HCMV-miR-US33-5p promotes apoptosis of aortic vascular smooth muscle cells by targeting EPAS1/SLC3A2 pathway

In patients with acute aortic dissection (AAD), increased vascular smooth muscle cell (VSMC) apoptosis has been found. Human cytomegalovirus (HCMV)-miR-US33-5p was significantly increased in the plasma of patients with AAD. However, the roles of miR-US33-5p in human aortic VSMC (HA-VSMC) apoptosis remain to be elucidated. In the current study, cell apoptosis was analyzed by flow cytometry, cell proliferation by CCK-8 assay, and differentially expressed genes by RNA sequencing. Luciferase reporter assay was used for binding analysis between miR-US33-5p and endothelial PAS domain protein 1 (EPAS1), and EPAS1 and amino acid transporter heavy chain, member 2 (SLC3A2). The enrichment degree of SLC3A2 promoter DNA was analyzed by chromatin immunoprecipitation assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting were performed for measuring messenger RNA (mRNA) and protein levels, respectively. It was found that HCMV infection inhibited proliferation but promoted HA-VSMC apoptosis by upregulating HCMV-miR-US33-5p. Transfection of HCMV-miR-US33-5p mimics the significant effect on several signaling pathways including integrin signaling as shown in the RNA sequencing data. Western blotting analysis confirmed that HCMV-miR-US33-5p mimics suppression of the activity of key factors of the integrin signal pathway including FAK, AKT, CAS, and Rac. Mechanistic study showed that HCMV-miR-US33-5p bound to the 3′-untranslated region of EPAS1 to suppress its expression, leading to suppression of SLC3A2 expression, which ultimately promoted cell apoptosis and inhibited cell proliferation. This was confirmed by the findings that silencing EPAS1 significantly reduced the SLC3A2 expression and inhibited proliferation and key factors of integrin signal pathway. HCMV-miR-US33-5p suppressed proliferation, key factors of integrin signal pathway, and EPAS1/SLC3A2 expression, but promoted HA-VSMC apoptosis. These findings highlighted the importance of HCMV-miR-US33-5p/EPAS1/SCL3A2 signaling and may provide new insights into therapeutic strategies for AAD.


Ultraviolet (UV) inactivation of viruses
HCMV AD169 and AD169 (ΔUS33) were grown in HELF cells and purified by ultracentrifugation on a 10-50% sucrose gradient according to Zucker's method [33]. The virus was titrated by plaque-forming assay using HELF cells and stored in small aliquots at −80 °C. Purified HCMV was irradiated with 5 × 10 3 µJ/m 2 using a UV crosslinker (Agilent Technologies, CA, USA). Under these conditions, no infectious virions were detected.

Cell transfection
The following were all purchased from GenePharma (Shanghai, China): HCMV-miR-US33-5p mimics (mimics sequence: GAU UGU GCC CGG ACC GUG GGCG), control miRNA (UCA CCG GGU GUA AAU CAG CUUG), EPAS1, and SLC3A2 small interfering RNAs and their corresponding controls (Table 1). Transfection was carried out in accordance with the Lipofectamine 3000 instructions (Invitrogene, CA, USA). The miRNA or siRNA and Lipofectamine 3000 were diluted with Opti-MEM ((Invitrogene) and incubated for 5 min. After mixing the two in proportion, they were incubated for 20 min to form an RNA/Lipofectamine 3000 complex. The complex was added to the cell culture medium, and the cells were harvested after 24-h incubation.

Cell viability assay
Cell viability was measured with CCK-8 (Beyotime Biotechnology, C0037, Shanghai). Each group's cells were trypsinized and then inoculated at 2000 cells/well in 96-well plates. At 0, 24, 48, and 72 h after cell inoculation, 10 μl CCK-8 solution was loaded and incubated for 1 h. The optical density at 450 nm (OD450) was recorded using a microplate reader.

Apoptosis detection
Apoptosis was measured with the Annexin V-PI analysis kit (Beyotime, C1062) on a flow cytometer (FACScan; BD). In short, the cells were harvested, washed, resuspended, mixed with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), incubated in the dark for 20 min, and analyzed in a flow cytometer (BD Biosciences, NJ, USA).

Caspase-3 activity
Caspase-3 activity was measured with the GreenNuc caspase-3 assay kit for live cells (Beyotime, C1168) by following the manufacturer's instructions. After treatment, the cells were collected, washed, and mixed with GreenNuc caspase-3 substrate. After 30-min incubation at 37 °C in the dark, the caspase-3 activity levels were determined by a fluorescence microplate reader. The relative caspase-3 activity was calculated according to the treated-to-control cell ratio.

RNA sequencing
Total RNA was isolated with TRIzol (Invitrogen

GTP-Rac assay
GTP-Rac quantitative analysis in different cell groups was carried out with the GTP-Rac detection kit (CST, #8815) as described previously [35]. Cells were lysed with a lysis buffer, and then the lysates were incubated in binding buffer containing 20 μg GST-p21-activated kinase binding domain and glutathione-agarose beads for 3 h at 4 °C. The beads were washed and eluted. Total Rac and bound Rac (GTP-Rac) were analyzed by immunoblotting using anti-Rac antibody (CST, #4651, 1:1000).

RNA binding protein immunoprecipitation (RIP) assay
The RIP experiment was performed according to the EZMagna RIP kit's instructions (Millipore). HCMV was used to infect HA-VSMCs at multiplicity of infection (MOI) of 5. After 72 h, the cells were lysed with RIP lysis buffer and incubated with magnetic beads bound to anti-Ago2 antibody (CST, 2897), or normal mouse immunoglobulin G (IgG). Proteins were removed by proteinase K at 55 °C for 30 min. RNA purity and content were evaluated by NanoDrop 1000 (Thermo Fisher Scientific, IL, USA). RNA was purified using RNeasy Micro kit (Qiagen, Germany, Dusseldorf ) and detected by fluorescence quantitative PCR.

Chromatin immunoprecipitation (ChIP)-qPCR assay
For ChIP analysis, SimpleChIP Plus sonication chromatin IP kit (CST, #9005) was used as described previously [36]. After the HCMV-infected HA-VSMCs were cross-linked with formaldehyde, the cells were sonicated for chromatin fragmentation, and then incubated overnight with anti-human EPAS1 antibody (CST, #59973) at 4 °C. Then, Protein G magnetic beads (CST, #9006) were added and incubated with shaking for 2 h at 4 °C. DNA on the beads was washed and eluted. A standard curve with 2% input chromatin DNA in serial dilutions (undiluted, 1:5, 1:25, and 1:125) was generated and used for measuring the enrichment degree of the SLC3A2 promoter DNA. PCR products were subjected to agarose gel electrophoresis.

Statistical analysis
Statistical analysis was done by SPSS version 17.0 (SPSS, Chicago, IL, USA), and data are expressed as mean ± standard deviation (SD). Differences between two groups and among more than two groups were measured by Student's t-test and one-way analysis of variance (ANOVA), respectively. P < 0.05 was defined as statistically significant.

Identification of differentially expressed genes (DEGs) following HCMV-miR-US33-5p transfection by RNA-seq analysis
To investigate how transfection of HCMV-miR-US33-5p affects HA-VSMC proliferation and apoptosis, high-throughput sequencing was used to find DEGs in HA-VSMCs  Table S1, Additional file 3: Table S2) using a cutoff of |log 2 FC| > 1.0, FDR < 0.05. GO and KEGG analysis of these DEGs showed that transforming growth factor (TGF)-β signaling, integrin signaling, and type II diabetes mellitus signaling pathway were the signaling pathways most affected by transfection of HCMV-miR-US33-5p mimics (Fig. 3C, D). The integrin signaling pathway, which plays critical roles in cell apoptosis and AAD, attracted our attention. Thus, its key factors were analyzed. The Western blot results also showed that transfection of HCMV-miR-US33-5p mimics decreased the phosphorylation of key factors of the integrin signal pathway, including FAK, AKT, and CAS ( Fig. 3E-G). Rac activation was also suppressed by HCMV-miR-US33-5p mimics (Fig. 3H). Together, these data suggest that HCMV-miR-US33-5p might suppress integrin signaling in HA-VSMCs.

HCMV-miR-US33-5p downregulates SLC3A2 by targeting EPAS1
High-throughput sequencing data analysis found that EPAS1 decreased significantly after transfection of HCMV-miR-US33-5p mimics (Fig. 5A), which was verified by fluorescence quantitative PCR and Western blot (Fig. 5B, C). The SLC3A2 promoter sequence was then analyzed, and a typical EPAS1 binding site sequence at positions −789 to −794 (Fig. 5D) was found. So, the interaction between EPAS1 and SLC3A2 was tested. Luciferase reporter assay showed that EPAS1 promoted upregulation of wild-type SLC3A2 promoter (ACG TGC ) activity but showed no effects on mutant SLC3A2 promoter (TAA AAA ) (Fig. 5E). ChIP-qPCR indicated that EPAS1 was significantly enriched in the SLC3A2 promoter region (Fig. 5F, G), indicating that EPAS1 HCMV-miR-US33-5p downregulates SLC3A2 by targeting EPAS1. A RNA sequencing data showing EPAS1 differential expression between the HCMV-miR-US33-5p and control groups. B, C HCMV-miR-US33-5p was transfected into HA-VSMCs. qRT-PCR (B) and Western blot (C) were used to analyze the EPAS1 expression level in HA-VSMCs. Western blot was performed three times; representative images are presented. Numbers above lanes represent the relative protein level, normalized relative to control samples. D Potential EPAS1 binding sites in SLC3A2 promoter region. E Luciferase reporter assay of the effect of EPAS1 on the SLC3A2 promoter. F ChIP assay showed that EPAS1 bound to SLC3A2 promoter. G ChIP-qPCR assay of the enrichment of EPAS1 to SLC3A2 promoter. H Potential HCMV-miR-US33-5p binding site in EPAS1 promoter. I Luciferase reporter assay of the effect of HCMV-miR-US33-5p on the EPAS1 promoter. J RIP analysis of the enrichment of Ago2 on 3′-UTR of EPAS1 and HCMV-miR-US33-5p. **P < 0.01, ***P < 0.001 (n = 3, ANOVA analysis for A and D; Student's t-test for F) bound to the SLC3A2 promoter. Bioinformatics predictions indicated that there are HCMV-miR-US33-5p binding sites in the 3′-UTR region of EPAS1 (Fig. 5H). So, the interaction between EPAS1 and HCMV-miR-US33-5p was tested. The luciferase reporter assay results indicated that HCMV-miR-US33-5p bound to 3′-UTR of EPAS1 to inhibit its expression (Fig. 5I). RIP test showed that that Ago2 antibody simultaneously enriched EPAS1 mRNA and HCMV-miR-US33-5p, indicating that the two can form RNA-induced silencing complex (Fig. 5J). These data indicate that HCMV-miR-US33-5p downregulates SLC3A2 by targeting EPAS1.

Discussion
HCMV is closely associated with various cardiovascular diseases, including hypertension, atherosclerosis, and graft vascular disease [37,38]. Here, it is reported that HCMV promotes HA-VSMC apoptosis, which is correlated with the pathogenesis of AAD [18][19][20], by upregulating an HCMV-encoded miRNA, HCMV-miR-US33-5p. It was also proved that HCMV-miR-US33-5p inhibited the key factors of the integrin signaling pathway in HA-VSMCs. Further studies indicated that miR-US33-5p targeted EPAS1 to suppress its expression, and downregulated EPAS1 resulted in SLC3A2 suppression. Knockdown of EPAS1 and SLC3A2 in HA-VSMCs inhibited the integrin signal pathway molecules' activity, and cell proliferation, but promoted cell apoptosis. For the first time, it was shown that HCMV-miR-US33-5p suppressed proliferation and promoted HA-VSMC apoptosis by targeting EPAS1/SLC3A2. HCMV is a double-stranded DNA virus. The positive rate of adult seroantibodies in the world is more than 60-90% [39]. Several studies have suggested that abnormally elevated VSMC apoptosis is correlated with the pathogenesis of AAD [18][19][20]. Evidence has shown that HCMV can induce apoptosis of several cell types, such as human astrocytes [40], myeloid progenitor cells [41], and neural stem/progenitor cells [42,43], although HCMV is known to block apoptosis in macrophages and cancer cells [44,45]. A previous study reported that HCMV can infect VSMCs and affect expression of genes related to cellular lipid metabolism (46). HCMV infection in rat VSMCs induced proinflammatory response through an IκB kinase-related pathway [47]. In the current study, HCMV infection in HA-VSMCs inhibited cell proliferation and promoted cell apoptosis, which may be related to the pathogenesis of AAD. Additionally, the roles of HCMV-encoded miRNAs in cell apoptosis have been described. Liang et al. indicated that HCMV-miR-UL112-3p suppresses the glioblastoma