Silencing of OIP5-AS1 Protects Endothelial Cells From ox-LDL-Triggered Injury by Regulating KLF5 Expression via Sponging miR-135a-5p

Background: Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. LncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been found to be associated with the development of atherosclerosis. In this study, we further investigated the molecular basis of OIP5-AS1 in atherosclerosis pathogenesis. Methods: Oxidative low-density lipoprotein (ox-LDL) was used to treat human umbilical vein endothelial cells (HUVECs). The levels of OIP5-AS1, miR-135a-5p, and Krüppel-like factor 5 (KLF5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell viability, migration, and apoptosis were evaluated using the Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and malondialdehyde (MDA) were determined with enzyme-linked immunosorbent assay (ELISA). Targeted interactions among OIP5-AS1, miR-135a-5p, and KLF5 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Animal studies were performed to assess the role of OIP5-AS1 in atherosclerosis progression in vivo. Results: Our data showed the significant upregulation of OIP5-AS1 in atherosclerosis serum and ox-LDL-stimulated HUVECs. The silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs and diminished lipids secretion in ApoE−/− mice. Moreover, OIP5-AS1 functioned as a molecular sponge of miR-135a-5p, and miR-135a-5p was a functional mediator of OIP5-AS1 in regulating ox-LDL-induced HUVEC injury. KLF5 was a direct target of miR-135a-5p, and the increased expression of miR-135a-5p alleviated ox-LDL-induced cytotoxicity by downregulating KLF5. Furthermore, OIP5-AS1 influenced KLF5 expression through sponging miR-135a-5p. Conclusion: The current work identified that the silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs at least in part by influencing KLF5 expression via acting as a miR-135a-5p sponge.


INTRODUCTION
Atherosclerosis, a chronic inflammatory disorder, is the pivotal pathological process of cardiovascular disease (CVD) (1,2). Dysfunction of endothelial cells plays an important role in the progression of atherosclerosis (3). Oxidative low-density lipoprotein (ox-LDL) enhances the occurrence and development of atherosclerosis through various mechanisms, including the induction of the dysfunction of endothelial cells (4). A clearer understanding of how ox-LDL drives endothelial cell injury is very important for developing the effective approaches to inhibit atherosclerosis progression.
Long non-coding RNAs (lncRNAs) are >200-nucleotide RNAs that are implicated in diverse biological and pathological processes (5). Many lncRNAs have recently been shown to be involved in atherosclerosis pathogenesis by functioning as microRNA (miRNA) sponges (6). For instance, Wu et al. demonstrated that the deficiency of taurine upregulated gene 1 (TUG1) protected against ox-LDL-triggered cytotoxicity in human umbilical vein endothelial cells (HUVECs) by targeting miR-148b (7). Li et al. reported that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acted as a miR-155 sponge to suppress atherosclerosis inflammatory response induced by ox-LDL (8). As for OIP5 antisense RNA 1 (OIP5-AS1), it was discovered to be associated with ox-LDL-mediated atherosclerosis progression in endothelial cells (9). Although the OIP5-AS1/miR-320a/lectin-like ox-LDL receptor 1 (LOX1) network has been highlighted in atherosclerosis pathogenesis (10), our understanding of the molecular basis of OIP5-AS1 remains limited.
MiRNAs are important regulators in atherosclerosis pathogenesis by silencing target genes (11,12). MiR-135a-5p, an underexpressed miRNA in atherosclerosis, was reported to exert a potential anti-atherosclerotic activity in HUVECs under ox-LDL stimulation (13). When we used computational methods to help identify the molecular basis of OIP5-AS1, we found two putative binding sequences among OIP5-AS1, miR-135a-5p, and Krüppel-like factor 5 (KLF5). For these reasons, we undertook to investigate the involvement of the lncRNA/miRNA/mRNA regulatory network in ox-LDL-triggered HUVEC damage.

Patient Cohort and Serum Samples
In this project, 41 patients with atherosclerosis were enrolled from Henan Provincial People's Hospital, and 25 healthy individuals without any history of cardiovascular, inflammatory, or other diseases were recruited as normal controls. The clinicopathologic features of these subjects were provided in Table 1. Five milliliters of peripheral blood was collected from each participant, and serum samples were stored at −80 • C. Ethics approval for the project was obtained from the Human Research Ethics Committee of Henan Provincial People's Hospital, and written informed consent was provided by all of the subjects.

Cell Culture and Treatment
HUVECs (ATCC R PCS-100-013 TM ) and human aortic endothelial cells (HAECs, ATCC R PCS-100-011) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and propagated in endothelial basal medium (EBM-2, Lonza, Basel, Switzerland) with 10% fetal bovine serum (EuroClone, Milan, Italy) at 37 • C in a 5% CO 2 humidified atmosphere. The sixth to seventh generations were used for our study. To generate the AS cell model in vitro, HUVECs of ∼50% confluence were cultured in the complete medium containing various concentrations (20,40, and 80 µg/ml) of ox-LDL (Yesen, Shanghai, China) for 24 h or 40 µg/ml of ox-LDL for 12, 24, and 48 h.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from serum samples (500 µl) and cultured cells using a modified TRIzol co-purification technique as previously reported (14). Briefly, for each 500 µl of serum and cell suspension, phase separation was done by adding 2 ml of TRIzol (Invitrogen, Hemel Hempstead, UK), followed by the addition of 200 µl of 1-bromo-4-methoxybenzene (Invitrogen). Total RNA was washed with 75% ethanol before solubilization with 50 µl of nuclease-free water. To quantify the expression of OIP5-AS1, KLF5, and endogenous control glyceraldehyde-3phosphate dehydrogenase (GAPDH), 1 µg of RNA was reverse transcribed to cDNA using the PrimeScript TM RT Reagent Kit (TaKaRa, Beijing, China), and qRT-PCR was performed using the One Step TB Green R PrimeScript TM PCR Kit (TaKaRa) as recommended by the manufacturers. The quantification of miR-135a-5p and U6 reference gene was performed using the Mir-X miRNA cDNA Synthesis Kit for cDNA synthesis and Mir-X miRNA qRT-PCR TB Green R Kit for qRT-PCR as per the protocols of the manufacturer (TaKaRa). The amplification profile was denatured at 95 • C for 10 min, followed by 40 cycles of 95 • C for 20 s and 60 • C for 1 min. All reactions were run in triplicate on a LightCycler 480 (Roche Diagnostic, Sussex, UK). The primers used in PCR amplification were provided in Table 2. Fold change of gene was calculated by the 2 − Ct method (15).

Subcellular Localization Assay
Total cytoplasmic RNA and nuclear RNA were isolated from HUVECs using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) as per the accompanying protocols. U6 and GAPDH were applied as the nuclear and cytoplasmic reference genes, respectively.

Transwell Migration Assay
Twenty-four Transwell inserts (8-µm pore size, BD Biosciences) were used for cell migration assays. Briefly, 2.5 × 10 4 treated HUVECs were plated in the top chamber, and a medium containing 10% serum was added in the lower chamber as a chemoattractant. The cells were stained with 0.5% crystal violet after 24 h of incubation and counted under a microscope at 100 × magnification.

Enzyme-Linked Immunosorbent Assay (ELISA)
The cellular levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were tested using human IL-6 and TNF-α ELISA kits, respectively, based on the suggestion of the manufacturer (Abcam, Cambridge, UK). Malondialdehyde (MDA) quantification in HUVECs was done with a Human MDA Assay Kit (Elabscience, Wuhan, China) as recommended by the manufacturers.

Animal Studies
All animal procedures were performed according to the Academia Sinica IACUC and Council of Agriculture Guidebook for the Care and Use of Laboratory Animals, and the study was approved by the Ethics Committee of Henan Provincial People's Hospital. Male 8-week-old ApoE −/− mice (n = 18) and wild-type C57BL/6J mice (n = 6) were purchased from the Vital River Laboratory (Beijing, China) and housed in a specificpathogen-free environment in the animal facility of the Institute of Biomedical Sciences, Academia China. ApoE −/− mice were fed with a high-fat diet (15.8% fat and 1.25% cholesterol), and wild-type C57BL/6J mice were fed with normal chow. In the 6th week, the ApoE −/− mice were randomly divided into three groups: control, sh-NC group, or sh-OIP5-AS1 group (n = 6 per group). The lentiviral particles (50 µl) were injected into the ApoE −/− mice by a caudal vein every week until the 12th week, and the control group was injected with the same volume of PBS. At the end of the experiments, blood samples were collected from these mice, and serum samples were harvested for further analyses. The expression levels of OIP5-AS1, miR-135a-5p, and KLF5 were gauged by qRT-PCR and western blot as above. The levels of total cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were detected using commercially available enzyme kits per the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Statistical Analysis
Data were expressed as the mean ± standard deviation (SD) from at least three independent biological replicates. Student's t-test or analysis of variance (ANOVA) was used to compare differences of different independent groups of quantitative data. The correlation of miR-135a-5p expression and OIP5-AS1 or KLF5 level was measured by the Spearman rank correlation. The clinical parameters between atherosclerosis patients and healthy controls were compared by a chi-square test. All P-values were two-tailed, and those < 0.05 were considered a statistically significant difference.

OIP5-AS1 Was Overexpressed in Atherosclerosis Serum and Ox-LDL-Stimulated HUVECs
Firstly, we evaluated the expression of OIP5-AS1 in the serum samples of atherosclerosis patients and ox-LDL-stimulated HUVECs. As demonstrated by qRT-PCR, OIP5-AS1 level was upregulated in atherosclerosis serum samples compared with the normal controls (P < 0.0001, Figure 1A). Moreover, ox-LDL stimulation led to an upregulation in the expression of OIP5-AS1 in HUVECs in dose-and time-dependent manners (P < 0.001 or P < 0.0001, Figures 1B,C). Furthermore, subcellular localization assays showed that OIP5-AS1 was mainly present in the cytoplasm of HUVECs ( Figure 1D).
Subsequently, we first identified KLF5 as a direct target of miR-135a-5p in HUVECs. KLF5, a zinc-finger transcription factor, is highly expressed in AS and is associated with the malignant symptomatology of atherosclerosis (31)(32)(33). In this report, we first showed the anti-atherosclerotic effect of miR-135a-5p overexpression in ox-LDL-induced HUVECs through KLF5. Similarly, Wang and colleagues reported that miR-152 protected against atherosclerosis malignant progression by directly targeting KLF5 (34). Our data also pointed out the role of OIP5-AS1 as a miR-135a-5p sponge to modulate KLF5 in HUVECs under ox-LDL. Similarly, smooth muscle-enriched lncRNA (SMILR) enhanced atherosclerosis development by modulating KLF5 expression by sponging miR-10b-3p (35). Zheng et al. reported that vascular smooth muscle cell-derived exosomes could mediate the transfer of KLF5-induced miR-155 from smooth muscle cells to endothelial cells, and the overexpression of miR-155 suppressed endothelial cell proliferation and migration, leading to atherosclerosis progression (36). Wang et al. demonstrated that KLF5 overexpression induced by hyperinsulinemia contributed to diabetic endothelial dysfunction by regulating endothelial nitric oxide synthase (eNOS) (37). Future work should build on these findings by determining precisely the molecular determinants of KLF5 in regulating ox-LDL-induced injury in HUVECs. Additionally, HAECs are atherosclerosis-related aortic endothelial cells that are widely used to establish the atherosclerosis in vitro model (38,39). Our data also showed that ox-LDL induced a strong increase in OIP5-AS1 expression in HAECs, and the reduced expression of OIP5-AS1 abolished ox-LDL-induced injury in HAECs (Supplementary Figures 1A-F), reinforcing OIP5-AS1 as a novel therapeutic target for atherosclerosis.
significantly overexpressed lncRNA in atherosclerosis, regulated ox-LDL-triggered HUVEC damage by targeting the miR-135a-5p/KLF5 axis, highlighting a promising molecular target for atherosclerosis management.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.