Abstract
Human corneal fibroblasts (HCFs) are implicated in corneal neovascularization (CRNV). The mechanisms underlying the inflammatory response in HCFs and the development of CRNV were explored in this study. Alkali burns were applied to the corneas of rats to establish a CRNV model. The expression of long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) and mRNA and protein levels of nuclear factor kappa B (NF-κB)- activating protein (NKAP) were examined by quantitative real-time (qRT-PCR) and Western blot methods, respectively. Lipopolysaccharide (LPS) is used to stimulate HCFs for inflammatory response. The level of inflammation factors in HCF supernatant was detected using an enzyme-linked immunosorbent assay (ELISA). Binding and interactions between NEAT1 and miRNA 1246 (miR-1246) were determined by RNA immunoprecipitation (RIP) and RNA pull-down assays in HCFs. Compared with the control group (n = 6), NEAT1 was upregulated in the corneas of the CRNV rat model (n = 6). The expression of NEAT1 in HCFs was upregulated by LPS. Downregulation of NEAT1 suppressed the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). NEAT1 could bind and interact with miR-1246. LPS regulated the expression of NKAP and NF-κB signaling via the NEAT1/miR-1246 pathway. Downregulation of NEAT1 in vivo inhibited CRNV progression in the CRNV rat model. The lncRNA NEAT1 induced secretion of inflammatory factors, mediated by NF-κB, by targeting miR-1246, thereby promoting CRNV progression.
Introduction
A healthy cornea is transparent and avascular, with a capillary network surrounding the corneal limbus. The development of new blood vessels in the cornea leads to pathological changes (i.e. corneal neovascularization (CRNV)) (Benayoun et al. 2015). Human corneal fibroblasts (HCFs) are essential for structural maintenance of the cornea (Wu et al. 2014), and they play a crucial role in modulating local immunity and the inflammatory response (Liu et al. 2008). Previous research demonstrated that HCFs contributed to the occurrence and progression of CRNV by secreting chemokines and proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8 (Lee et al. 2016). Lipopolysaccharide (LPS) is the main component of the cell wall in gram-negative bacteria, and it induces inflammation by promoting the secretion of cytokines, including TNF-α, IL-6 and vascular endothelial growth factor (Marton et al. 2014, Yang et al. 2014). LPS is often used to stimulate HCFs for inflammatory response. The nuclear transcription factor kappa B (NF-κB) is a well-known regulator, which mediates the transcription and expression of multiple inflammatory mediators, thereby adjusting and controlling the pathological microenvironment and cell function (DiDonato et al. 2012). As previous research showed that NF-κB played a vital regulatory role in cytokine and chemokine secretion of HCFs (Orita et al. 2013), implying that NF-κB may participate in the development of CRNV via the regulation of HCFs.
The role of curcumin in suppressing CRNV development by inhibiting angiogenic growth of human umbilical vein endothelial cells has been demonstrated (Pradhan et al. 2015). We showed that curcumin inhibited angiogenesis by upregulation of miRNA-1246 (miR-1246), which repressed NF-κB by targeting the NF-κB-activating protein (NKAP) (Bai et al. 2016). This finding revealed the inhibitory effect of miR-1246 on angiogenesis and CRNV progression. Using a bioinformatics method, we detected complementary base pairs between miR-1246 and the long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1), thereby pointing to potential binding and interactions between them.
The role of NEAT1, a nuclear-restricted lncRNA, as a key transcriptional regulator in cancer cell growth has been demonstrated previously (Cao et al. 2016). NEAT1 was recently discovered to be downregulated in plasma of HIV-1-infected patients and that its expression was correlated with the CD4 T-cell count (Jin et al. 2016). In contrast, it was upregulated in patients with multiple sclerosis, which is an inflammatory disease of the central nervous system (Santoro et al. 2016). Moreover, NEAT1 regulated the expression of matrix metalloprotease 13, IL-6 and IL-8, in osteoarthritis (Wang et al. 2017). In systemic lupus erythematosus, NEAT1 acted as a positive regulator in Toll-like receptor 4 signaling (Zhang et al. 2016). Toll-like receptor 4 was activated in CRNV, and it promoted neovascularization (Yang et al. 2015). The latter points to the involvement of NEAT1 in the inflammatory reaction of CRNV. Based on the literature, we speculated that NEAT1 may influence CRNV progression by regulating miR-1246, which mediates the inflammatory response of HCFs by targeting NKAP. This study was carried out to explore this regulatory mechanism and to provide insights and novel targets for therapeutic interventions.
Materials and methods
Corneal neovascularization (CRNV) model in rats
The animal experiments were performed in accordance with the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and all animal experimental procedures were approved by the Experimental Animal Ethics Committee of Zhengzhou University (No.1701976). Wistar rats (180–220 g; 8 weeks old; male; n = 12) were purchased from the laboratory animal center of Zhengzhou University and randomly allocated into an experimental group (CRNV, n = 6) and a sham group (n = 6). The animals were housed in a temperature-, humidity- and light-controlled room, with access to food and water ad libitum. All the rats were confirmed to be free of ocular diseases before experimentation.
The rats were anesthetized with 40 mg/kg of pentobarbital by intraperitoneal injection, followed by topical administration of one drop of tetracaine (Han et al. 2015). A round filter paper (3.5 mm in diameter) that had been soaked in 1 mol/L of NaOH was then placed on the center of the corneal surface for 30 s to induce an alkali burn. The ocular surface was then rinsed with 10 mL of phosphate buffered saline (PBS). The centers of the corneal surfaces of the rats in the sham group were treated with filter paper that had been soaked in 1mol/L of PBS, and the filter paper was applied for 30 s. On postoperative day 7, the rats were killed with an overdose of pentobarbital sodium administered via an intraperitoneal injection. The eyeballs were removed, and the corneas were taken for total RNA and protein extraction.
Real-time quantitative RT-PCR (qRT-PCR)
The qRT-PCR was performed for gene expression analysis. Total RNA was extracted from the corneas of rats using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Quantity and quality of RNA were evaluated using the Nanodrop technology (Thermo Scientific) with A260/A280 ratios between 1.9 and 2.1. In total, 2 μg of total RNA was taken for cDNA synthesis using a SuperScript Reverse Transcription Kit (Invitrogen). The qRT-PCR was completed on an ABI PRISM7900 Sequence Detection System (Applied Biosystems) with SYBR Green Master Mix (Applied Biosystems). Oligonucleotide primers (Table 1) for target genes were designed and synthesized by Sangon Biotech (Shanghai, China), and U6 served as the internal control of miR-1246. The relative expression was analyzed using the 2−∆∆Ct method.
The primer pair sequences used in this study.
| Targets genes | Primer pair sequences |
|---|---|
| Neat1 | F: 5′-TGGCTAGCTCAGGGCTTCAG-3′ R: 5′-TCTCCTTGCCAAGCTTCCTTC-3′ |
| miR-1246 | F: 5′-TGAAGTAGGACTGGGCAGAGA-3′ R: 5′-TGTTTGCAATAGCCCTTTGAG-3′ |
| Nkap | F: 5′-GAATTCATGGCTCCTGTATCGGGCTC-3′ R: 5′-GGATCCTCACTTGTCATCCTTCCCTTTG-3′ |
| U6 | F: 5′-CTCGCTTCGGCAGCACA-3′ R: 5′-AACGCTTCACGAATTTGCGT-3′ |
| β-actin | F: 5′-ATCGTGCGTGACATTAAGGAGAAG-3′ R: 5′-AGGAAGGAAGGCTGGAAGAGTG-3′ |
| Tnf-α | F: 5′-CCTCTCTCTAATCAGCCCTCTG-3′ R: 5′-GAGGACCTGGGAGTAGATGAG-3′ |
| Il6 | F: 5′-GAGGATACCACTCCCAACAGACC-3′ R: 5′-AAGTGCATCATCGTTGTTCATACA-3′ |
Western blotting
For soluble protein extraction, corneas were treated with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime), and the concentration of protein was measured using a BCA protein assay kit (Beyotime). Total protein was separated with 12% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad). The membrane was then blocked with Tris-buffered saline Tween-20 containing 5% nonfat milk for 1 h at 25°C and then incubated with the primary antibodies of NKAP (1:1000, ab229096, Abcam), p65 (1:250, ab16502, Abcam), p-p65 (1:500, ab86299, Abcam) and β-actin (1:2000, ab6276, Abcam) overnight at 4°C. The membrane was then incubated with horseradish peroxidase bound to secondary antibody (1:2000 for goat anti-rabbit immunoglobulin G (IgG, ab6721, Abcam) for 2 h at 25°C. Subsequently, the proteins were detected using an enhanced chemiluminesence (ECL) Plus Western Blotting Substrate (Thermo Fisher) and blots were exposed to BioMax-XAR film (Kodak, Rochester, NY, USA). The Western blot results were quantified by gray value analysis using Quantity One 4.6.2 software (Bio-Rad) and β-actin was used as the internal control. All the raw and unedited Western blot figures for this manuscript were cropped and displayed in the Supplementary Western blot figures (see section on supplementary data given at the end of this article).
Culture and treatment of HCFs
HCFs were purchased from ScienCell (San Diego, CA, USA) and cultured in fibroblast medium Dulbecco Modified Eagle Medium (DMEM; Invitrogen) supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin solution and maintained at 37°C in 5% CO2. The HCFs at 70% confluence were treated for 24 h with 100 ng/mL of LPS (O11:B4; L4391, Sigma-Aldrich) in DMEM/F12 serum-free media (SFM) in accordance with standard protocols to stimulate the inflammatory response in HCFs.
Enzyme-linked immunosorbent assay
The concentrations of TNF-α (Catalog: MTA00B) and IL-6 (Catalog: D6050) were measured using ELISA system kits (R&D Systems). Monoclonal antibodies were coated onto standard ELISA plates in a volume of 100 μL/well overnight at 4°C. Uncoated sites were blocked with 100 μL of 1% bovine serum albumin in PBS for 1 h at 25°C. The supernatant of the HCF culture was added and incubated for 2 h at 37°C, followed by biotinylated anti-TNF-α or anti-IL-6 (Biosynthesis Biotechnology, Beijing, China) for 1 h at 37°C. Streptavidin–horseradish peroxidase (1:1000, Biosynthesis Biotechnology) was then added and incubated at 37°C for 30 min. A color reaction was developed with 100 μL of tetramethylbenzidine substrate (Tiangen Biotechnology, China) for 30 min, and the reaction was terminated by adding 2 M sulfuric acid (Sigma). Finally, the absorbance at 450 nm was measured in triplicate using a microplate ELISA reader. Each sample was measured in triplicate.
Cell transfection
To analyze the regulation of gene expression, siRNA targeting NEAT1 (si-NEAT1) and its negative control (si-control) were purchased from GenePharma Co. Ltd (Shanghai, China). They were transfected into HCF cells using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. After cell transfection for 48 h, the expression of NEAT1 was evaluated by qRT-PCR.
RNA pull-down assay
Binding sites between NEAT1 and miR-1246 were predicted using DIANA tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-predicted). An RNA pull-down assay was conducted to determine the interaction between lncRNA NEAT1 and miR-1246 in HCFs. Briefly, a DNA probe complementary to NEAT1 was synthesized and biotinylated by GenePharma Co., Ltd. (Shanghai, China). The RNA pull-down assay was carried out using a Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the manufacturer’s protocol. NEAT1 (3 µg) labeled with streptavidin was diluted into 100 µL with structure buffer and then kept at 90°C for 5 min. After the pretreated RNA was incubated at 25°C for 1 h with agitation, 60 µL of streptavidin beads washed by cell lysates were added and incubated at 25°C for 1 h with agitation. Subsequently, the beads were washed with 1 mL of low-concentration salt solution twice and then washed with 1 mL of a high-concentration salt solution twice. The RNA-binding protein complexes were washed and eluted for Western blot or qRT-PCR analysis.
RNA immunoprecipitation
RNA immunoprecipitation (RIP) was performed using an RNA-Binding Protein Immunoprecipitation Kit (Millipore). The HCFs were lysed with lysis buffer, and the cell lysis solutions were incubated with Argonaute2 (AGO2) antibody or normal mouse IgG. RNA–protein complexes were immunoprecipitated with protein A agarose beads, and the bead-bound complexes were immobilized with magnet. After the unbound materials were washed off with wash buffer, RNA was extracted using TRIzol (Invitrogen). Immunoprecipitation Western blots (IP-Western) were used to detect the AGO2 protein, and qRT-PCR was performed to quantify NEAT1 and miR-1246.
Interference of NEAT1 in the CRNV rat model
After construction of the CRNV model, the rats were randomly divided into an si-control (n = 6) and si-NEAT1 (n = 6) group, and siRNA was administered through a subconjunctival injection (Rho et al. 2015). The length and area of CRNV were observed on days 3, 5 and 7 with slit-lamp microscopy for evaluation of CRNV. The length was considered the longest blood vessel that developed in the cornea. The area of CRNV (A) was calculated using the following formula: A = C/12 × π × [r 2−(r−l)2], where C refers to the number of clock hours of limbus involved, l is the radius to the border of the vessel and r is the radius of the cornea (Han et al. 2015). The rats were killed with an overdose of pentobarbital sodium by an intraperitoneal injection on postoperative day 7, and the corneas were dissected for detection of the expression of NEAT1, miR-1246 mRNA and NKAP. The sequences of the si-control and si-NEAT1 were as follows: si-control, 5′-CACUGAUUUCAAAUGGUGCUAUU-3′ and si-NEAT1, 5′-GUGAGAAGUUGCUUAGAAAUU-3′.
Statistical analysis
SPSS 21.0 software (SPSS Inc.) was used for all statistical analyses, and data were presented as mean ± standard deviation. Comparisons between two groups were performed using the Student’s t-test and an ANOVA for more than two groups. A level of P < 0.05 was considered significant.
Results
NEAT1 was upregulated in the corneas of the CRNV rat model
The expression of NEAT1, miR-1246 and NKAP were determined in corneas from CRNV rat models (CRNV, n = 6) and the sham group (sham, n = 6). Compared with the sham group, the expression of NEAT1 (Fig. 1A) and NKAP mRNA (Fig. 1C) was upregulated in corneas with CRNV, whereas that of miR-1246 (Fig. 1B) was downregulated, and the NKAP protein level (Fig. 1D; Supplementary Fig. 2A) was decreased. The results showed altered expression of NEAT1, miR-1246 and NKAP in corneal tissue of CRNV, although their impact on CRNV progression remained uncertain.
Upregulation of NEAT1 in neovascularized corneas of the CRNV rat model. Corneas from CRNV rat models (CRNV, n = 6) and the sham group (sham, n = 6) were taken for gene expression analysis. The expression of NEAT1 (A) and miR-1246 (B) was quantified by qRT-PCR. The expression of NKAP mRNA (C) and protein (D) was examined by qRT-PCR and Western blot, respectively. *P < 0.05 vs sham. **P < 0.01 vs sham.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Upregulation of NEAT1 in neovascularized corneas of the CRNV rat model. Corneas from CRNV rat models (CRNV, n = 6) and the sham group (sham, n = 6) were taken for gene expression analysis. The expression of NEAT1 (A) and miR-1246 (B) was quantified by qRT-PCR. The expression of NKAP mRNA (C) and protein (D) was examined by qRT-PCR and Western blot, respectively. *P < 0.05 vs sham. **P < 0.01 vs sham.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Upregulation of NEAT1 in neovascularized corneas of the CRNV rat model. Corneas from CRNV rat models (CRNV, n = 6) and the sham group (sham, n = 6) were taken for gene expression analysis. The expression of NEAT1 (A) and miR-1246 (B) was quantified by qRT-PCR. The expression of NKAP mRNA (C) and protein (D) was examined by qRT-PCR and Western blot, respectively. *P < 0.05 vs sham. **P < 0.01 vs sham.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
The expression of NEAT1 in HCFs was upregulated by LPS
LPS (100 ng/mL) was used to stimulate HCFs. After treatment with LPS for 24 h, the expression of NEAT1 and miR-1246, in addition to the mRNA and protein levels of NKAP in HCFs, was evaluated. As shown by the results, LPS markedly promoted the expression of NEAT1 (Fig. 2A) and NKAP (Fig. 2C) but repressed the expression of miR-1246 (Fig. 2B). NKAP protein level was also increased by LPS (Fig. 2D; Supplementary Fig. 2B). Furthermore, the levels of TNF-α (Fig. 2E) and IL-6 (Fig. 2F) were significantly elevated by LPS. The results indicated that LPS promoted the secretion of inflammatory factors including TNF-α and IL-6, as well as altering the expression of various genes in HCFs.
The expression of NEAT1 in HCFs was upregulated by LPS. LPS (100 ng/mL) was used to stimulate HCFs. After treatment with LPS for 24 h, the expression of NEAT1 and miR-1246, in addition to the mRNA and protein levels of NKAP in HCFs, was evaluated. (A) The expression of NEAT1 was quantified by qRT-PCR. (B) The expression of miR-1246 was quantified by qRT-PCR. The levels of NKAP mRNA (C) and protein (D) were determined by qRT-PCR and Western blot, respectively. The levels of TNF-α (E) and IL-6 (F) were detected by an ELISA. *P < 0.05 vs control. **P < 0.01 vs control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
The expression of NEAT1 in HCFs was upregulated by LPS. LPS (100 ng/mL) was used to stimulate HCFs. After treatment with LPS for 24 h, the expression of NEAT1 and miR-1246, in addition to the mRNA and protein levels of NKAP in HCFs, was evaluated. (A) The expression of NEAT1 was quantified by qRT-PCR. (B) The expression of miR-1246 was quantified by qRT-PCR. The levels of NKAP mRNA (C) and protein (D) were determined by qRT-PCR and Western blot, respectively. The levels of TNF-α (E) and IL-6 (F) were detected by an ELISA. *P < 0.05 vs control. **P < 0.01 vs control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
The expression of NEAT1 in HCFs was upregulated by LPS. LPS (100 ng/mL) was used to stimulate HCFs. After treatment with LPS for 24 h, the expression of NEAT1 and miR-1246, in addition to the mRNA and protein levels of NKAP in HCFs, was evaluated. (A) The expression of NEAT1 was quantified by qRT-PCR. (B) The expression of miR-1246 was quantified by qRT-PCR. The levels of NKAP mRNA (C) and protein (D) were determined by qRT-PCR and Western blot, respectively. The levels of TNF-α (E) and IL-6 (F) were detected by an ELISA. *P < 0.05 vs control. **P < 0.01 vs control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Downregulation of NEAT1 suppressed the secretion of TNF-α and IL-6
To investigate the relationship between NEAT1 expression and the secretion of inflammatory factors, HCFs were divided into four groups: control, LPS, LPS + si-control and LPS + si-NEAT1. As shown in the figures, NEAT1 (Fig. 3A) and NKAP mRNA (Fig. 3C) were upregulated by LPS, and this increase was reversed by si-NEAT1 transfection. In contrast, miR-1246 was downregulated by LPS, and this decrease was reversed by si-NEAT1 transfection (Fig. 3B). In addition, the LPS-induced increase in the secretion of TNF-α (Fig. 3D) and IL-6 (Fig. 3E) was facilitated by LPS but decreased by si-NEAT1 transfection, which was in accordance with the expression of NEAT1. NKAP protein level was also increased by LPS treatment but decreased by NEAT1 interference (Fig. 3F; Supplementary Fig. 2C). These findings revealed that downregulation of NEAT1 suppressed the secretion of inflammatory factors, including TNF-α and IL-6, induced by LPS.
Downregulation of NEAT1 suppressed the secretion of TNF-α and IL-6. HCFs were divided into four groups: control, LPS, LPS + si-control, and LPS + si-NEAT1. (A) The expression of NEAT1 was quantified by qRT-PCR. (B) The expression of miR-1246 and NKAP mRNA (C) was quantified by qRT-PCR. The levels of TNF-α (D)and IL-6 (E) were detected by an ELISA. (F) The expression of NKAP protein was measured with Western blot. *P < 0.05 vs control or si-control. **P < 0.05 vs control or si-control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Downregulation of NEAT1 suppressed the secretion of TNF-α and IL-6. HCFs were divided into four groups: control, LPS, LPS + si-control, and LPS + si-NEAT1. (A) The expression of NEAT1 was quantified by qRT-PCR. (B) The expression of miR-1246 and NKAP mRNA (C) was quantified by qRT-PCR. The levels of TNF-α (D)and IL-6 (E) were detected by an ELISA. (F) The expression of NKAP protein was measured with Western blot. *P < 0.05 vs control or si-control. **P < 0.05 vs control or si-control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Downregulation of NEAT1 suppressed the secretion of TNF-α and IL-6. HCFs were divided into four groups: control, LPS, LPS + si-control, and LPS + si-NEAT1. (A) The expression of NEAT1 was quantified by qRT-PCR. (B) The expression of miR-1246 and NKAP mRNA (C) was quantified by qRT-PCR. The levels of TNF-α (D)and IL-6 (E) were detected by an ELISA. (F) The expression of NKAP protein was measured with Western blot. *P < 0.05 vs control or si-control. **P < 0.05 vs control or si-control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Interaction between NEAT1 and miR-1246
Bioinformatics tools (DIANA) predicted binding sites between NEAT1 and miR-1246 (Fig. 4A), thereby implying potential binding and interactions between them. An RIP assay was used to verify the binding between NEAT1 and miR-1246 in HCFs, and AGO2 was detected with the IP-Western method. Compared with IgG, high levels of NEAT1 and miR-1246 were detected in the AGO2 antibody precipitate (Fig. 4B), thereby confirming binding between NEAT1 and miR-1246. In an RNA pull-down assay, the AGO2 level in the pull-down complex of NEAT1 was analyzed by Western blot. Compared with its negative control (NC), strongly expressed miR-1246 was detected in the pull-down complex of NEAT1 (Fig. 4C and D). This finding confirmed the interaction between miR-1246 NEAT1 in HCFs. The proposed relationship between NEAT1 and miR-1246 was further confirmed using a luciferase reporter assay. The results are displayed in Supplementary Fig. 1.
Interaction between NEAT1 and miR-1246. Interaction between NEAT1 and miR-1246 in HCFs was verified. (A) The binding site between NEAT1 and miR-1246 was predicted by bioinformatics software (DIANA tools). (B) The binding relationship between NEAT1 and miR-1246 was determined using the RNA immunoprecipitation (RIP) assay. The expression of NEAT1 (C) and miR-1246 (D) was quantified by qRT-PCR. (E) The interaction between NEAT1 and miR-1246 was measured by the RNA pull-down assay. (F) The expression of miR-1246 was detected by qRT-PCR. ***P < 0.05 vs IgG or negative control (NC).
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Interaction between NEAT1 and miR-1246. Interaction between NEAT1 and miR-1246 in HCFs was verified. (A) The binding site between NEAT1 and miR-1246 was predicted by bioinformatics software (DIANA tools). (B) The binding relationship between NEAT1 and miR-1246 was determined using the RNA immunoprecipitation (RIP) assay. The expression of NEAT1 (C) and miR-1246 (D) was quantified by qRT-PCR. (E) The interaction between NEAT1 and miR-1246 was measured by the RNA pull-down assay. (F) The expression of miR-1246 was detected by qRT-PCR. ***P < 0.05 vs IgG or negative control (NC).
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Interaction between NEAT1 and miR-1246. Interaction between NEAT1 and miR-1246 in HCFs was verified. (A) The binding site between NEAT1 and miR-1246 was predicted by bioinformatics software (DIANA tools). (B) The binding relationship between NEAT1 and miR-1246 was determined using the RNA immunoprecipitation (RIP) assay. The expression of NEAT1 (C) and miR-1246 (D) was quantified by qRT-PCR. (E) The interaction between NEAT1 and miR-1246 was measured by the RNA pull-down assay. (F) The expression of miR-1246 was detected by qRT-PCR. ***P < 0.05 vs IgG or negative control (NC).
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
LPS regulated the expression of NKAP and NF-κB signaling via the NEAT1/miR-1246 pathway
Subsequently, we explored the regulatory effects of LPS on NEAT1 and miR-1246 expression and on the secretion of inflammatory factors. HCFs were divided into six groups: control, LPS, LPS + si-control, LPS + si-NEAT1, LPS + si-NEAT1 + NC and LPS + si-NEAT1 + miR-1246 inhibitor. The results revealed that LPS clearly promoted the NKAP protein expression and phosphorylation of p65 (p-p65), and these were reversed by si-NEAT1 transfection, but inhibition of miR-1246 finally increased the NKAP protein expression and p-p65 level (Fig. 5A and B). In addition, LPS inhibited miR-1246 expression, and this effect was reversed by si-NEAT1 transfection, but miR-1246 inhibitor reduced miR-1246 expression (Fig. 5C). Furthermore, LPS clearly increased the secretion of inflammatory factors, including TNF-α and IL-6, which was reversed by si-NEAT1 transfection, but the levels of TNF-α and IL-6 was increased with miR-1246 inhibitor transfection (Fig. 5D and E).
LPS regulated the expression of NKAP and NF-κB signaling via the NEAT1/miR-1246 pathway. HCFs were divided into six groups: control, LPS, LPS + si-control, LPS + si-NEAT1, LPS + si-NEAT1 + NC and LPS + si-NEAT1 + miR-1246 inhibitor. The protein level of NKAP (A) and p-p65 (B) was determined using Western blot. (C) The expression of miR-1246 was quantified by qRT-PCR. The levels of TNF-α (D) and IL-6 (E) were assessed with ELISA. *P < 0.05 vs control. **P < 0.05 vs si-control. ***P < 0.05 vs negative control of miR-1246 inhibitor (NC).
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
LPS regulated the expression of NKAP and NF-κB signaling via the NEAT1/miR-1246 pathway. HCFs were divided into six groups: control, LPS, LPS + si-control, LPS + si-NEAT1, LPS + si-NEAT1 + NC and LPS + si-NEAT1 + miR-1246 inhibitor. The protein level of NKAP (A) and p-p65 (B) was determined using Western blot. (C) The expression of miR-1246 was quantified by qRT-PCR. The levels of TNF-α (D) and IL-6 (E) were assessed with ELISA. *P < 0.05 vs control. **P < 0.05 vs si-control. ***P < 0.05 vs negative control of miR-1246 inhibitor (NC).
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
LPS regulated the expression of NKAP and NF-κB signaling via the NEAT1/miR-1246 pathway. HCFs were divided into six groups: control, LPS, LPS + si-control, LPS + si-NEAT1, LPS + si-NEAT1 + NC and LPS + si-NEAT1 + miR-1246 inhibitor. The protein level of NKAP (A) and p-p65 (B) was determined using Western blot. (C) The expression of miR-1246 was quantified by qRT-PCR. The levels of TNF-α (D) and IL-6 (E) were assessed with ELISA. *P < 0.05 vs control. **P < 0.05 vs si-control. ***P < 0.05 vs negative control of miR-1246 inhibitor (NC).
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Downregulation of NEAT1 inhibited CRNV progression in the CRNV rat model
To verify the effect of NEAT1 expression on CRNV progression in vivo, the CRNV rat models were administered si-control (n = 6) or si-NEAT1 (n = 6) by a subconjunctival injection, and the length and area of CRNV were evaluated. The representative pictures of slit-lamp microscope in the two groups were provided (Fig. 6A). The results showed that the length (Fig. 6B) and area (Fig. 6C) of CRNV were reduced in the si-NEAT1 injection group. As compared with the si-control group, the expression of NEAT1 (Fig. 6D) and that of NKAP protein (Fig. 6F; Supplementary Fig. 2D) were decreased in the si-NEAT1 injection group, whereas the expression of miR-1246 was increased (Fig. 6E). The findings revealed that downregulation of NEAT1 inhibited CRNV progression in this rat model.
Downregulation of NEAT1 inhibited CRNV progression in the CRNV rat model. CRNV rat models were administered si-control (n = 6) or si-NEAT1 (n = 6) by a subconjunctival injection. (A) The representative pictures of slit-lamp microscope in the two groups were taken for evaluation of CRNV. The length (B) and area (C) of CRNV were evaluated on days 3, 5 and 7 with slit-lamp microscopy. The expression of NEAT1 (D) and miR-1246 (E) in corneas was quantified by qRT-PCR. (F) NKAP protein level was determined with Western blot. *P < 0.05 vs si-control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Downregulation of NEAT1 inhibited CRNV progression in the CRNV rat model. CRNV rat models were administered si-control (n = 6) or si-NEAT1 (n = 6) by a subconjunctival injection. (A) The representative pictures of slit-lamp microscope in the two groups were taken for evaluation of CRNV. The length (B) and area (C) of CRNV were evaluated on days 3, 5 and 7 with slit-lamp microscopy. The expression of NEAT1 (D) and miR-1246 (E) in corneas was quantified by qRT-PCR. (F) NKAP protein level was determined with Western blot. *P < 0.05 vs si-control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Downregulation of NEAT1 inhibited CRNV progression in the CRNV rat model. CRNV rat models were administered si-control (n = 6) or si-NEAT1 (n = 6) by a subconjunctival injection. (A) The representative pictures of slit-lamp microscope in the two groups were taken for evaluation of CRNV. The length (B) and area (C) of CRNV were evaluated on days 3, 5 and 7 with slit-lamp microscopy. The expression of NEAT1 (D) and miR-1246 (E) in corneas was quantified by qRT-PCR. (F) NKAP protein level was determined with Western blot. *P < 0.05 vs si-control.
Citation: Journal of Molecular Endocrinology 61, 4; 10.1530/JME-18-0098
Discussion
In this study, NEAT1 was upregulated in neovascularized rat corneas and by LPS in HCFs. NEAT1 activated the NF-κB signaling pathway and induced a downstream inflammatory reaction by targeting miR-1246, thus promoting CRNV occurrence. For the first time, the regulatory effects of NEAT1 on the inflammatory response in HCFs and its role in CRNV development were highlighted. These results confirm the proinflammatory property of NEAT1 in human disease and the significance of the inflammatory reaction mediated by HCFs in CRNV.
The role of noncoding transcripts lncRNAs in modulating many biological processes, including cell pluripotency, cell cycle regulation, cell function, dosage compensation and gene imprinting, was demonstrated previously (Zhu et al. 2013, Li et al. 2014). Abnormal lncRNA expression has been associated with the occurrence and progression of various diseases, such as cancer, cardiovascular diseases and neurological disorders, as well as aging (Kazemzadeh et al. 2015). To reveal the potential influence of lncRNAs in CRNV, differentially expressed lncRNAs in normal and vascularized corneas were identified by a microarray analysis, and 154 lncRNAs (60 downregulated and 94 upregulated) were identified (Huang et al. 2015). This finding implied that lncRNAs may be promising targets for the prevention and treatment of CRNV. Furthermore, studies showed that lncRNA H19 negatively regulated corneal epithelial proliferation and adhesion (Klein et al. 2016) and that lncRNA MIAT promoted neurovascular remodeling in eyes and brain (Jiang et al. 2016). The above findings pointed to the potential roles of lncRNAs in the pathogenesis of CRNV. A previous study identified the role of NEAT1 as a key regulator in the pathogenesis of CRNV. The authors showed that NEAT1 mediated the inflammatory response in HCFs through the miR-1246/NKAP/NF-κB pathway and that it played a critical role in modulating the secretion of inflammatory factors in HCFs. The present study revealed the function of NEAT as a competing endogenous RNA of miR-1246 to free the NKAP, which led to activation of the NF-κB signaling pathway and subsequent inflammatory reactions.
miRNAs are a class of noncoding RNAs that negatively regulate gene expression by directly targeting mRNA. Several miRNAs, including miR-204, miR-184 and miR-206, have been implicated in CRNV progression, with these miRNA regulating different targets (Kather et al. 2014, Li et al. 2015, Zong et al. 2016). A previous study reported that miR-1246 was a target of the p53 family and that it was closely related to cancer by promoting cancer cell proliferation, invasion and migration (Chen et al. 2014a ). Furthermore, miR-1246 overexpression aggravated the decrease in cell viability induced by LPS, as well as increasing apoptosis and overproduction of proinflammatory factors in a chondrogenic cell line, ATDC5, by downregulation of hepatocyte nuclear factor 4γ and suppression of the PI3K/AKT and JAK/STAT pathways (Wu et al. 2017). These finding demonstrated the impact miR-1246 on inflammatory reactions and inflammatory diseases. In a previous study, we showed that miR-1246 repressed NF-κB by targeting NKAP and inhibiting the proliferation of human umbilical vein endothelial cells (Bai et al. 2016). This finding suggested that miR-1246 may be promising target for CRNV treatment. In the present study, we identified miR-1246 as a novel target of NEAT1, which influences the secretion of inflammatory factors by HCFs, mediated by downregulation of NKAP and NF-κB signaling. These findings emphasize the role of NEAT1 in inflammatory regulation of CRNV.
The NF-κB transcription factor family regulates the expression of multiple genes that are critical for cell proliferation, apoptosis, tumorigenesis, viral replication, inflammation and various autoimmune disorders (Tobon-Velasco et al. 2014, Chen et al. 2014b ). Activation of NF-κB is thought to be part of a stress response, as NF-κB is activated by a variety of stimuli, which include growth factors, cytokines, lymphokines, ultraviolet, pharmacological agents and stress (Baldwin 1996). NF-κB is considered the central mediator of the inflammatory process and a key participant in innate and adaptive immune responses (DiDonato et al. 2012). NF-κB often mediates the inflammatory reaction by activating inflammation-promoting genes or induces cytokines, such as TNF-α, IL-1, IL-6 and IL-8, that regulate the immune response, as well as adhesion molecules, which lead to the recruitment of leukocytes to sites of inflammation (Hoesel & Schmid 2013). NKAP is a transcriptional repressor that mediates TNF-α- and IL-1-induced NF-κB activation (Chen et al. 2003). It is also required for invariant natural killer T-cell proliferation and differentiation and subsequent generation of natural killer T17 cells (Thapa et al. 2016). The aforementioned findings demonstrate the key role of NF-κB in immunity and inflammatory processes. In the present study, NF-κB was activated by NKAP and evoked an inflammatory reaction by inducing the secretion of inflammatory factors of HCFs, which resulted in CRNV development.
In conclusion, we postulate that lncRNA NEAT1 induces an inflammatory reaction, mediated by NF-κB via the miR-1246/NKAP pathway, thereby promoting CRNV occurrence. This study revealed the regulatory mechanism of the inflammatory response in CRNV. The results offer novel insights for developing promising therapeutic strategies for CRNV and related eye diseases.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JME-18-0098.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the grants from the National Natural Science Foundation of China (No. 81700798 and No. 81300656) and the Bethune-Merk Diabetes Research Fund (No. G2017044).
References
Bai Y, Wang W, Sun G, Zhang M & Dong J 2016 Curcumin inhibits angiogenesis by up-regulation of microRNA-1275 and microRNA-1246: a promising therapy for treatment of corneal neovascularization. Cell Proliferation 49 751–762. (https://doi.org/10.1111/cpr.12289)
Baldwin AS Jr 1996 The NF-kappa B and I kappa B proteins: new discoveries and insights. Annual Review of Immunology 14 649–683. (https://doi.org/10.1146/annurev.immunol.14.1.649)
Benayoun Y, Petellat F, Leclerc O, Dost L, Dallaudière B, Reddy C, Robert PY & Salomon JL 2015 Current treatments for corneal neovascularization. Journal Francais Dophtalmologie 38 996–1008. (https://doi.org/10.1016/j.jfo.2015.09.006)
Cao J, Zhang Y, Yang J, He S, Li M, Yan S, Chen Y, Qu C & Xu L 2016 NEAT1 regulates pancreatic cancer cell growth, invasion and migration though mircroRNA-335-5p/c-met axis. American Journal of Cancer Research 6 2361–2374.
Chen D, Li Z, Yang Q, Zhang J, Zhai Z & Shu HB 2003 Identification of a nuclear protein that promotes NF-kappaB activation. Biochemical and Biophysical Research Communications 310 720–724. (https://doi.org/10.1016/j.bbrc.2003.09.074)
Chen J, Yao D, Zhao S, He C, Ding N, Li L & Long F 2014a MiR-1246 promotes SiHa cervical cancer cell proliferation, invasion, and migration through suppression of its target gene thrombospondin 2. Archives of Gynecology and Obstetrics 290 725–732. (https://doi.org/10.1007/s00404-014-3260-
Chen L, Zhang X, Chen J, Zhang X, Fan H, Li S & Xie P 2014b NF-kappaB plays a key role in microcystin-RR-induced HeLa cell proliferation and apoptosis. Toxicon 87 120–130. (https://doi.org/10.1016/j.toxicon.2014.06.002)
DiDonato JA, Mercurio F & Karin M 2012 NF-kappaB and the link between inflammation and cancer. Immunological Reviews 246 379–400. (https://doi.org/10.1111/j.1600-065X.2012.01099.x)
Han Y, Shao Y, Liu T, Qu YL, Li W & Liu Z 2015 Therapeutic effects of topical netrin-4 inhibits corneal neovascularization in alkali-burn rats. PLoS ONE 10. (https://doi.org/10.1371/journal.pone.0122951)
Hoesel B & Schmid JA 2013 The complexity of NF-kappaB signaling in inflammation and cancer. Molecular Cancer 12 86. (https://doi.org/10.1186/1476-4598-12-86)
Huang J, Li YJ, Liu JY, Zhang YY, Li XM, Wang LN, Yao J, Jiang Q & Yan B 2015 Identification of corneal neovascularization-related long noncoding RNAs through microarray analysis. Cornea 34 580–587. (https://doi.org/10.1097/ICO.0000000000000389)
Jiang Q, Shan K, Qun-Wang X, Zhou RM, Yang H, Liu C, Li YJ, Yao J, Li XM, Shen Y, et al.2016 Long non-coding RNA-MIAT promotes neurovascular remodeling in the eye and brain. Oncotarget 7 49688–49698. (https://doi.org/10.18632/oncotarget.10434)
Jin C, Peng X, Xie T, Lu X, Liu F, Wu H, Yang Z, Wang J, Cheng L & Wu N 2016 Detection of the long noncoding RNAs nuclear-enriched autosomal transcript 1 (NEAT1) and metastasis associated lung adenocarcinoma transcript 1 in the peripheral blood of HIV-1-infected patients. HIV Medicine 17 68–72. (https://doi.org/10.1111/hiv.12276)
Kather JN, Friedrich J, Woik N, Sticht C, Gretz N, Hammes HP & Kroll J 2014 Angiopoietin-1 is regulated by miR-204 and contributes to corneal neovascularization in KLEIP-deficient mice. Investigative Ophthalmology and Visual Science 55 4295–4303. (https://doi.org/10.1167/iovs.13-13619)
Kazemzadeh M, Safaralizadeh R & Orang AV 2015 LncRNAs: emerging players in gene regulation and disease pathogenesis. Japanese Journal of Genetics 94 771–784. (https://doi.org/10.1007/s12041-015-0561-6)
Klein RH, Stephens DN, Ho H, Chen JK, Salmans ML, Wang W, Yu Z & Andersen B 2016 Cofactors of LIM domains associate with estrogen receptor alpha to regulate the expression of noncoding RNA H19 and corneal epithelial progenitor cell function. Journal of Biological Chemistry 291 13271–13285. (https://doi.org/10.1074/jbc.M115.709386)
Lee SH, Kim KW, Joo K & Kim JC 2016 Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts. BMC Ophthalmology 16 57. (https://doi.org/10.1186/s12886-016-0235-z)
Li X, Wu Z, Fu X & Han W 2014 lncRNAs: insights into their function and mechanics in underlying disorders. Mutation Research/Reviews in Mutation Research 762 1–21. (https://doi.org/10.1016/j.mrrev.2014.04.002)
Li X, Zhou H, Tang W, Guo Q & Zhang Y 2015 Transient downregulation of microRNA-206 protects alkali burn injury in mouse cornea by regulating connexin 43. International Journal of Clinical and Experimental Pathology 8 2719–2727.
Liu Y, Kimura K, Yanai R, Chikama T & Nishida T 2008 Cytokine, chemokine, and adhesion molecule expression mediated by MAPKs in human corneal fibroblasts exposed to poly(I:C). Investigative Ophthalmology and Visual Science 49 3336–3344. (https://doi.org/10.1167/iovs.07-0972)
Marton A, Kolozsi C, Kusz E, Olah Z, Letoha T, Vizler C & Pecze L 2014 Propylene-glycol aggravates LPS-induced sepsis through production of TNF-alpha and IL-6. Iranian Journal of Immunology 11 113–122. (https://doi.org/IJIv11i2A6)
Orita T, Kimura K, Nishida T & Sonoda KH 2013 Cytokine and chemokine secretion induced by poly(I:C) through NF-kappaB and phosphoinositide 3-kinase signaling pathways in human corneal fibroblasts. Current Eye Research 38 53–59. (https://doi.org/10.3109/02713683.2012.721044)
Pradhan N, Guha R, Chowdhury S, Nandi S, Konar A & Hazra S 2015 Curcumin nanoparticles inhibit corneal neovascularization. Journal of Molecular Medicine 93 1095–1106. (https://doi.org/10.1007/s00109-015-1277-z)
Rho CR, Choi JS, Seo M, Lee SK & Joo CK 2015 Inhibition of lymphangiogenesis and hemangiogenesis in corneal inflammation by subconjunctival Prox1 siRNA injection in rats. Investigative Ophthalmology and Visual Science 56 5871–5879. (https://doi.org/10.1167/iovs.14-14433)
Santoro M, Nociti V, Lucchini M, De Fino C, Losavio FA & Mirabella M 2016 Expression profile of long non-coding RNAs in serum of patients with multiple sclerosis. Journal of Molecular Neuroscience 59 18–23. (https://doi.org/10.1007/s12031-016-0741-8)
Thapa P, Chen MW, McWilliams DC, Belmonte P, Constans M, Sant’Angelo DB & Shapiro VS 2016 NKAP regulates invariant NKT cell proliferation and differentiation into ROR-gammat-expressing NKT17 cells. Journal of Immunology 196 4987–4998. (https://doi.org/10.4049/jimmunol.1501653)
Tobon-Velasco JC, Cuevas E & Torres-Ramos MA 2014 Receptor for AGEs (RAGE) as mediator of NF-kB pathway activation in neuroinflammation and oxidative stress. CNS and Neurological Disorders /Drug Targets 13 1615–1626. (https://doi.org/10.2174/1871527313666140806144831)
Wang Q, Wang W, Zhang F, Deng Y & Long Z 2017 NEAT1/miR-181c Regulates osteopontin (OPN)-Mediated synoviocyte Proliferation in osteoarthritis. Journal of Cellular Biochemistry 118 3775–3784. (https://doi.org/10.1002/jcb.26025)
Wu J, Du Y, Mann MM, Funderburgh JL & Wagner WR 2014 Corneal stromal stem cells versus corneal fibroblasts in generating structurally appropriate corneal stromal tissue. Experimental Eye Research 120 71–81. (https://doi.org/10.1016/j.exer.2014.01.005)
Wu DP, Zhang JL, Wang JY, Cui MX, Jia JL, Liu XH & Liang QD 2017 MiR-1246 promotes LPS-induced inflammatory injury in chondrogenic cells ATDC5 by targeting HNF4gamma. Cellular Physiology and Biochemistry 43 2010–2021. (https://doi.org/10.1159/000484162)
Yang HL, Huang PJ, Liu YR, Kumar KJ, Hsu LS, Lu TL, Chia YC, Takajo T, Kazunori A & Hseu YC 2014 Toona sinensis inhibits LPS-induced inflammation and migration in vascular smooth muscle cells via suppression of reactive oxygen species and NF-kappaB signaling pathway. Oxidative Medicine and Cellular Longevity 2014 901315. (https://doi.org/10.1155/2014/901315)
Yang S, Yang TS, Wang F & Su SB 2015 High-mobility group box-1-Toll-Like receptor 4 axis mediates the recruitment of endothelial progenitor cells in alkali-induced corneal neovascularization. International Immunopharmacology 28 450–458. (https://doi.org/10.1016/j.intimp.2015.07.013)
Zhang F, Wu L, Qian J, Qu B, Xia S, La T, Wu Y, Ma J, Zeng J, Guo Q, et al.2016 Identification of the long noncoding RNA NEAT1 as a novel inflammatory regulator acting through MAPK pathway in human lupus. Journal of Autoimmunity 75 96–104. (https://doi.org/10.1016/j.jaut.2016.07.012)
Zhu J, Fu H, Wu Y & Zheng X 2013 Function of lncRNAs and approaches to lncRNA-protein interactions. Science in China 56 876–885. (https://doi.org/10.1007/s11427-013-4553-6)
Zong R, Zhou T, Lin Z, Bao X, Xiu Y, Chen Y, Chen L, Ma JX, Liu Z & Zhou Y 2016 Down-regulation of MicroRNA-184 is associated with corneal neovascularization. Investigative Ophthalmology and Visual Science 57 1398–1407. (https://doi.org/10.1167/iovs.15-17417)
