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MiR-21-3p modulates lipopolysaccharide-induced inflammation and apoptosis via targeting TGS4 in retinal pigment
Jiong Liu1, Zhizhong Ma2, Zhenlong Ran3
1Department of Ophthalmology, Beijing Jishuitan Hospital, Beijing, China.
Abstract:
Age-related macular degeneration (AMD) is a major reason of blindness in the elderly. MicroRNAs are implicated in various pathological processes, including inflammation and apoptosis. In this study, we aim to investigate the biological functions of miR-21-3p in inflammation and apoptosis caused by lipopolysaccharide (LPS) in human retinal pigment epithelial (ARPE-19) cells. The miR-21-3p inhibitor and mimic were transfected into ARPE-19 cells for 48 hours, followed by exposed to LPS (10 μg/mL) for 24 hours. The mRNA and protein expression of IL-6 and MCP-1 were measured using real-time PCR (RT-PCR) and enzyme-linked immunosorbent assays. Cell viability, apoptosis, caspase 3 activity, cleaved caspase-3 and cleaved-PARP protein levels were detected to evaluate the effects of miR-21-3p on apoptosis. Additionally, the target relationship between miR-21-3p and regulator of G-protein signalling 4 (RGS4) was verified by dual luciferase reporter assay. RT-PCR analysis demonstrated that LPS induced miR-21-3p expression. Inhibition of miR-21-3p reduced the mRNA and protein levels of IL-6 and MCP-1. Apoptosis, caspase-3 activity, and cleaved-caspase 3 and cleaved PARP protein levels were repressed by the miR-21-3p inhibitor. However, overexpression of miR-21-3p showed the opposite results. Furthermore, we identified that miR-21-3p directly targeted the 3' untranslated region of RGS4. MiR-21-3p negatively regulated the expression of RGS4 both in mRNA and protein levels. Silencing RGS4 reduced the anti-inflammatory and anti-apoptotic effects of miR-21-3p inhibitor. Our results revealed that miR-21-3p inhibition targeted RGS4 to attenuate inflammatory responses and apoptosis caused by LPS in ARPE-19 cells.
Insights
Inhibition of miR-21-3p reduces inflammation and apoptosis in retinal cells by targeting RGS4. This finding offers potential therapeutic strategies for age-related macular degeneration (AMD).
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Age-related macular degeneration (AMD) is a leading cause of vision loss in older adults.
- MicroRNAs play crucial roles in cellular processes like inflammation and apoptosis, relevant to AMD pathogenesis.
- Retinal pigment epithelial cells (ARPE-19) are central to retinal health and disease.
Purpose of the Study:
- To investigate the role of miR-21-3p in lipopolysaccharide (LPS)-induced inflammation and apoptosis in ARPE-19 cells.
- To elucidate the molecular mechanisms underlying miR-21-3p's function, including its potential targets.
- To assess the therapeutic potential of modulating miR-21-3p in a cellular model of AMD-related inflammation.
Main Methods:
- ARPE-19 cells were transfected with miR-21-3p inhibitors or mimics, followed by LPS stimulation.
- Quantitative real-time PCR (RT-PCR) and ELISA were used to measure inflammatory markers (IL-6, MCP-1).
- Apoptosis, caspase-3 activity, and protein levels of cleaved caspase-3 and PARP were assessed. A dual luciferase reporter assay identified RGS4 as a direct target of miR-21-3p.
Main Results:
- LPS treatment increased miR-21-3p expression in ARPE-19 cells.
- Inhibiting miR-21-3p significantly reduced LPS-induced IL-6 and MCP-1 expression, mitigating inflammation.
- miR-21-3p inhibition suppressed apoptosis, caspase-3 activity, and levels of cleaved caspase-3 and PARP.
- miR-21-3p directly targets the 3' UTR of RGS4, negatively regulating its expression.
- Silencing RGS4 diminished the anti-inflammatory and anti-apoptotic effects of miR-21-3p inhibition.
Conclusions:
- miR-21-3p promotes inflammation and apoptosis in ARPE-19 cells, contributing to LPS-induced cellular damage.
- The anti-inflammatory and anti-apoptotic effects of miR-21-3p inhibition are mediated through its direct targeting of RGS4.
- Modulating miR-21-3p and its target RGS4 presents a potential therapeutic avenue for AMD and related inflammatory eye conditions.
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