Background. Although long non-coding RNAs (lncRNAs) have been reported to serve as potential biomarkers of atherosclerosis (AS), the role of lncRNA small nucleolar RNA host gene 12 (SNHG12) in AS still remains to be elucidated.
Objectives. The present study aimed to investigate the regulatory effects and potential mechanisms of SNHG12 in human vascular smooth muscle cells (hVSMCs).
Materials and methods. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was employed to determine the expression of SNHG12, miR-766-5p and eukaryotic translation initiation factor 5A (EIF5A) in oxidized low-density lipoprotein (ox-LDL)-induced hVSMCs. After transfection with short hairpin RNA (shRNA)-SNHG12, cell viability was estimated using the Cell Counting Kit-8 (CCK-8) assay. Wound healing and transwell assays were used for evaluating migratory capacities of hVSMCs. To further investigate the regulatory mechanisms, binding sites between SNHG12 and miR-766-5p, and EIF5A and miR-766-5p were predicted using starBase database and validated using luciferase reporter gene assays. Moreover, cell viability and migration were detected following EIF5A overexpression and SNHG12-knockdown.
Results. SNHG12 was significantly upregulated in ox-LDL-induced hVSMCs. SNHG12 silencing inhibited ox-LDL-induced proliferation and migration of hVSMCs. Moreover, SNHG12 acted as a sponge of miR-766-5p, and miR-766-5p also interacted with EIF5A. EIF5A plasmids promoted the capacities of proliferation and migration in ox-LDL-induced hVSMCs. However, shRNA-SNHG12 counteracted the facilitation of EIF5A plasmids on hVSMCs biological behaviors.
Conclusions. Taken together, these findings demonstrated that silencing of SNHG12 blocks the proliferation and migration of hVSMCs via targeting the miR-766-5p/EIF5A axis.
Key words: migration, SNHG12, human vascular smooth muscle cells, miR-766-5p, eukaryotic translation initiation factor 5A
Long non-coding RNAs (lncRNAs) are a heterogeneous class of non-coding RNAs greater than 200 nucleotides in length without protein-coding capacity.1 Recently, studies have found that lncRNAs emerge as crucial regulators of atherosclerosis (AS).2, 3 Atherosclerosis is commonly recognized as a lipid-induced chronic inflammation of the vascular wall associated with activation and dysfunction of resident vascular cells4 and contributes to stenosis of internal arteries due to plaque accumulation.5 The number of lncRNAs was reported to be implicated in regulating cholesterol and lipid metabolism, and they also play diverse roles in a variety of atherosclerotic processes including cell proliferation, migration, inflammation, differentiation, and apoptosis.6
Small nucleolar RNA host gene 12 (SNHG12) is one of the classes of SNHGs.7 Studies revealed that SNHG12 regulates cell proliferation, migration, invasion, and metastasis in several cancers,8 indicating a potential target for cancer-directed interventions.13 Except for its role in cancers, SNHG12 could also ameliorate brain microvascular endothelial cell injury.14 To date, a number of well-studied lncRNAs gave us important clues about their potential for AS treatment.15 For instance, lincRNA-p21 is downregulated in atherosclerotic plaques of ApoE(−/−) mice, and it can suppress vascular smooth muscle cell (VSMC) proliferation and induce apoptosis.16 HIF1α-AS1 regulates the proliferation and apoptosis of VSMCs.17 The expression of H19 is higher in serum of AS patients,18 serving as a potential biomarker for diagnosing AS. However, the status, biological function and regulatory mechanisms of SNHG12 in AS are still unknown.
We examined the expression of SNHG12 in human VSMCs (hVSMCs) exposed to oxidized low density lipoprotein (ox-LDL) and evaluated the influence of SNHG12 on cell migration. Furthermore, the regulatory mechanisms of SNHG12 on hVSMCs were explored.
Materials and methods
Cell lines and transfection
The hVSMCs (Cell Bank of the Shanghai Institute of Cell Biology, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; ProCell, Wuhan, China) containing 10% fetal bovine serum (FBS; Gibco, Waltham, USA) under an atmosphere of 95% air and 5% CO2 at 37°C. The ox-LDL (Solarbio, Beijing, China) was used to stimulate hVSMCs for 48 h. miR-766-5p mimic, miR-NC (negative control), and 2 pairs of short hairpin RNA (shRNA)-SNHG12 (sh-SNHG12-1, sh-SNHG12-2) were obtained from GenePharma Co., Ltd. (Shanghai, China). Overexpression plasmids of EIF5A and the negative control were generated with the help of Sangon Biotech (Shanghai, China). Cells were collected 24 h following transfection, and transfection efficiency was evaluated using reverse-transcription quantitative polymerase chain reaction (RT-qPCR).
Total RNA was harvested (TRIzolTM Plus RNA Purification Kit; Invitrogen, Carlsbad, USA) and reverse transcribed into cDNA (M-MLV Reverse Transcriptase; Promega, Madison, USA). TaqMan MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Waltham, USA) was employed to quantify miR-766-5p, the relative expression of miR-766-5p was normalized to U6, and others were normalized to GAPDH based on the 2−ΔΔCt method.19 The primers used in this study were as follows: SNHG12, forward: 5′-GTGATACTGAGGAGGTGAG-3′ and reverse: 5′-CCTTCTGCTTCCCATAGAG-3′; EIF5A, forward: 5′-AGGCCATGGCAAAATAACTG-3′ and reverse: 5′-GGGTGGGGAAAACCAAAATA-3′; GAPDH, forward: 5′-AGCCTCCCGCTTCGCTCTCTGC-3′ and reverse: 5′-ACCAGGCGCCCAATACGACCAAA-3′; miR-766-5p, forward: 5′-TCGAGTACTTGAGATGGAGTTTT-3′ and reverse: 5′-GGCCGCGTTGCAGTGAGCCGAG-3′; U6, forward: 5′-CTCGCTTCGGCAGCACA-3′ and reverse: 5′-AACGCTTCACGAATTTGCGT-3′.
Cell viability assay
The hVSMCs were seeded into a 96-well plate, then cells were incubated with 10 μL Cell Counting Kit-8 (CCK-8) solution (Beyotime, Jiangsu, China) at 24 h, 48 h and 72 h. Absorbance values were recorded on a BioTek microplate reader (BioTek, Winooski, USA) at 450 nm.
Wound healing assay
An amount of 1 × 105 of hVSMCs were plated into each well of a 12-well plate. When 100% confluence was achieved, the culture medium was removed and drew straight from the plate using a 200 μL plastic pipette. The sample was washed gently to remove the floating cells, then serum-free medium was added and maintained in the incubator for 24 h. Samples were photographed at 0 h and 24 h under a microscope (Axioscope 5; Carl Zeiss, Oberkochen, Germany).
Transwell migration assay
For the transwell migration assay, serum-free media containing 5 × 104 of hVSMCs were seeded into the upper chamber of a 24-well transwell filter with 8-µm pore size. The lower chamber was filled with media supplemented with 10% FBS. Cells were allowed to transgress through the porous filters for 24 h at 37°C. Then, VSMCs were fixed with 4% paraformaldehyde for 20 min. Cells that migrated through the pores of the filter were stained with 1% crystal violet for 30 min. The images were photographed under a fluorescence microscope (BX51; Olympus Corp., Tokyo, Japan), and the number of migrated cells was calculated using ImageJ software (National Institutes of Health, Bethesda, USA).
Luciferase reporter gene assay
SNHG12 or EIF5A sequences containing the wild-type (WT) binding site or mutated-type (Mut) binding site for miR-766-5p were synthesized by Vigorous Biotechnology Beijing Co. Ltd. (Beijing, China) and cloned into the pmiR-GLO vector (Promega). Prior to transfection, cells were seeded into 24-well plates (5 × 103 cells/well) and cultured for 24 h. Afterward, the WT or Mut of SNHG12 was transiently co-transfected with miR-766-5p mimics or miR-NC using Lipofectamine 3000 reagent for another 48 h. The firefly luciferase activity normalized to Renilla represented the value of relative luciferase activity. Likewise, EIF5A WT or Mut co-transfected with miR-766-5p mimic or miR-NC was similar to the above method.
Total protein from treated cells was extracted using a radio immunoprecipitation assay lysis buffer containing proteinase inhibitors (Beyotime). After the determination of protein concentrations, equal protein samples (40 µg) were loaded on SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Madison, USA). Then, the membranes were blocked with 5% non-fat milk for 2 h and incubated with primary antibodies against EIF5A and GAPDH (both obtained from Cell Signaling Technology, Inc., Danvers, USA) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, USA) was used to incubate membranes for 2 h at room temperature. The blots were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, USA) and subsequently quantified using ImageJ software v. 1.52r (National Institutes of Health).
All data were presented as the mean ± standard deviation (SD). The results were analyzed using GraphPad Prism v. 6.0 (GraphPad Software, Inc., San Diego, USA). An unpaired student’s t-test was employed to evaluate differences between 2 groups, and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for comparison of differences between 3 or more groups. A value of p < 0.05 was considered statistically significant.
Interference with SNHG12 inhibits proliferation and migration
of ox-LDL-induced hVSMCs
We first investigated the expression of SNHG12. The hVSMCs were stimulated with different concentrations of ox-LDL, and, as shown in Figure 1A, ox-LDL could promote the expression of SNHG12 in a dose-dependent manner. A volume of 100 mg/L ox-LDL was considered an optimal concentration to induce the transcription of SNHG12. To elucidate the function of SNHG12 in hVSMCs, a loss-of-function study was performed via transfecting sh-SNHG12 into cells. It was identified that sh-SNHG12-1 presented a better outcome for silencing SNHG12 (Figure 1B). Afterward, we estimated the cell viability of hVSMCs in the absence of SNHG12, and results showed that sh-SNHG12-1 transfection significantly inhibited the increased cell proliferation caused by ox-LDL stimulation (Figure 1C). Moreover, the wound healing assay and transwell migration assay indicated that ox-LDL-triggered cell migration was overturned by silencing of SNHG12 (Figure 1D–G). These results suggest that disturbing the expression of SNHG12 could inhibit the viability and migration of hVSMCs induced by ox-LDL.
SNHG12 functions as a sponge
The LncRNAs are considered competing endogenous RNAs (ceRNAs) to bind with miRNAs and modulate gene expression.20 Jia et al. demonstrated that miR-766-5p participated in cell proliferation, migration and invasion in colorectal cancer.21 Of note, binding sites between SNHG12 and miR-766-5p were predicted using starBase v. 2.0 (http://starbase.sysu.edu.cn) (Figure 2A), and miR-766-5p mimic was validated to be effective to elevate the expression of miR-766-5p (Figure 2B). The luciferase reporter gene assay demonstrated that miR-766-5p mimic inhibited luciferase activity in hVSMCs transfected with SNHG12-WT (Figure 2C). Additionally, it was found that sh-SNHG12-1 elevated the expression of miR-766-5p (Figure 2D). Moreover, the level of miR-766-5p in hVSMCs treated with ox-LDL was notably decreased (Figure 2E). Collectively, this data reveal that miR-766-5p is remarkably downregulated in ox-LDL-treated hVSMCs, and SNHG12 directly targeted miR-766-5p.
EIF5A is a direct target gene of miR-766-5p
As mentioned above, SNHG12 directly targeted miR-766-5p and served as a ceRNA to bind with miR-766-5p. The ceRNA activity forms a large-scale cross-talk network among the transcriptome. The miRNAs are generally regarded as active regulatory elements which reduce the stability of target RNAs or inhibit their translation.22 Therefore, target mRNAs are considered as silencing objects of miRNAs. EIF5A is a small molecule protein in eukaryotic cells, which plays an important role in cell growth, survival and senescence. It is especially essential for cell proliferation.23 Of note, EIF5A was predicted as a potential target of miR-766-5p (Figure 3A). Luciferase reporter gene analysis was employed to test the potential interaction between them. It was observed that miR-766-5p mimic apparently decreased the luciferase activity of EIF5A-WT in hVSMCs, and mutation of EIF5A abrogated the function of miR-766-5p mimic (Figure 3B). Subsequently, overexpression of miR-766-5p reduced the transcription and translation of EIF5A (Figure 3C,D). All of these data indicate that EIF5A may be a target mRNA of miR-766-5p.
SNHG12 regulates the proliferation
and migration of ox-LDL-induced hVSMCs via regulating EIF5A
In order to further explore the interaction between EIF5A and SNHG12, gain-of-function and loss-of-function studies were applied in subsequent experiments. Overexpression plasmids were constructed and transfected into hVSMCs with or without sh-SNHG12-1, and high-expression of EIF5A validated the plasmids could overexpress EIF5A successfully. However, sh-SNHG12-1 drastically impeded the mRNA and protein levels of EIF5A (Figure 4A,B). Cell viability was elevated in EIF5A overexpression group, while the effect was abolished by knockdown of SNHG12 (Figure 4C). Migratory capacity represented by wound width illustrated that EIF5A promoted hVSMCs migration, while SNHG12 knockdown exhibited an inhibition of cell migration (Figure 4D,E). Consistently, transwell migration assays showed a similar result with that of the wound healing assay (Figure 4F,G). Taken together, these results indicate that SNHG12 mediated the migratory capacities of hVSMCs through regulating EIF5A.
In recent years, numerous studies have demonstrated that lncRNAs regulate various cellular process including cell proliferation, migration, invasion, and apoptosis.26 It was reported that lncRNA MIAT activates the PI3K/Akt signaling pathway, thereby exacerbating atherosclerotic damage in AS mice.3 LncRNA activated by transforming growth factor (TGF) expression is significantly higher in AS patients compared with healthy patients, and it could enhance the expression of caspase-3 in human vascular endothelial cells (HUVECs).27 Furthermore, the proliferation and migration of VSMCs were promoted by lncRNA 430945.28
The hVSMCs are the major cell type observed in blood vessel walls, and play a considerable role in the regulation of multiple physiological and pathological situations.29 Aberrant proliferation and migration of VSMCs are key events in the progression of AS and restenosis after percutaneous coronary intervention.30 A large amount of studies have suggested that ox-LDL exert a promotion effect in the development of AS by stimulating the proliferation of hVSMCs within the vessel wall; therefore, ox-LDL was widely used to stimulate hVSMCs for investigating the related mechanisms of AS.31, 32 Studies have implicated SNHG12 in various cancers, and it functions as a potential candidate for cancer-directed interventions.33, 34 The altered expression of SNHG12 is associated with cell viability, proliferation, metastasis, and invasion, thereby affecting the progression and diagnosis of cancer.13 However, the function of SNHG12 in AS has not yet been clearly elucidated. In this study, it was found that ox-LDL facilitated the expression of SNHG12 in hVSMCs. Deletion of SNHG12 impeded cell migration induced by ox-LDL.
Previous reports have described that lncRNAs interact with miRNA as ceRNAs and protect miRNAs from binding to and repressing target RNAs,22, 35 suggesting a complicated crosstalk among diverse RNA species. Accumulating reports have been made to understand the effect of miRNAs in VSMC biology, especially in cellular proliferation and migration.36, 37, 38 In our study, luciferase reporter gene assays revealed an interplay between SNHG12 and miR-766-5p, and SNHG12 knockdown enhanced the expression of miR-766-5p. To further examine the target RNA regulated by lncRNA-miRNA, binding sites between miR-766-5p and EIF5A sequence were predicted using starBase v. 2.0. Subsequently, the interaction between miR-766-5p and EIF5A was further validated using luciferase reporter gene assay and RT-qPCR. Finally, we found that overexpression of EIF5A expedited the proliferation and migration of hVSMCs, whereas the effect was reversed by SNHG12 silencing.
The present study illustrates that SNHG12 was highly expressed in ox-LDL-challenged hVSMCs. An intricate interplay among SNHG12, miR-766-5p and EIF5A was discovered, and all of these results indicated that SNHG12-knockdown inhibited the proliferation and migration of hVSMCs through targeting the miR-766-5p/EIF5A axis. Further research is necessary for investigating the impact of SNHG12/miR-766-5p/EIF5A signaling pathway on other pathological alterations in AS progression.
The data supporting our findings are available from the corresponding author upon reasonable request.