lncRNA H19 sponging miR‑93 to regulate inflammation in retinal epithelial cells under hyperglycemia via XBP1s
Rong Luo1· Fan Xiao1 · Pei Wang1 · Yu‑Xiang Hu1
Abstract
Objective Diabetic retinopathy (DR) is a major complication of both type 1 and type 2 diabetes. Recently, inflammation was found to play an important role in DR pathogenesis. But the mechanism has not been fully understood.
Methods ARPE-19 cells were cultured under normal condition and high-glucose condition, then the expressions of miR-93, XBP1s and lncRNA H19 were measured using RT-qPCR or western blots. Besides, the mRNA level of eIF2α and GRP78 and protein level of p-eIF2α and GRP78 were measured by RT-qPCR or western blots. In addition, RT-qPCR and ELISA were adopted to examine the expression and secretion of cytokine factors in these conditions. Dual-luciferase reporter gene assay was used to elucidate the binding and regulation among XBP1s, miR-93 and H19. RNA immunoprecipitation was also performed to verify the interaction between H19 and miR-93. The expressions of DNAJC3 and DNAJB9, the downstream targets of XBP1s, were detected by RT-qPCR.
Results We identified that H19 and XBP1s were down-regulated in ARPE-19 cells under high-glucose condition, while miR-93 was up-regulated. ER stress inducers TM and IRE1 inhibitor STF-083010 were adopted and data suggest that ER stress could be induced during high-glucose treatment. In addition, the altered expressions of miR-93, XBP1s and H19 might mediate the increased level of pro-inflammatory cytokines. Furthermore, miR-93 interacted with either lncRNA H19 or XBP1s then modulating the inflammatory processes.
Conclusions H19 played an important role in regulating inflammatory processes in retinal endothelial cells under high- glucose condition through modulating miR-93/XBP1s axis.
Keywords Long non-coding RNA · H19 · MicroRNA · miR-93 · Diabetic retinopathy · Inflammation
Background
Diabetic retinopathy (DR) is a frequent problem in diabetic patients, which almost all type 1 diabetes patients and more than 60% of type 2 diabetes patients have retin- opathy [1]. With the increasing prevalence of diabetes, DR becomes the principal reason for visual impairment and blindness among adults [2]. DR is characterized by the ret- inal microvascular disorders, accompanied with increased leukocyte adhesion and blood-retinal barrier breakdown [3]. It has been revealed that inflammatory processes play a central role in the development of diabetic [1, 3, 4]. The therapeutic approaches that inhibit production of inflam- matory mediators showed beneficial effects on the devel- opment of DR, especially at the early stages [5].
X-box-binding protein 1 (XBP1), a transcription fac- tor activated by endoplasmic reticulum (ER) stress, is critical for various cellular processes, including apoptosis and inflammation [6]. The XBP1(U) is encoded by the unspliced mRNA of XBP1, while the isoform XBP1(S) is generated from an unconventional spliced mRNA in which 26 nt would be spliced. Besides, XBP1(U) is constitutively expressed while XBP1(S) is induced under ER stress [7]. It has been reported that ER stress could up-regulate the expression of TNF-α and mediate the retinal inflamma- tion during the development of diabetic retinopathy [8]. On the other hand, XBP1 activation was found to partici- pate in the regulation of VEGF signaling pathway during angiogenesis [9]. However, whether XBP1 influences the inflammation of DR pathogenesis remains unknown.
MicroRNAs (miRNAs) are non-coding RNAs with the length of about 20–22 nucleotides and modulate gene expression and, therefore, regulate a variety of fundamen- tal cellular processes. Several miRNAs have been discov- ered to be up-regulated in diabetic retinal endothelial cells (RECs) [10]. The elevated level of miR-93 in plasma has been revealed to be associated with high risk of developing diabetic retinopathy [11]. Besides, miR-93 was shown to regulate VEGF function in hyperglycemic conditions [12]. We identified that XBP1 might be the target of miR-93, but the regulation and the mechanism involved in the inflam- mation of DR need to be explored further.
Similarly, long non-coding RNAs (lncRNAs), the non- coding RNAs more than 200 nucleotides, also contribute to regulate gene expression at transcriptional or post-tran- scriptional level. Increasing evidences have shown that lncRNAs take part in modulating critical processes in DR development. Myocardial infarction-associated transcript (MIAT) is a highly expressed lncRNA in retinal precur- sor cells and was found to be up-regulated in the diabetic retinas [13]. Antisense RNA to INK4 locus (ANRIL) regu- lates the expression and function of VEGF in DR which might be mediated by miR-200b [14]. H19 was reported to be down-regulated in human retinal endothelial cells under high-glucose conditions [15], but the mechanism has not been fully explored. We discovered the binding site between H19 and miR-93 through bioinformatics predic- tion, but whether they are involved in the inflammation of DR has not been investigated. In the present study, we elucidated that H19 and miR-93 could regulate the inflam- matory cytokine production via XBP1.
Methods
Cell culture and transient transfection
Retinal epithelial cells ARPE-19 were purchased from American Type Culture Collection ATCC (Manassas, VA, USA) and cultured in Dulbecco’s minimum essential medium (DMEM): F12 medium (5 mM (D-) glucose for normal condition and 25 mM (D-) glucose for high-glu- cose condition) supplemented with 10% fetal bovine serum (FBS). Cells were incubated in 5% CO2 condition at 37 °C. For passaging, cells were digested with trypsin–EDTA solution. For transient transfection, cells in logarithmic growth period were digested, collected and inoculated in 10 cm dishes. When cell density reaches 50–60%, transfection was carried out with Lipofectamine2000 (Invitrogen, Burl- ington, ON, Canada) as the guideline suggests.
RNA extraction and quantitative polymerase chain reaction (RT‑qPCR)
The total RNAs of ARPE-19 cells were extracted using Trizol reagent (Invitrogen, Burlington, ON, Canada). After concentration determination, RNA was reversely transcribed into complementary DNA (cDNA) with high- capacity cDNA reverse transcription kit (Applied Bio- systems, Burlington, ON, Canada). Then, RT-qPCR was performed using SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA). The primers used in RT-qPCR are listed in Table 1. GAPDH and U6 were used as the internal control for mRNA/lncRNA and miRNA, respectively.
Western blots
After transfection or treatments, ARPE-19 cells were lysed with radioimmune precipitation assay (RIPA) buffer and centrifuged at 12,000 g for 5 min. The supernatants were used for western blots. Protein was separated by 10% polyacrylamide gel electrophoresis (PAGE) with a 5% stacking gel and then transferred onto nitrocellulose (NC) membrane. After blocked with skimmed milk at room temperature for 1 h, the membrane was incubated with the anti-XBP1 (1:1000, 647502, Biolegend, San Diego, CA) or β-Actin (1:1000, #4967, CST, Danvers, MA, USA) specific antibody at 4 °C overnight. Next, the membrane was washed with PBS/Tween-20 (PBST) and followed by incubation with HRP-labeled secondary antibody for 1 h at room temperature in the dark. Finally, the protein bands were visualized in a gel imaging system. Gray values of bands were analyzed using the ImageJ software and the relative expression level of XBP1 was calculated as the level of Actin used as the internal reference.
Enzyme‑linked immunosorbent assays (ELISA)
After transfection of designated time, ARPE-19 cells were collected for RNA extraction, while the culture media were collected for cytokine examination by ELISA as ELISA kit guideline suggested. ELISA kits for human TNF-α, IL-6 and IL-1β were used for TNF-α, IL-6 and IL-1β detection.
RNA‑binding protein immunoprecipitation (RIP)
RIP assay was performed with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Briefly, after transfection, ARPE-19 cells were lysed with the lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 2 mM ethylene diamine tetraacetic acid, 1 mM NaF, and 0.5 mM dithiothreitol) containing RNasin and protease inhibitors. After centrifugation, the supernatant was col- lected and incubated with protein G agarose beads (Invitro- gen) pre-coated with anti-Ago2 antibody or anti-immuno- globulin G (IgG) (ab5072, Abcam, Cambridge, MA, USA). After incubation at 4 °C overnight, the beads were centri- fuged and washed for three times. Next, the RNA binding onto the beads was extracted with Trizol reagent and fol- lowed by RT-qPCR detection as described above.
Dual‑luciferase reporter assay
The 3′-untranslated region (3′-UTR) of XBP1 or H19 was cloned into the plasmid pmirGLO vectors (Promega, Madi- son, WI) to generate wild-type or mutated XBP1 and H19 firefly luciferase plasmids. The renilla luciferase reporter vector was used as the internal control. Cells were seeded at a density of 2 × 105 cells/well in 24-well plates. 0.2 μg of firefly luciferase reporter plasmid, 0.002 μg of renilla lucif- erase plasmid (pRL-CMV, Promega, Madison, WI, USA) and 50 nM of miR-93 mimics, or miR-93 mimics NC were transfected to each well using Lipofectamine 2000 (Invitro- gen, Carlsbad, CA, USA). 48 h after transfection, the relative luciferase activity was confirmed following the Dual-Lucif- erase Reporter Assay Kit instructions (Promega, Madison, WI, USA). The luciferase signal was detected at 560 nm for firefly luciferase and 465 nm for renilla luciferase, respec- tively. The ratio of firefly luciferase signal to renilla lucif- erase signal was calculated as relative luciferase activity.
Statistical analysis
All experiments were performed at least for three times and the data were analyzed with GraphPad prism software. The data are presented as mean ± standard deviation (SD). The difference between two groups was compared using inde- pendent sample t test, while the comparisons among multi- ple groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Differences were taken as significant when the P value was lower than 0.05. *, **, and *** denoted significance at 0.05, 0.01, and 0.001 level, respectively.
Results
The expressions of lncRNA H19, XBP1s and miR‑93 were altered in ARPE‑19 cells under high‑glucose condition
To explore the role of lncRNA H19, XBP1s and miR-93 in diabetic retinal endothelial cells, we examined the expres- sions of lncRNA H19, XBP1s and miR-93 in ARPE-19 cells under high-glucose condition. As shown in Fig. 1a, compared with cells under normal condition, the expres- sions of H19 and XBP1s were reduced in ARPE-19 cells cultured in high-glucose medium, while the level of miR- 93 was increased under high-glucose medium. Besides, the expression of XBP1s was confirmed by western blots and the suppressed expression of XBP1s protein was observed. Then, we sought to investigate whether H19, XBP1s and miR-93 participate in the inflammation of DR pathogenesis in the following studies.
ER stress was induced in ARPE‑19 cells under high‑glucose condition
XBP1 was reported to be a key factor for ER stress [16] and ER stress was observed under high-glucose condition [9], thus we sought to test whether ER stress could be induced in ARPE-19 cells under high-glucose condition. ARPE-19 cells were cultured in medium containing high glucose, and the expressions of p-eIF2α and GRP78 were detected after 0, 2, 4, 8 and 12 h using RT-qPCR and Western blots. As shown in Fig. 2a–c, the expressions of p-eIF2α and GRP78 were both increased after cultured in high-glucose medium, indi- cating that ER stress was induced in ARPE-19 cells under high-glucose condition. Besides, not only the mRNA level of eIF2α/GRP78 and protein level of p-eIF2α/GRP78 were elevated by ER stress inducer TM and suppressed by IRE1 inhibitor STF-083010, we also observed that the expression of XBP1s was increased by TM and decreased by STF- 083010 (Fig. 2d–g), suggesting that XBP1s was involved in the ER stress induced by high-glucose condition. Further- more, we found that the expressions of p-eIF2α and GRP78 were down-regulated by XBP1 shRNA under TM treatment and enhanced by XBP1 overexpression under STF-083010 treatment (Fig. 2d–g). These date suggest that ER stress was induced in ARPE-19 cells under high-glucose condition and during which the expression XBP1s was enhanced, which indicates the important role of XBP1s in ER stress process.
miR‑93 promotes the production of inflammatory cytokines under high‑glucose condition
Then, we detected the inflammatory cytokines produced by ARPE-19 cells under high-glucose condition. After trans- fected with control miRNA (miR-NC), miR-93 mimics, con- trol inhibitor (inhibitor NC) or miR-93 inhibitor, the mRNA and protein were extracted from ARPE-19 cells. As shown in Fig. 3a, compared with control and miR-NC groups, the mRNA level of miR-93 was remarkably elevated in miR-93 mimics group and reduced in miR-93 inhibitor group, sug- gesting that the expression of miR-93 was altered by the treatment. Next, we examined the production of TNF-α, IL-6 and IL-1β in these conditions by RT-qPCR and ELISA assay. As shown in Fig. 3b, the mRNA levels of TNF-α, IL-6 and IL-1β were enhanced significantly in miR-93 mimics group, while suppressed in miR-93 inhibitor group. Besides, the same result was obtained by ELISA assay which showed that the protein levels of TNF-α, IL-6 and IL-1β were altered in the same way (Fig. 3c). These data suggest that miR-93 might promote the inflammatory processes in ARPE-19 cells under high-glucose condition.
miR‑93 targets and modulates XBP1s expression to modulate inflammatory processes
Through sequence alignments in starbase, we identified that the 17–26 nt of miR-93 sequence is identical to a region of XBP1 genes (Fig. 4a), suggesting that miR- 93 might bind to XBP1 gene and regulate its expres- sion. Then, we investigated whether miR-93 modulates XBP1s expression in ARPE-19 cells. We first performed dual-luciferase activity reporter gene assay and observed that miR-93 mimics inhibited the luciferase activity of wild-type XBP1s reporter, but showed limited effect on the luciferase activity of mutated XBP1s reporter (Fig. 4b, c). Furthermore, ARPE-19 cells were transfected with miR-93 mimics or miR-93 inhibitor and the expression of XBP1s was examined. The mRNA level of XBP1s was robustly repressed upon miR-93 mimics expression and highly elevated by miR-93 inhibitor treatment (Fig. 4d), suggest- ing that miR-93 could regulate XBP1s gene transcription.
lncRNA H19 is relevant to miR‑93‑mediated inflammation caused by high glucose
Next, we identified the high similarity between H19 and miR-93 sequence (Fig. 5a), then we conducted dual-lucif- erase activity reporter gene assay to test the reciprocal regu- lation between H19 and miR-93. We generated luciferase reporter constructs harboring the full sequence of wild-type 3′-UTR of H19, or the binding site-mutated 3′-UTR of H19 western blots (e). f–g miR-93 inhibitor suppressed the production of TNF-α, IL-6 and IL-1β, while XBP1s knockdown eliminated the inhibition, which is detected by RT-qPCR (f) and ELISA (g). Error bars showed mean ± SD, N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA (Fig. 5b), which were then transfected into ARPE-19 cells with miR-93 mimics or its negative control, respectively. The results demonstrated that miR-93 mimics significantly reduced the luminescence of luciferase when co-transfection with wild-type H19 (Fig. 5c). In contrast, binding site muta- tion strongly abolished this suppression caused by miR-93 mimics (Fig. 5b), which robustly suggested that miR-93 is a binding target for H19. In addition, we performed RIP to confirm these results. The result showed that lncRNA H19
was enriched in AGO2 precipitation (Fig. 5d). Besides, the expression of H19 was enhanced in the lysate and AGO2 precipitation of cells transfected with miR-93 mimics com- pared to the mimics NC transfected cells (Fig. 5d, e). These data demonstrated that H19 and miR-93 might be located in the same RNA-induced silencing complex (RISC). To explore whether H19 influences, the expressions of miR- 93, ARPE-19 cells were transfected with H19 and reduced expression of miR-93 was observed in cells transfected with H19 (Fig. 5f). Next, we explored whether H19 was involved in the inflammatory processes under high-glucose condi- tion. We found that ARPE-19 cells transfected with H19 showed reduced level of TNF-α, IL-6 and IL-1β detected by RT-qPCR and ELISA (Fig. 5g, h). Besides, the suppres- sion of the production of inflammatory cytokines could be rescued by miR-93 expression (Fig. 5g, h). Furthermore, DNAJC3 and DNAJB9, the downstream targets of XBP1s were detected by RT-qPCR. As shown in Fig. 5i, the expres- sions of DNAJC3 and DNAJB9 were enhanced by ER stress inducer TM and suppressed by STF-083010. In addition, the expressions of DNAJC3 and DNAJB9 were reduced with the treatment of miR-93 mimics and this effect could be partial reversed by OE-H19. Taken together, these data suggested that H19 might contribute to the inflammatory processes through sponging miR-93 and affect the regulation function of XBP1.
H19 enhances the expression of XBP1s and regulates inflammation via miR‑93 inhibition
Then, we tested whether H19 could modulate XBP1s expres- sion and function via miR-93. First, we detected whether the expression of XBP1s could be modulated by H19. Upon H19 overexpression, the level of XBP1s mRNA and protein were both increased remarkably, which could be eliminated by XBP1s shRNA (Fig. 6a, b), suggesting that H19 might be involved in the regulation of XBP1s expression. The expres- sions of inflammatory factors including TNF-α, IL-6 and IL-1β induced by high glucose were significantly reduced in overexpression H19 cells, however, the suppressed produc- tion of inflammatory cytokines induced by H19 expression was rescued by XBP1s silence detected by qPCR (Fig. 6c) and ELISA assay (Fig. 6d). On the other hand, we detected the expressions of DNAJC3 and DNAJB9. As shown in Fig. 6e, the expressions of DNAJC3 and DNAJB9 were reduced by XBP1s shRNA treatment which could be rescued by H19 overexpression under no matter normal condition, or TM or STF-083010 treatment. These data indicated that H19 suppressed high glucose-induced inflammation by up- regulating XBP1s, which was mediated by the restrained miR-93 expression.
Discussion
The duration of diabetes has been reported to be the con- sistent risk factor for DR and the present interventions could only limit visual loss [17]. In this study, we identi- fied the dysregulated expressions of lncRNA H19, XBP1s and miR-93 in the epithelial cells under high-glucose con- dition. Furthermore, we elucidated the mechanism that H19 played an important role in regulating inflammatory processes in retinal endothelial cells under high glucose condition through modulating miR-93/XBP1s axis.
miRNAs and lncRNAs are newly identified regulators of gene transcription and expression. The expression of miR-93 was reported to be altered in the chemical-induced inflammation, indicating that miR-93 might play a role in the inflammatory process [18]. On the other hand, the dysfunction of XBP1 was extensively investigated in intes- tinal inflammation, including inflammatory bowel dis- ease [19, 20]. XBP1/inositol-requiring protein-1 (IRE1) α signaling could be up-regulated by high glucose [9]. Our results showed that miR-93 could decrease the level of XBP1 under high-glucose condition and play a critical role in the inflammatory induced by high-glucose medium, which is consistent with the investigation between XBP1 and miRNA reported previously [21, 22].
MicroRNA and XBP1 was reported to be activated during endoplasmic reticulum (ER) stress [23] and in this study, we suggest the mechanism that lncRNA H19 might up-regu- late the expression of XBP1 by sponging XBP1 suppressor miR-93 during the ER stress induced by high-glucose condi- tion, which is similar mechanism with previous reports [24, 25]. When ER stress is induced, PERK, IRE and ATF6 are activated. In our study, we found that XBP1 could be sup- pressed using IRE inhibitor. However, we have not examined whether PERK and ATF6 participate in this process which needs to be elucidated further. As H19 was described to be expressed mainly in cytoplasm, giving the possibility that it may regulate gene expression at post-transcriptional level [26], but its role in DR development has not been explored. We found that H19 was down-regulated in the retinal epithe- lial cells under high-glucose condition and may contribute to the decreased expression of XBP1s. Overexpression of lncRNA H19 resulted in inhibitory effects of inflammation of ARPE-19 cells, suggesting that H19 suppressed the pro- cess of DR. Most importantly, H19 was predicted to bind with miR-93 in our study, and their interaction was further confirmed by dual-luciferase reporter and RIP assays. More- over, miR-93 mimics and sh-XBP1s were revealed to reverse the inhibitory effects of H19 expression on ARPE-19 cell inflammation. lncRNA H19/miR-93/XBP1s axis might play an important regulatory in the pathogenesis of DR.
Conclusions
In summary, we found that H19 might regulate the inflammatory processes of ARPE-19 cells under high- glucose condition via enhancement of the expression of XBP1s through inhibiting miR-93. Our study expands the landscape of association between XBP1s and non-coding RNAs, however, whether the regulation of H19 on XBP1s and miR-93 plays a role in DR should be examined in animal model further.
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