Fa-Qing Long, Qing-Jie Su, Jing-Xia Zhou, De-Sheng Wang, Peng-Xiang Li, Chao-Sheng Zeng, Yi Cai

The Second Af filiated Hospital of Hainan Medical University, Haikou, Hainan Province, China

Abstract Long non-coding RNAs regulate brain microvascular endothelial cell death, the in flammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation (OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pcDNA-control, pcDNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the in flammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12,and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the in flammatory response,and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.

Key Words: nerve regeneration; ischemic stroke; microRNA; brain microvascular endothelial cell death; in flammatory response; angiogenesis;oxygen-glucose deprivation/reoxygenation; ischemia/reperfusion; therapeutic targets; neural regeneration; gene regulation; neural regeneration

Introduction

Ischemic stroke is the primary cause of death and permanent disability in adults worldwide and occurs when blood flow to the brain is blocked, causing immediate deprivation of oxygen and nutrients (Fisher, 2009; Jauch et al., 2013).Despite advances in medical and endovascular recanalization therapy, treatment choices for ischemic stroke are still very limited (Furlan et al., 2003; Lakhan et al., 2009); therefore,novel and effective treatments for ischemic stroke are urgently needed. Undoubtedly, a good understanding of pathogenesis will facilitate the development of such therapies.

Many mechanisms are known to be involved in the pathogenesis of ischemic stroke. Among these, it is increasingly appreciated that brain microvascular endothelial cell(BMEC) death induced by ischemia/reperfusion (I/R) injury is the initial stage of blood-brain barrier disruption and leads to a poor prognosis for ischemic stroke patients (Hawkins and Davis, 2005; Date et al., 2006; Lakhan et al., 2013; Zhang et al., 2017a). It is also known that endothelial in flammation and subsequent impairment of endothelial function contribute to ischemic brain injury and determine stroke outcome(Anwaar et al., 1998; Stanimirovic and Satoh, 2000; Zoppo et al., 2000; Hassan et al., 2003; Muir et al., 2007; Pei et al.,2015). Angiogenesis after ischemic stroke is also clinically significant; it improves recovery from stroke by promoting neurogenesis and enhancing neuronal and synaptic plasticity (Wang et al., 2012; Liu et al., 2014; Alhusban et al., 2015;Hui et al., 2017; Li et al., 2017).

Non-coding RNAs (ncRNAs) are generally defined as untranslated regulatory RNA molecules. Among ncRNAs,microRNAs (miRNAs, usually 18–25 nucleotides in length)and long ncRNAs (lncRNAs, usually ＞ 200 nucleotides in length) regulate various biological processes, including cell growth, apoptosis, differentiation, the in flammatory response and angiogenesis, and are involved in various diseases (Pillai,2005; Park et al., 2014; Schaukowitch and Kim, 2014; Asghari et al., 2016; Ninova et al., 2016; Tian et al., 2016; Kondo et al.,2017; Qiu et al., 2017). miRNAs regulate gene expression and function via translation inhibition, mRNA degradation, or both (Pillai, 2005). Accumulated evidence is overwhelming for miRNAs playing a role in stroke. For example, overexpression of circulating miR-223 is associated with the pathogenesis of acute ischemic stroke, inhibition of miR-155 expression facilitates recovery after experimental stroke in mice,and miR-497 modulates neuronal death after transient focal cerebral ischemia in mice (Yin et al., 2010; Wang et al., 2014;Caballero-Garrido et al., 2015).

LncRNAs can modulate gene expression at transcriptional and post-transcriptional levels. Recently, lncRNAs were reported to regulate BMEC survival, the inflammatory response and angiogenesis during and after ischemic stroke.For example, Liu et al. (2016) reported that downregulation of the lncRNA, Meg3, increases angiogenesis after ischemic stroke by activating Notch signaling. Meanwhile, Zhang et al. (2017) reported that the lncRNA, Malat1, is remarkably upregulated after I/R or oxygen-glucose deprivation/reoxygenation (OGD/R) insult and that Malat1 plays a critical role in protecting the cerebral microvasculature by promoting BMEC survival and inhibiting endothelial in flammation after I/R or OGD/R insult. SNHG12 is also dramatically upregulated by I/R or OGD/R (Liu et al., 2016). In addition,SNHG12 can promote cell proliferation and angiogenesis in various types of cancer (Ruan et al., 2016; Lan et al., 2017;Wang et al., 2017). However, the function of SNHG12 in ischemic stroke remains unknown. This study investigated the effects of SNHG12 on BMEC death, angiogenesis and the in flammatory response after OGD/R, and the molecular mechanisms underlying these effects.

Materials and Methods

Cell culture and OGD/R

Mouse primary BMECs were purchased from Cell Biologics,Inc. (Chicago, IL, USA) and were grown to approximately 90% confluence before use. To mimic OGD ischemia-like conditions in vitro, BMECs were cultured in deoxygenated glucose-free Hanks’ Balanced Salt Solution (Invitrogen,Carlsbad, CA, USA) gassed with 95% N2/5% CO2, in an anaerobic chamber (Forma Scientific, Marietta, OH, USA) for different lengths of time. Control BMECs were not exposed to OGD. For reoxygenation with glucose reintroduction,cells were cultured in standard medium under a humidified 95% air/5% CO2atmosphere for 4 hours.

Cell transfection

Plasmid pcDNA3.1, containing SNHG12 (pcDNASNHG12), siRNAs targeting SNHG12 (si-SNHG12) and their respective controls (pcDNA-control and si-control), were obtained from Sigma-Aldrich (St. Louis, MO, USA). An miR-199a mimic (miR-199a) and an miR-control were obtained from GenePharma Co., Ltd (Shanghai, China). BMECs were plated in 96-well plates at 2 × 105cells per well and incubated overnight. Transfection was performed using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. pcDNA-control, pcDNA-SNHG12, si-control, si-SNHG12, miR-control, and miR-199a groups were established by transection of BMECs with pcDNA-control,pcDNA-SNHG12, si-control, si-SNHG12, miR-control and miR-199a, respectively. The miR-199a + pcDNA-control group was established by cotransfection of BMECs with miR-199a mimic and pcDNA-control, while the miR-199a +pcDNA-SNHG12 group was established by cotransfection of BMECs with miR-199a mimic and pcDNA-SNHG12.

Quantitative real-time polymerase chain reaction(qRT-PCR)

At 48 hours post-transfection or after exposure to OGD for indicated times, total RNA was extracted from BMECs using Trizol reagent (Invitrogen), and reverse transcribed into cDNA using Taq-Man Reverse Transcription reagents(Applied Biosystems, Foster City, CA, USA). The cDNA was used for qRT-PCR using a SYBR PrimeScript RT-PCR kit (TaKaRa, Dalian, China). GAPDH, a constitutively expressed gene, was used as an internal control. miR-199a expression was detected with a TaqMan microRNA real-time RT-PCR kit (Applied Biosystems), and U6 small nuclear RNA was used as a loading control. Fold change in gene expression was calculated using the 2-ΔΔCtmethod. Primer sequences are listed in Table 1.

Cell growth assay

After exposure to OGD for 16 hours, BMEC viability and death were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and trypan blue exclusion assays, respectively, as previously described (Hopkins-Donaldson et al., 2003). Brie fly, for the MTT assay, 1× 105cells per well in 96-well plates were exposed to OGD for 16 hours, and then cultured in standard medium under a humidified 95% air/5% CO2atmosphere for 4 hours, after which 20 μL of MTT (5 mg/ml) solution was added to each well, and the cultures incubated for a further 2 hours. The culture medium was then discarded and 100 μL of dimethyl sulfoxide (Sigma) added per well. The plates were shaken todissolve the crystals and absorbance was determined with an enzyme-linked immunosorbent assay plate reader (BIOTEK, Winooski, VT, USA) at 570 nm. For the trypan blue exclusion assay, 1 × 106BMECs per well in six-well plates were exposed to OGD for 16 hours, and then cultured in standard medium for 4 hours. After trypsinizing and washing in PBS, the cells were resuspended in 0.4% trypan blue.Dead and live cells were counted using a hemocytometer,and the percentage of dead cells was calculated.

Table 1 Primer sequences used for quantitative real-time polymerase chain reaction

Western blot assay

After exposure to OGD for 16 hours, western blot assays were performed as described previously (Lou et al., 2012).Briefly, total proteins were extracted from BMECs using lysis buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer of proteins to membranes, and immune detection were then sequentially performed. Primary antibodies against vascular endothelial growth factor (VEGF)A(diluted 1:200; Abcam, Cambridge, UK), fibroblast growth factor (FGF) b (diluted 1:500; Abcam), E-selectin (diluted 1:500; Abcam), monocyte chemotactic protein 1 (MCP1)(diluted 1:100; Chemicon, Temecula, CA, USA), interleukin-6 (IL-6) (diluted 1:100; Chemicon), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (diluted 1:1000;Abcam), and horseradish peroxidase-conjugated secondary antibody (diluted 1:5000; Abcam) were employed. Membranes were incubated with primary antibodies for 1 hour at 37°C. After washing, the membranes were incubated with the secondary antibody for 2 hours at room temperature.Protein bands were visualized using a Supersignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL,USA). Image analysis was performed to quantify the integrated density of the individual bands using Image J software (NIH, Bethesda, USA).

Capillary-like tube formation assay

After exposure to OGD for 16 hours, an in vitro angiogenesis assay was performed as previously described (Haralabopoulos et al., 1994). Brie fly, 0.8 mL of growth factor-reduced Matrigel (Becton-Dickinson, San Jose, CA, USA) was added to a 35-mm Petri dish and allowed to polymerize at 37°C for 3 hours. BMECs (2 × 104cells) were added and cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL,Rockville, MD, USA) for 6 hours. To quantify tube formation, five random areas were photographed and the total tube length was measured using cellSens Standard 1.6 image software (Olympus, Napa, CA, USA).

Statistical analysis

All data are presented as the mean ± SEM from at least three independent experiments. All statistical analyses were performed with SPSS 17.0 software (SPSS, Chicago, IL, USA).The differences between groups were analyzed by Student’s two-tailed t-test or one-way analysis of variance followed by the Bonferroni post hoc test. A value of P ＜ 0.05 was considered statistically significant.

Results

Upregulated SNHG12 inhibited BMEC death under OGD/R conditions

After exposure to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours, SNHG12 levels in BMECs was determined by qRT-PCR. SNHG12 expression increased with respect to OGD exposure time (Figure 1A). To investigate the effect of SNHG12 on BMEC growth, BMECs were transfected with pcDNA-control, pcDNA-SNHG12, si-control,or si-SNHG12, and then analyzed by MTT and trypan blue exclusion assays. qRT-PCR assays showed that SNHG12 levels were higher in the pcDNA-SNHG12 group than in the pcDNA-control group, and that SNHG12 levels were lower in the si-SNHG12 group than in the si-control group, which confirmed successful transfection (Figure 1B). The MTT and trypan blue exclusion assays showed that SNHG12 upregulation increased BMEC viability, and inhibited BMEC death, while SNHG12 knockdown had the opposite effect under OGD/R conditions (Figure 1C, D). Taken together,these results indicate that SNHG12 is upregulated and inhibits BMEC death under OGD/R conditions.

SNHG12 inhibited the in flammatory response under OGD/R conditions

To investigate the effect of SNHG12 on the in flammatory response of BMECs, BMECs transfected with pcDNA-control,pcDNA-SNHG12, si-control or si-SNHG12 were exposed to OGD for 16 hours and subsequently cultured in standard medium for 4 hours. qRT-PCR and western blot assays were then performed. SNHG12 upregulation decreased mRNA and protein levels of the proinflammatory cytokines-E-selectin, MCP1, and IL-6, and SNHG12 knockdown increased mRNA and protein levels of these proinflammatory cytokines under OGD/R conditions (Figure 2A, B). Taken together, these results indicate that SNHG12 plays an anti-infl ammatory role in BMECs under OGD/R conditions.

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Figure 1 SNHG1 was upregulated and inhibited BMEC death under OGD/R conditions.

Figure 2 SNHG12 inhibited the in flammatory response under OGD/R conditions.

SNHG12 promoted BMEC angiogenesis after OGD insult

To investigate the effects of SNHG12 on angiogenesis, we detected mRNA and protein levels of the proangiogenic factors,VEGFA and FGFb, under OGD/R conditions, and the formation of capillary-like tubes after OGD/R insult. SNHG12 upregulation increased levels of VEGFA and FGFb mRNA and protein, while SNHG12 knockdown decreased VEGFA and FGFb mRNA and protein levels under OGD/R conditions (Figure 3A, B). The capillary-like tube formation assay showed that SNHG12 upregulation promoted capillary-like tube formation, and that SNHG12 knockdown inhibited capillary-like tube formation (Figure 3C). Taken together, these data indicate that SNHG12 promotes BMEC angiogenesis.

SNHG12 directly targeted miR-199a in BMECs

To further investigate the molecular mechanism of SNHG12 on BMEC death, angiogenesis, and the inflammatory response, bioinformatic analysis of miRNA recognition sequences in SNHG12 was performed using miRcode. miR-199a was identified as being capable of binding to complementary sequences in SNHG12 (Figure 4A). miR-199a levels were determined in BMECs exposed to OGD for 0, 2, 4, 8, 16 or 24 hours. and were shown to decrease with respect to OGD exposure time, which was opposite to SNHG12 expression(Figure 4B). In addition, the regulatory effect of SNHG12 on miR-199a expression was also determined. SNHG12 upregulation decreased miR-199a expression, and SNHG12 knockdown increased miR-199a expression (Figure 4C). Taken together, these data indicate that SNHG12 inhibits miR-199a expression by directly targeting miR-199a.

Figure 3 SNHG12 promotes BMEC angiogenesis after OGD.

SNHG12 inhibited BMEC death, the in flammatoryresponse, and anti-angiogenesis by targeting miR-199a

Considering the inhibitory action of SNHG12 on miR-199a expression in BMECs, we further explored whether SNHG12 inhibited the function of miR-199a. Cell growth, the in flammatory response, and angiogenesis assays were performed.The transfection of miR-199a mimic decreased BMEC viability, and promoted BMEC death, while cotransfection of miR-199a + pcDNA-SNHG12 abolished the effects induced by miR-199a (Figure 5A, B). In the in flammatory response assay, transfection of the miR-199a mimic promoted mRNA and protein expression of E-selectin, MCP1, and IL-6, and the cotransfection of miR-199a + pcDNA-SNHG12 reversed the effects induced by miR-199a (Figure 5C, D). The angiogenesis assay showed that transfection of the miR-199a mimic inhibited mRNA and protein expression of VEGFA and FGFb, and capillary-like tube formation, while cotransfection of miR-199a + pcDNA-SNHG12 mitigated the effect induced by miR-199a (Figure 5C–E). These data indicate that SNHG12 targets miR-199a to inhibit BMEC death and the in flammatory response, and to promote angiogenesis.

Discussion

Ischemic stroke begins with serious focal hypoperfusion,and results in excitotoxicity and oxidative damage, which in turn cause microvascular damage, blood-brain barrier dysfunction and the initiation of in flammation. These events can further worsen the initial damage, and result in permanent brain damage and even death (Lo et al., 2003; Sandoval and Witt, 2008; Brouns and De Deyn, 2009; Lakhan et al.,2009). Therefore, it is essential for the development of effective ischemic stroke therapies to understand the molecular mechanism of microvascular damage, blood-brain barrier dysfunction and initiation of in flammation after ischemia.

Figure 4 SNHG12 directly targeted miR-199a in BMECs.

Figure 5 SNHG12 inhibited BMEC death, the in flammatory response, and promoted angiogenesis by targeting miR-199a.

LncRNAs were initially regarded as products generated from transcriptional background noise (Hung et al., 2011;Hudson and Ortlund, 2014). However, it has become increasingly clear that lncRNAs are key regulators of physiological and pathological responses, and are involved in various biological processes, such as cell death, the inflammation response, and angiogenesis. For example, lncRNA GAS5 regulates prostate cancer cell apoptosis (Pickard et al., 2013).LncRNA IL7R knockdown reduces trimethylation of histone H3 and is involved in the regulation of the inflammatory response (Cui et al., 2014) and lncRNA MIAT knockdown remarkably ameliorates diabetes mellitus-induced retinal microvascular dysfunction in vivo (Yan et al., 2015). In recent years, the involvement of lncRNAs in cerebrovascular pathophysiology, such as stroke, has been increasingly investigated.For example, lncRNA N1LR inhibits p53 phosphorylation and enhances neuroprotection against ischemic stroke (Wu et al., 2016). LncRNA TUG1 promotes neuronal apoptosis by upregulating Bcl2l11 expression under ischemia (Chen et al., 2017). LncRNA FosDT increases ischemic brain injury by interaction with REST-associated chromatin-modifying proteins (Mehta et al., 2015). In addition, recent studies report that upregulation of Malat1 and downregulation of Meg3 after I/R and OGD/R insults can regulate BMEC death and the inflammatory response, and angiogenesis, respectively(Liu et al., 2016; Zhang et al., 2017). LncRNA SNHG12 is also upregulated in BMECs after I/R or OGD/R insult. However,its role remains unknown. In this study, SNHG12 inhibited BMEC death and the inflammatory response under OGD/R conditions and promoted BMEC angiogenesis after OGD/R by suppressing miR-199a expression. This study provides new clues for understanding the molecular mechanism of microvascular damage, blood-brain barrier dysfunction and the initiation of in flammation after ischemic stroke.

SNHG12 was initially discovered as an oncogene that promotes the initiation and development of many tumors. Ruan et al. (2016) found that SNHG12 promotes human osteosarcoma cell proliferation and migration by increasing angiomotin gene expression. Wang et al. (2017) found that overexpression of SNHG12 promotes cell proliferation and migration and inhibits apoptosis in triple-negative breast cancer. Lan et al. (2017) found that SNHG12 promotes carcinogenesis in hepatocellular carcinoma by targeting miR-199a/b-5p. However, to date, the role of SNHG12 upregulation in BMECs after I/R or OGD/R insult has not been studied. Here, a series of in vitro cell-based experiments were performed to investigate the role of SNHG12 during and after OGD/R insult. SNHG12 inhibited BMEC death and the expression of the proin flammatory cytokines, E-selectin, MCP1, and IL6, and promoted the expression of proangiogenic factors, VEGFA and FGFb under OGD/R conditions. In addition, SNHG12 also promoted capillary-like tube formation after OGD/R. Capillary-like tube formation is the reorganization stage of angiogenesis, involving cell adhesion, migration, differentiation, and growth.These findings indicate that SNHG12 inhibits BMEC death and the in flammatory response under OGD/R conditions and promotes BMEC angiogenesis after OGD/R insult. Enhancing SNHG12 expression may, therefore, improve outcomes for ischemic stroke patients.

The molecular mechanism by which SNHG12 functions in BMECs was further investigated. miR-199a was identified as a potential target of SNHG12 by bioinformatic prediction using miRcode software. miR-199a, a cancer-related miRNA, regulates cell growth, the in flammatory response, and angiogenesis. Xu et al. (2012a) found that miR-199a increases the inhibition of cell proliferation induced by cisplatin.Zhang et al. (2015) found that downregulation of miRNA-199a-5p reduces hyper-in flammation by normalizing CAV1 mRNA levels in cystic fibrosis macrophages. Raimondi et al. (2014) found that miR-199a suppresses myeloma-related angiogenesis in vitro and in vivo. Decreased miR-199a expression was recently reported to play a neuroprotective role in ischemic tolerance induced by 3-nitropropionic acid in rat brain (Xu et al., 2012b). The above findings prompted our further explorations into miR-199a. Our results confi rmed that SNHG12 can directly target miR-199a, and that the effects of miR-199a overexpression on BMEC death, the in flammatory response, and angiogenesis can be mitigated by SNHG12. These results indicate that SNHG12 exerts its function on BMECs by suppressing miR-199a.

Author contributions: FQL and YC designed the study. FQL, QJS, JXZ and DSW performed the experiments. PXL and CSZ analyzed the data. All authors approved the final version of the paper.

Conflicts of interest: The authors declare no competing financial interests.Financial support: This study was supported by the Natural Science Foundation of Hainan Province of China, No. 817334. The funding body played no role in the study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.

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Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

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Open peer reviewers: Bensu Karahalil, Gazi Universitesi, Toxicology, Gazi University, Turkey; Byung G. Kim, Anjou University School of Medicine, Korea.Additional file: Open peer review reports 1 and 2.