lncRNA-ES3/miR-34c-5p/BMF axis is involved in regulating high-glucose-induced calcification/senescence of VSMCs

The expression of miR-34c-5p was decreased in high glucose-induced HA-VSMCs calcification/senescence

Mineralized nodule formations revealed by Alizarin Red S staining were increased greatly in high glucose (HG)-induced HA-VSMCs for 14 days (Figure 1A), and SA-β-gal staining showed that the SA-β-gal positive cells were significantly increased as well (Figure 1B). Osmolarity control (OC), however, had no significant effects on calcification/senescence of HA-VSMCs (Figure 1A and 1B). qRT-PCR revealed that the level of miR-34c-5p was strikingly decreased in HA-VSMCs treated with HG (Figure 1C). Nevertheless, the level of miR-34c-3p had no significant difference between HA-VSMCs treated with normal glucose (NG) and those with HG (Figure 1D). Moreover, OC had no significant effect on expression of both miR-34c-5p and miR-34c-3p compared with NG. These results suggested that miR-34c-5p might be involved in regulating HA-VSMCs calcification/senescence.

Figure 1. The expression of miR-34c-5p in HG-induced HA-VSMCs. (A) HA-VSMCs were treated with NG, OC, or HG for 14 days and then subjected to Alizarin Red S staining. The calcium content was extracted with cetylpyridinium chloride and quantified by spectrophotometry. Representative pictures are shown and the scale bar is 100 μm. (B) HA-VSMCs were treated with NG, OC, or HG for 72 hours and then subjected to SA-β-gal staining. Semi-quantitative analysis of SA-β-gal positive cells were performed using Image J. (C and D) qRT-PCR showing the expression of miR-3c-5p and miR-34c-3p in the above three groups. The data are expressed as mean ± SD, n=3, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose.

miR-34c-5p was involved in attenuating calcification/senescence of HA-VSMCs

In order to demonstrate whether miR-34c-5p was involved in regulating calcification/senescence of HA-VSMCs, the gain- and loss-of-function approach were used to overexpress or inhibit the expression of miR-34c-5p, respectively. MiR-34c-5p mimics and inhibitor were transfected into HA-VSMCs to overexpress or inhibit expression of miR-34c-5p. The qRT-PCR results showed that the miR-34c-5p mimics induced miR-34c-5p levels by about 50-fold and the miR-34c-5p inhibitor decreased the expression of miR-34c-5p significantly (Figure 2A). Meanwhile, the ALP activity, OC secretion, and Runx2 protein levels were significantly decreased when overexpressing miR-34c-5p, whereas the opposite occurred when inhibiting its expression (Figure 2B–2D). In addition, the overexpression of miR-34c-5p significantly decreased the level of senescence markers p16 and p21 proteins, whereas inhibiting the expression of miR-34c-5p produced the opposite results (Figure 2D). Therefore, these data indicated that miR-34c-5p plays a negative role in the process of calcification/senescence of HA-VSMCs.

Figure 2. miR-34c-5p inhibiting the calcification/senescence of HA-VSMCs. (A) HA-VSMCs was transfected with miRNA NC, miR-34c-5p mimics, and miR-34c-5p inhibitor, and subjected to qRT–PCR analysis of miR-34c-5p. (B–D) HA-VSMCs were transfected with miRNA NC, miR-34c-5p mimics, and miR-34c-5p inhibitor, respectively. Then, ALP activity, OC secretion, and Runx2, p16, and p21 protein levels were measured. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NC: negative control.

lncRNA-ES3 suppressed miR-34c-5p expression by direct interaction

Bioinformatics analysis showed that some complementary sites existed between miR-34c-5p and lncRNA-ES3 (Figure 3A). Moreover, qRT-PCR verified that the level of lncRNA-ES3 was markedly increased in HA-VSMCs treated with HG (Figure 3B). However, OC did not influence the expression of lncRNA-ES3. Hence, we intended to further explore whether miR-34c-5p could directly interact with lncRNA-ES3. Firstly, overexpression of miR-34c-5p by transfecting miR-34c-5p mimics to HA-VSMCs significantly reduced lncRNA-ES3 expression (Figure 3C). When introducing shlncRNA-ES3 to knockdown lncRNA-ES3 expression, we found that the shlncRNA-ES3-2 variant was the most effective to inhibit the expression of lncRNA-ES3 (Figure 3D), hence we chose it for the downstream study. In the knockdown study, the expression of lncRNA-ES3 elevated the expression of miR-34c-5p (Figure 3E). Subsequently, the luciferase reporter assay showed that the overexpression of miR-34c-5p significantly decreased the relative luciferase activity of the WT-lncRNA-ES3 reporter, but miR-34c-5p had no effect on luciferase activity of Mut-lncRNA-ES3 reporter, in which the putative binding sites between lncRNA-ES3 and miR-34c-5p were mutant (Figure 3F). Lastly, biotin-labeled miR-34c-5p pull-down assay was performed to verify that the sequence of miR-34c-5p could highly bind with lncRNA-ES3 (Figure 3G). Additionally, RIP assay was performed using Ago2 antibody to explore whether miR-34c-5p and lncRNA-ES3 were involved in RNA-induced silencing complex (RISC). The results showed that miR-34c-5p and lncRNA-ES3 were substantially enriched by Ago2 antibody compared with control IgG antibody (Figure 3H), which suggested that miR-34c-5p and lncRNA-ES3 were present in RISC. Based on these data, we could draw a conclusion that lncRNA-ES3 and miR-34c-5p could combine with each other in HA-VSMCs.

Figure 3. lncRNA-ES3 suppressed miR-34c-5p expression by direct interaction. (A) Schematic representation of the putative binding sites between lncRNA-ES3 and miR-34c-5p, and the mutant sites in Mut-lncRNA-ES3 reporter were underlined. (B) qRT-PCR showed the expression of lncRNA-ES3 in HA-VSMCs cultured with NG, OC, and HG. (C) HA-VSMCs was transfected with miRNA NC and miR-34c-5p mimics and harvested for the examination of lncRNA-ES3 by qRT-PCR. (D) The inhibitory efficiency of shRNAs targeting lncRNA-ES3 was verified by qRT-PCR. (E) HA-VSMCs were transfected with shRNA NC and shRNA-ES3-2, and the expression of miR-34c-5p was detected by qRT-PCR. (F) The WT-lncRNA-ES3 3’UTR and the Mut-lncRNA-ES3 3’UTR reporters were co-transfected with miR-34c-5p mimics or control oligos into HA-VSMCs. Forty-eight hours after transfection, luciferase activities were measured. (G) The expression of lncRNA-ES3 was detected by qRT-PCR after biotin-labeled miR-34c-5p pull-down assay. (H) RIP and qRT-PCR assays were performed to explore the binding efficiency of miR-34c-5p and lncRNA-ES3 to Ago2 protein in HA-VSMCs. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose; NC: negative control.

BMF was the target of miR-34c-5p

Bioinformatics analysis showed that the 3’UTR region of BMF was predicted to contain some potential binding sites for miR-34c-5p (Figure 4A). In cultured HA-VSMCs, overexpression of miR-34c-5p significantly reduced both the mRNA and protein level of BMF, while miR-34c-5p inhibitor dramatically increased them (Figure 4B and 4C). The following luciferase assay demonstrated that enforced expression of miR-34c-5p markedly suppressed the luciferase activity of WT-BMF reporter, while no change was observed in the luciferase activity of Mut-BMF reporter after miR-34c-5p overexpression (Figure 4D). In addition, in cultured HA-VSMCs, qRT-PCR assay revealed that the expression of BMF was markedly increased in HA-VSMCs induced by HG compared with NG (Figure 4E). Western blot demonstrated that HG also induced BMF protein level in HA-VSMCs (Figure 4F). Nevertheless, OC had no effect on both the level of mRNA and protein of BMF. Thus, the expression trend of BMF was consistent with that of lncRNA-ES3 in HA-VSMCs under HG. Western blot further showed that silencing of lncRNA-ES3 prominently inhibited BMF expression (Figure 4G). Collectively, these data indicated that BMF was the target of miR-34c-5p and that lncRAN-ES3 acted as a ceRNA of miR-34c-5p to regulate expression of the target gene, BMF, in HA-VSMCs.

Figure 4. BMF was the target of miR-34c-5p. (A) Schematic representation of the miR-34c-5p putative target sites in BMF 3’UTR and alignment of miR-34c-5p with WT and Mut BMF 3’UTR showing pairing. The mutated nucleotides were underlined. (B and C) HA-VSMCs were transfected with miRNA NC, miR-34c-5p mimics, and miR-34c-5p inhibitors, and harvested for the examination of BMF mRNA and protein. (D) The WT-BMF 3’UTR and Mut-BMF 3’UTR were co-transfected with miR-34c-5p mimics or control oligos into HA-VSMCs. Forty-eight hours after transfection, luciferase activities were measured. (E and F) qRT-PCR and Western blot analysis showed the expression level of BMF in HA-VSMCs cultured with NG, OC, and HG. (G) HA-VSMCs were transfected with shRNAs NC and shlncRNA-ES3-2, and the protein level of BMF was detected by Western blot. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose; NC: negative control.

miR-34c-5p inhibited whereas lncRNA-ES3 and BMF promoted calcification/senescence of HA-VSMCs

The biological roles of miR-34c-5p in calcification/senescence of HA-VSMCs were explored. Herein, overexpression of miR-34c-5p attenuated calcification of HA-VSMCs induced by HG, which were verified by a significant decrease of ALP activity, OC secretion, Runx2 expression, and the formation of mineralized nodules (Figure 5A–5E). Meanwhile, the decreased expression of p16 and p21, as well as the staining of SA-β-gal positive cells, confirmed that the senescence of HA-VSMCs transfected with miR-34c-5p mimics was also significantly alleviated (Figure 5C, 5F–5G). To evaluate the effect of lncRNA-ES3 and BMF on the calcification/senescence of HA-VSMCs, the expression of lncRNA-ES3 and BMF in HA-VSMCs were knocked down using shlncRNA-ES3 and siBMF, respectively. The results demonstrated that silencing of lncRNA-ES3 or BMF markedly attenuated the high glucose-stimulated HA-VSMCs calcification/senescence, which were confirmed by the decrease in ALP activity, OC secretion, Runx2, p16, and p21 expression, and mineralized nodules, as well as staining of SA-β-gal positive cells (Figure 5A–5G). Interestingly, the inhibitory effect of lncRNA-ES3 deficiency on HA-VSMCs calcification/senescence was greatly abrogated by miR-34c-5p inhibitor, as presented by the increase in ALP activity, OC secretion, Runx2, p16, and p21 expression, and mineralized nodules, as well as staining of SA-β-gal positive cells (Figure 5A–5G). Taken together, these results indicated that miR-34c-5p downregulation contributed to the calcification/senescence of HA-VSMCs, and that the mechanism might be mediated by lncRNA-ES3 and BMF in high glucose-induced HA-VSMCs.

Figure 5. miR-34c-5p inhibited whereas lncRNA-ES3 and BMF promoted the calcification/senescence of HA-VSMCs. (A and B) The ALP activity and OC secretion were detected in HA-VSMCs with different treatment, respectively. (C) Representative images of Western blot analyses of p16, p21, Runx2, and BMF in HA-VSMCs with different treatment are shown. (D and E) Alizarin Red S staining showed the mineralized nodules in HA-VSMCs, and the calcium content was extracted with cetylpyridinium chloride and quantified by spectrophotometry. Representative pictures are shown and the scale bar is 100 μm. (F and G) SA-β-gal staining showed the senescent cells of HA-VSMCs with different treatment, and the quantification of SA-β-gal-stained positive cells is shown. Representative pictures are shown and the scale bar is 100 μm. (H) The model proposed to explain the mechanism of miR-34c-5p in inhibiting VSMC calcification/senescence is shown. Herein, lncRNA-ES3 inhibits miR-34c-5p expression, enhances the expression of BMF, and finally promotes calcification/senescence of VSMCs. The data are expressed as mean ± SD, n=3, *p<0.05. NG: normal glucose; HG: high glucose; NC: negative control.

Cell culture

HA-VSMCs were purchased from ATCC (ATCC-CRL-1999). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 5% CO₂. HA-VSMCs were incubated at various times with DMEM plus 10% FBS in the presence of 5 mM glucose or 30 mM glucose. In some experiments, 30 mM mannitol was used as OC. The medium was refreshed every 2 days and cells were passaged every 3–4 days.

Alizarin Red S staining

Alizarin Red S staining was done as previously described [2, 23]. Briefly, HA-VSMCs cultured with 5 or 30 mM glucose or 30 mM mannitol for 14 days were fixed in 4% paraformaldehyde for 30 minutes at room temperature and then stained with 1% (pH 4.2) Alizarin Red S for 1–2 minutes at 37°C. The stained matrix was assessed and photographed using a digital microscope.

SA-β-gal staining

SA-β-gal staining was performed using a Senescence-associated β-Galactosidase Staining kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s protocol. Briefly, HA-VSMCs cultured with 5 or 30 mM glucose or 30 mM mannitol for 72 hours were fixed in β-galactosidase fixation solution (2% formaldehyde/0.2% glutaraldehyde in PBS) for 5 min and then washed three times with PBS. The cells were stained in SA-β-gal staining solution (pH 6.0) overnight at 37°C. The intensity of positive SA-β-gal staining was determined as previously described [25].

Measurement of ALP activity and osteocalcin

The confluent HA-VSMCs were washed with PBS twice then the cell layers were scraped into a solution. The cell lysates were homogenized and measured for ALP activity by ALP assay kit (A059-2, Jiancheng, Nanjing) and for osteocalcin secretory by osteocalcin assay kit (H152, Jiancheng, Nanjing) according to the manufacturer’s instructions. ALP activity and osteocalcin were normalized to total cellular protein of the cell layers by the Bradford protein assay as previously described [39].

Gene expression determined using qRT-PCR

Total RNA was extracted from cultured HA-VSMCs using Trizol Reagent (Invitrogen, 15596-026) [12]. For BMF mRNA and lncRNA-ES3 detection, cDNA was synthesized from 1 μg of total RNA using RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, K1631). Then, a 20-μl reverse-transcription reaction was carried out for 60 minutes at 42°C, followed by a second step of 10 minutes at 70°C and a final hold at 4°C. Quantitative PCR analysis was performed with SYBR Green PCR Master Mix (ABI 4309155) in a real-time fluorescence quantitative PCR instrument (ABI, 7900, USA). For qPCR analysis, 25-μl reactions were incubated in a 96-well optical plate at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. Data were normalized to GAPDH values.

For miR-34c analysis, total RNA was purified by miRNeasy Mini Kit (Qiagen, 217004), and miRNA Q-PCR Detection Kit (Genecopoiea, R0101L) was used as described by the manufacturer’s protocol using U6 snRNA as the reference. Briefly, a 25-μl reverse-transcription reaction was carried out for 60 minutes at 37°C, 5 minutes at 85°C, and a hold at 4°C. qPCR was performed for 5 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C, 20 seconds at 60°C, and 10 seconds at 72°C. All of the PCR primers used in this study are shown in Supplementary Table 1. The relative standard curve method (2^-△△CT) was used to determine the relative mRNA and miRNA expression. Results were expressed as fold change relative to the appropriate control. The qPCRs were run in triplicate and results are presented as the mean ± standard error of samples.

RNA interference

MiR-34c-5p mimics, inhibitor, and their negative controls were purchased from RiboBio (Guangzhou, China). ShlncRNA-ES3 (shlncRNA-ES3 #1,2,3,4), si-BMF, and their negative controls (si-con) were designed and synthesized by GenePharma Co. Ltd (Shanghai, China). The sequences of shlncRNA-ES3 and siBMF used in this study are shown in Supplementary Table 2. The cells were transfected using Lipofectamine 3000 (Invitrogen, USA). Lastly, the inhibitory efficiency of siRNAs was verified by qRT-PCR and the most effective shlncRNA-ES3 was used for the downstream functional experiments.

Western blot analysis

Protein expression analysis was detected by Western blot analysis as previously described [29, 40]. The membranes were incubated with primary antibodies, including anti-BMF (ab9655, 1:1000, Abcam), anti-Runx2 (ab76956, 1:1000, Abcam), anti-p16 (10883-1-AP, 1:1000, Proteintech), anti-p21 (10355-1-AP, 1:1000, Proteintech), and anti-GAPDH (sc-365062, 1:3000, Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with the horseradish peroxidase-conjugated goat anti-rabbit (sc-2004, 1:5000, Santa Cruz Biotechnology) or goat anti-mouse (sc-2005, 1:5000, Santa Cruz Biotechnology) secondary antibodies at 37°C for 1 hour. The immunoreactive bands were visualized using the ECL Plus Western blot detection kit (Amersham Biosciences U.K. Ltd) and densitometric quantification of band intensity from three independent experiments was carried out with the Image-Pro Plus 6.0 software. The relative expression level of target protein was normalized to the intensity of the GAPDH band.

Luciferase reporter assay

Luciferase reporter assay was performed to determine the binding of lncRNA-ES3 and miR-34c-5p as well as miR-34c-5p and BMF. Partial fragments of lncRNA-ES3 and BFM 3’UTR containing the predicted binding sites of miR-34c-5p were amplified by PCR and cloned into XbaI-FseI restriction sites of the pGL3 luciferase reporter vector (Promega, Madison, WI, USA). Meanwhile, the QuikChange Multi Site-Directed Mutagenesis kit (Stratagene, Lajolla, CA, USA) was employed to construct a mutant 3’UTR of lncRNA-ES3 and BMF. HA-VSMCs were co-transfected with a luciferase reporter carrying wild type lncRNA-ES3 3’UTR (WT-pGL3-lncRNA-ES3), mutant lncRNA-ES3 3’UTR (Mut-pGL3-lncRNA-ES3), wild type BMF 3’UTR (WT-pGL3-BMF), mutant BMF 3’UTR (Mut-pGL3-BMF), and miR-34c-5p mimics or scrambled oligos, respectively. Then, 48 hours after transfection, luciferase activities in cells were quantified via a luciferase reporter assay system (Promega) according to the protocols of the manufacturer.

RNA pull-down assay

RNA pull-down assays were performed using Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, 2016420). Briefly, biotin-labeled RNAs were transfected into HA-VSMCs and the cells were lysed using Thermo Scientific Pierce IP lysis buffer. Biotinylated RNAs were mixed and incubated with HA-VSMCs cell lysates. Streptavidin magnetic beads were added to each binding reaction and the magnetic beads were washed twice with 0.1 M NaOH and 50 mM NaCl. The magnetic beads were then resuspended in equal volume of 20 mM Tris (pH 7.5) and incubated with RNA-protein binding reaction buffer for 60 minutes at 4°C. Then, the bound RNA-protein complexes were washed and eluted from the magnetic beads. Lastly, RNAs in the complexes were purified and qRT-PCR assay was employed to measure the enrichment patterns of lncRNA-ES3 and miR-34c-5p.

RNA immunoprecipitation (RIP) assay

RIP assay were performed using EZ-Magna RIP kit (No. 17-701, Millipore, Billerica, MA, USA) and Argonaute 2 (Ago2) antibody (Abcam, ab32381) to explore whether miR-34c-5p and lncRNA-ES3 existed in RNA-induced silencing complex (RISC). Anti-SNRNP70 was taken as the positive control. Briefly, HA-VSMCs cells were lysed in RIP lysis buffer, followed by the incubation of protein A/G magnetic beads and antibody against rabbit IgG or Ago2. Then, RNAs in magnetic beads-binding complexes were purified. Lastly, qRT-PCR assay was employed to measure the enrichment patterns of miR-34c-5p and lncRNA-ES3 by IgG or Ago2 antibody.

Statistical analysis

The data are presented as mean ± standard deviation (SD) and were analyzed using GraphPad Prism software (GraphPad Prism version 6.0). The normality of data distribution was assessed before analysis. Student’s t-test was used to compare normally distributed data between two different groups, while one-way analysis of variance (ANOVA) was used for multiple groups. A level of p<0.05 was considered statistically significant. All experiments were repeated at least three times, and representative experimental results are shown in the figures.