Major Depressive Disorder (MDD) is increasingly recognized as a multifactorial disorder involving dysregulation of neuroinflammatory signaling , glial activation , and non-coding RNA (ncRNA)-mediated gene expression . Recent studies published in International Journal of Biological Macromolecules (2024) , Journal of Extracellular Vesicles (2022) , Molecular Psychiatry (2020) , and Biological Psychiatry (2020) have provided mechanistic insights into how circular RNAs (circRNAs) modulate microglial and astrocytic function through miRNA sponging, epigenetic regulation, and extracellular vesicle (EV)-mediated intercellular communication.

This article delves into :

• The functional roles of specific circRNAs—circDYM , circHIPK2 , circSTAG1
• Their interactions with miRNAs such as miR-9 , miR-16-5p , and miR-34a
• Neuroinflammatory pathways regulated by NLRP3 inflammasome , m6A methylation , and astrocyte dysfunction
• Applications in RT-PCR detection kits and precision psychiatry

In Molecular Psychiatry (2020) , it was demonstrated that circDYM levels are significantly reduced in both chronic unpredictable stress (CUS) mouse models and LPS-treated BV2 microglial cells . Lentiviral overexpression of circDYM attenuated LPS-induced LC3B-II accumulation and cleaved caspase-3 expression , indicating a protective effect against neuroinflammation and apoptosis.

Mechanistically, circDYM functions as a miR-9 sponge , preventing miR-9-mediated suppression of HECT domain-containing E3 ubiquitin protein ligase 1 (HECTD1) . HECTD1 stabilizes HSP90 ubiquitination , thereby reducing microglial activation and neuroinflammatory cytokine release.

These findings suggest that circDYM downregulation contributes to glial hyperactivation in depression, and its restoration may serve as a therapeutic strategy.

The Microbiome (2019) study revealed that circHIPK2 expression in the hippocampus and plasma is elevated following CUS exposure in wild-type (WT) microbiota recipient mice. However, transplantation of NLRP3 knockout (KO) microbiota significantly suppressed circHIPK2 upregulation and alleviated depressive-like behaviors.

Using GFP-labeled lentivirus microinjection , the authors showed that circHIPK2 knockdown in astrocytes mimicked the antidepressant effects observed in NLRP3 KO microbiota recipients.

These data support a model where circHIPK2 mediates astrocytic dysfunction downstream of gut-derived inflammatory signals , positioning it as a gut-brain axis-linked biomarker in MDD.

According to Biological Psychiatry (2020) , circSTAG1 is downregulated in MDD patients and correlates with symptom severity. circSTAG1 interacts with ALKBH5 , an m6A demethylase, and modulates FAAH expression—a key enzyme in endocannabinoid metabolism.

This interaction affects m6A RNA methylation status , which in turn influences astrocyte transcriptomic profiles and synaptic plasticity .

Notably, circSTAG1 loss leads to increased m6A methylation at FAAH mRNA , enhancing its stability and contributing to endocannabinoid system dysregulation —a known feature of depression pathophysiology.

The Int J Biol Macromol (2024) paper reported that circ_0000831 exhibits anti-inflammatory properties in cerebral ischemia models by acting as a miR-16-5p sponge . Overexpression of circ_0000831 reduced TNF-α , IL-6 , and IL-1β levels in BV2 microglial cells treated with LPS.

While this study focused on vertigo and stroke, the conserved miR-16-5p binding motif suggests potential relevance in MDD-associated neuroinflammation , particularly in comorbid conditions involving vascular pathology or chronic inflammation.

The J Extracell Vesicles (2022) study described the use of rabies virus glycoprotein (RVG)-modified EVs to deliver circDYM-GFP into the central nervous system (CNS).

Engineered EVs were isolated from HEK293T cells transfected with RVG-Lamp2b plasmid and circDYM-GFP lentivirus , then purified using Exo-spin™ SEC columns .

These EVs efficiently delivered circDYM to primary mouse microglia , as confirmed by absolute qPCR and fluorescence imaging . The delivery resulted in significant reductions in LPS-induced pro-inflammatory cytokines , demonstrating the feasibility of circRNA-based EV therapeutics in neuroinflammatory disorders.

Across these studies, the following techniques were used to validate circRNA function:

• Quantitative real-time PCR (qPCR) using divergent primers to detect back-spliced junctions
• Western blotting to assess protein expression of HECTD1, HSP90, LC3B, cleaved caspase-3
• Immunofluorescence staining with GFAP (astrocytes), IBA1 (microglia), NeuN (neurons)
• Behavioral assays : Sucrose preference test (SPT), tail suspension test (TST), forced swim test (FST)

These methods provide a robust framework for studying circRNA dynamics in vivo and in vitro .

The integration of non-coding RNA biology , neuroinflammatory signaling , and extracellular vesicle engineering offers a transformative approach to understanding and treating Major Depressive Disorder . circRNAs such as circDYM , circHIPK2 , circSTAG1 , and circ_0000831 represent promising diagnostic markers and therapeutic targets , particularly when analyzed using RT-PCR detection kits and validated via behavioral and biochemical assays .

As our understanding of these molecular mechanisms deepens, so too does the potential for personalized treatment strategies based on individual circRNA profiles, paving the way toward precision psychiatry .