µl of the above cell suspensions was seeded per well in 96-well plates in triplicate. RPM (Selleck Chemicals, Houston, TX, USA) solutions at serials of concentrations (0, 1, 10 and 100 nM) were added to the cells. After incubation for another 48 h, a Cell Counting Kit-8 (CCK8) assay was used to determine the appropriate RPM concentration for subsequent experiments.

The appropriate intervention time for RPM treatment was then determined. HUVECs were treated with the selected RPM concentration determined by the above CCK8 experiment and were cultured for 0, 24, 48 and 72 h. The control group treated with an equal volume of pbs instead at each time point was normally cultured and treated with no RPM. The CCK8 assay was then performed.

A CCK8 assay was performed according to the supplier's instructions (Beyotime Institute of Biotechnology, Haimen, China). Specifically, 10 µl CCK-8 and 90 µl serum-free culture medium were added to each well and cultured at 37°C and 5% CO₂ for 1 h. The absorbance at 450 nm wavelength was measured with a plate reader (DNM-9602, Beijing, China) and the value of each well was recorded.

The mRNA from each group of cells was extracted and reverse transcribed into cDNA using reverse transcription kit (Fermentas; Thermo Fisher Scientific, Inc.). The resultant cDNA was used as a template for quantitative polymerase chain reaction (qPCR) detection. The primers for lncRNAs MALAT1 and SENCR are listed in Table I. β-actin was used as the reference gene. qPCR procedure followed the standard procedure of the SYBR-Green PCR kit (Thermo Fisher Scientific, Inc.): 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 45 sec, then 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. The results were analyzed with real-quantitative PCR & the 2^−ΔΔCt method.

The plasmid pcDNA 3.1+ (1.52 µg/µl; Invitrogen; Thermo Fisher Scientific, Inc.) was used to construct the overexpression vector for lncRNA SENCR. Specifically, EcoR1 and Not1 restriction enzyme sites were introduced to clone SENCR which was synthesized in vitro and this created a EcoR1-SENCR-Not1 fragment, then the full-length SENCR sequence was ligated to pcDNA 3.1+ vector with DNA ligase. Finally, the recombinant plasmid was verified by sequencing analysis. The transfection was performed according to the recommended protocol of Lipofectamine^™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as follows: i) HUVEC cells in the logarithmic growth phase were cultured at 5×10⁵ cells per well in a 6-well culture plate and incubated at 37°C and 5% CO₂ for 24 h; ii) the culture medium was changed to serum-free opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) 2 h before transfection; iii) the transfection reagent Lipofectamine^™ 2000 was diluted in opti-MEM at 1:20, and the mixture was kept at room temperature for 5 min; iv) Lipofectamine 2000 and lncRNA SENCR-overexpression plasmid (2.5 µg/well, empty plasmid vector with equivalent concentration was used as control in the transfection experiment) were mixed gently and kept at room temperature for 20 min; v) the mixture was added to the wells with 200 µl per well and the plate was cultured in the incubator at 37°C and 5% CO₂ for 6 h; vi) the mixture was aspirated and replaced with a complete medium and vii) the plate was cultured overnight at 37°C and 5% CO₂.

In all the proliferation, migration and angiogenesis experiments, cells were firstly treated by control medium, lncRNA SENCR overexpression (lncRNA SENCR OE), RPM (100 nM) or RPM plus lncRNA SENCR OE (RPM+lncRNA SENCR OE) for 24 h. For the detection of proliferation, CCK8 assay was used and the procedure was the same as described above. For the analysis of cell migration, scratch test and transwell test were performed. The wound was measured at 0, 8 and 12 h in the scratch test. In the transwell test, the above treated cells were collected and reseeded into the up chamber of the transwell insert (24-well plate; Corning Incorporated, Corning, NY, USA) at the density of 2×10⁵ cells per well with 200 µl serum free basic medium. 500 µl complete endothelial cell growth medium was placed in the lower chamber. The cells were incubated for 12 h at 37°C. Cells migrated to the lower surface of the filter were fixed with 70% methanol and stained with 0.5% crystal violet solution and then were imaged (magnification, ×100). For the quantification of the migrated cells, cell numbers were counted in 4 randomly selected fields (magnification, ×100). For angiogenesis detection, Matrigel was dissolved overnight at 4°C before being used. A total of 50 µl per well was added and incubated at 37°C for 1 h. The above treated cells were collected and reseeded into the Matrigel-coated wells at a density of 10⁴ cells per well. After 4 and 8 h of incubation at 37°C, 4 fields of view (magnification, ×200) were selected per well to analyze the tube length and the number of branches of the blood vessels.

After 24 h treatment with RPM, the cell cycle stage was detected in the HUVECs by flow cytometry. The cells were collected and stained by PI (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) staining. Specifically: i) the cells were washed by PBS twice and the cell concentration was set at 1×10⁶/ml; ii) the cell suspension was fixed at 70% ethanol at −20°C for 24 h and washed with PBS before staining; iii) 100 µg/ml RNase A was added and the mixture was incubated at 37°C for 30 min; iv) 50 µg/ml PI solution was added and the mixture was incubated at 4°C for 30 min in the dark; v) the sample was run on machine and the recording excitation wavelength was 488 nm and vi) cell cycle analysis was performed with FLOWJO software (v7.6.2).

Total mRNA was extracted and then reverse transcribed into cDNA. The resultant cDNA was used as the template for qPCR detection. The qPCR primers for VEGFA, VCAM-1 and p21 are listed in Table II. β-actin was used as the reference gene. The qPCR procedure followed the standard thermocycling protocol: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 45 sec, then 95°C for 15 sec; 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. The results were analyzed with the real-quantitative PCR & the 2^−ΔΔCq method (18).

HUVECs were cultured in 6-well plates for 24 h, then the total protein was extracted. Western blotting was performed to detect the protein expression levels of phosphorylated (p-ERK1/2, ERK1/2, p-mTOR and mTOR, with GAPDH as the reference gene. The procedure of western blotting followed the standard protocol. In brief, about 10⁶ cells were taken and lysed and the total protein was obtained; a 10% SDS-PAGE gel was prepared; 30 µg total protein was loaded in each lane and gel electrophoresis was performed at 60 V for 30 min and then at 90 V for 1 h; the protein bands on the gel were transferred to a polyvinylidene difluoride (PVDF) membrane under a steady current of 200 mA for 120 min; the PVDF membrane was blocked with 5% skimmed milk powder for 2 h, then the membrane was incubated with appropriate antibodies for p-ERK1/2 (Affinity, AF1014), ERK1/2 (Affinity, AF0155), p-mTOR (Affinity, AF3308), mTOR (Affinity, AF6308) with a dilution ratio of 1:1,000 and GAPDH (ab37168; Abcam) as the reference with a dilution ratio of 1:2,000. For chemiluminescence detection, an ECL western blotting kit (Thermo Fisher Scientific, Inc.) was used to react with the interest proteins. Tanon Automatic Chemiluminescence Image Analysis System (5200, China) was used to scan the light bands 5 min after the reaction and the matching software accompanying the machine was used for analysis.

Data were analyzed with SPSS v19 statistical software (SPSS, Inc., Chicago, IL, USA). All data are presented in the form of mean ± standard deviation. Comparisons among >2 groups were performed with one-way analysis of variance followed by the least significant difference post hoc method, and comparisons between two groups were performed with Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To confirm the effect of RPM on the proliferation of vascular endothelial cells, a CCK8 assay was performed on the HUVECs. As shown in Fig. 1, compared with the control group (0 nM RPM treatment), the proliferative capacity of HUVECs was significantly inhibited in a concentration-dependent manner. Specifically, the rate of inhibition was about 50% when the RPM concentration was 100 nM. Since the suppression was the most effective at this concentration, we selected 100 nM RPM to be taken forward in the present study. An obviously inhibited HUVECs proliferation after RPM treatment (100 nM) at all the time points could be observed (Fig. 1B). Therefore, to study the relationship between lncRNA SENCR and RPM treatment on HUVEC behavior, treatment with 100 nM RPM for 24 h was used in the follow-up experiments.

To study the relationship between RPM treatment and the endogenous expression of the two vascular endothelial cell function-related lncRNAs, MALAT1 and SENCR, in HUVECs, qPCR analysis was performed. Fig. 2 showed that both lncRNAs MALAT1 (P<0.01) and SENCR (P<0.01 in 24 h and P<0.001 in 48 h) genes were significantly downregulated in HUVECs after exposure to 100 nM RPM. It was noted that lncRNA SENCR showed a more remarkable downregulation than lncRNA MALAT1 after RPM treatment in 48 h. Therefore, lncRNA SENCR was selected to study the effect of lncRNA on the cellular functions of vascular endothelial cells after RPM treatment.

After the construction and transfection of the lncRNA SENCR-overexpression plasmid, we verified the overexpression of lncRNA SENCR in HUVECs. As shown in Fig. 3A, compared with the control group, the relative expression of the SENCR gene in transfected HUVECs was significantly increased, indicating the successful overexpression of lncRNA SENCR in HUVECs (P<0.001).

To study the effects of RPM treatment and overexpression of lncRNA SENCR on the functions of vascular endothelial cells, proliferation, migration and angiogenesis analysis of HUVECs was performed in detail. Firstly, the proliferation of HUVECs in Fig. 3B showed that compared with the control group, the lncRNA SENCR overexpression group enhanced and RPM treatment (100 nM for 24 h) decreased proliferation of HUVECs. However, RPM treatment associated with SENCR overexpression (RPM+lncRNA-SENCR OE) showed increased proliferation compared with RPM treatment alone. These results indicate that RPM could inhibit the proliferation of HUVEC cells and overexpression of lncRNA SENCR could alleviate the inhibitory effect of RPM on the proliferation of HUVECs.

We then examined the migration profile of HUVECs. The results of the scratch assay showed that lncRNA SENCR overexpression obviously decreased the scratch width of HUVECs, at either 8 or 12 h (P<0.001 vs. control; Fig. 4A and B) and RPM treatment significantly inhibited the cell migration, which got effectively relieved by high level of lncRNA SENCR (P<0.01 vs. RPM at 8 h, P<0.05 vs. RPM at 12 h; Fig. 4A and B). However, interestingly, the RPM+lncRNA-SENCR OE group demonstrated a smaller scratch width than the RPM group. Transwell assay provided a similar data (Fig. 4C and D) and it revealed that lncRNA SENCR upregulation partly reversed the inhibitory effect on cell migration in presence of RPM. These results suggest that RPM inhibits the migration of HUVECs and overexpression of the lncRNA SENCR could alleviate the inhibitory effect of RPM on the migration of HUVECs.

The angiogenesis detection was performed using the Matrigel method. The results in Fig. 5 are after cultivation for 4 and 8 h. Compared with the control group, the number and length of branches were significantly increased in the lncRNA-SENCR OE group and significantly reduced in the RPM treatment group at 8 h (P<0.01). However, consistent with the aforementioned cellular function results, the RPM+lncRNA-SENCR OE group showed an increased cell branch number and increased vessel length compared with the RPM treatment group alone, indicating that the overexpression of lncRNA SENCR relieves the inhibitory effect of RPM treatment on angiogenesis in HUVECs.

The effect of RPM treatment and overexpression of lncRNA SENCR on cell cycle progression was detected by flow cytometry. The flow cytometry analysis in Fig. 6 showed that the lncRNA-SENCR OE group had a reduced proportion of G0/G1 phase cells and the RPM treatment group had an increased proportion of G0/G1 phase cells compared with the control group. Not unexpectedly and also consistent with the above results, there was a decreased proportion of G0/G1 phase cells in the RPM+lncRNA-SENCR OE group. These data demonstrate that RPM treatment (100 nM for 24 h) blocks the proliferation of HUVECs in the G0/G1 stage, and overexpression of lncRNA SENCR could reverse the blocking effect of RPM on HUVEC proliferation and decreased the percentage of G0/G1 cells from 78.9 to 60.4%.

In order to further investigate the mechanism that supports the above results, we performed analysis by qPCR of the level of related genes in HUVECs after different treatments. Fig. 7 showed that the VEGFA and VCAM-1 genes were significantly downregulated after RPM treatment, however the p21 gene was remarkably upregulated (P<0.01). Moreover, RPM+lncRNA-SENCR OE treatment reversed the expression of VEGFA almost to control cells (P=0.053; Fig. 7A) and significantly relieved the upregulation of the p21 gene and downregulation of the VCAM-1 gene caused by RPM treatment (P<0.05; Fig. 7B and C).

p21 is a negative regulator of the cell cycle, which can arrest the cell cycle in the G1 phase and inhibit DNA replication. VEGFA and VCAM-1 are related genes for vascular endothelial growth and cell adhesion. These results demonstrate that RPM treatment could inhibit angiogenesis and cell proliferation, and the overexpression of the lncRNA SENCR could alleviate the inhibitory effect of RPM on the angiogenesis and proliferation of HUVECs.

As the ERK1/2 pathway is one of the most influential pathways associated with cellular responses to various external stimuli and mTOR has an important role as the mammalian target of RPM, we next examined the involvement of these proteins during the RPM treatment and lncRNA SENCR overexpression in HUVECs. Fig. 8A showed the expression of several proteins involved in the RPM-related signaling pathway in HUVECs, demonstrating that the phosphorylation of ERK1/2 and mTOR proteins decreased upon RPM treatment. However RPM+lncRNA-SENCR OE treatment alleviated the downregulation of p-ERK1/2 and p-mTOR proteins caused by RPM treatment. Specifically, RPM+lncRNA-SENCR OE treatment reversed the decrease of the p-ERK1/2/ERK1/2 ratio caused by RPM treatment to the normal level (P<0.01, vs. RPM group; Fig. 8B). It was noted that RPM+lncRNA-SENCR OE treatment also significantly increased the ratio of p-mTOR/mTOR (P<0.05 vs. RPM group; Fig. 8C). These results suggested that, as shown in Fig. 9, RPM treatment could inhibit the activation of the ERK1/2 and mTOR pathway, and overexpression of lncRNA SENCR could relieve the inhibitory effect of RPM on the ERK1/2 and mTOR pathway.