Protective Effect of lncRNA FGD5-AS1 Against Brain Injury After Intracerebral Hemorrhage by Targeting miR-93-5p

Summary

This study investigated the interaction between FGD5-AS1 and miR-93-5p in experimental intracerebral hemorrhage models and found that FGD5-AS1 knockdown was associated with reduced neurological injury, inflammation, and oxidative stress.

Abstract

Intracerebral hemorrhage (ICH) is a neurological disorder associated with high mortality and disability rates and can lead to severe neurological injury. This study investigated the role and underlying mechanism of FGD5-AS1 in ICH. In vitro and in vivo models of ICH were established. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure FGD5-AS1 and miR-93-5p expression. Neurological deficits were assessed using the modified Neurological Severity Score (mNSS), and cerebral edema was evaluated using the dry-wet weight method. Enzyme-linked immunosorbent assay (ELISA) was used to measure inflammatory cytokines and oxidative stress markers. Cell viability was evaluated using a cell viability and proliferation assay kit. The interaction between FGD5-AS1 and miR-93-5p was examined using dual-luciferase reporter assays and Pearson correlation analysis. Experimental ICH was accompanied by elevated FGD5-AS1 expression in both animal and cellular models. Suppression of FGD5-AS1 alleviated brain swelling, improved neurological performance, reduced inflammatory and oxidative injury, and enhanced endothelial cell survival. Inhibition of miR-93-5p diminished these protective effects, supporting a regulatory interaction between the two molecules. FGD5-AS1 negatively regulated miR-93-5p expression, and inhibition of miR-93-5p attenuated the effects observed following FGD5-AS1 knockdown. These results indicate that FGD5-AS1 may contribute to ICH-associated brain injury through regulation of miR-93-5p and that the FGD5-AS1/miR-93-5p axis may represent a potential target for further investigation in experimental ICH.

Introduction

Intracerebral hemorrhage (ICH) is a neurological disorder with high mortality and disability rates, accounting for 10% to 20% of all stroke cases^1,2. Due to the lack of effective therapeutic targets, treatment for ICH is currently limited to surgery and supportive care³. Pinho et al. validated predictions of ICH mortality, functional outcomes, and hematoma growth fraction, providing a new perspective for healthcare professionals⁴. Despite progress in ICH treatment, patient outcomes remain poor. Therefore, identifying novel therapeutic targets for ICH is important for improving long-term prognosis.

Long non-coding RNAs (lncRNAs) comprise a class of non-protein-coding transcripts exceeding 200 nucleotides in length that regulate gene expression through diverse mechanisms, including transcriptional, post-transcriptional, and epigenetic processes⁵. Accumulating evidence has demonstrated that aberrant lncRNA expression is implicated in the pathogenesis of a range of neurological disorders, such as stroke, traumatic brain injury, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage⁶. Among these regulatory molecules, FGD5-AS1 has been linked to several cellular processes, including proliferation, apoptosis, inflammatory signaling, and oxidative stress responses. Through mechanisms such as competing endogenous RNA activity, transcriptional regulation, and epigenetic modification, FGD5-AS1 influences the expression of multiple downstream targets. Dysregulated FGD5-AS1 expression has been reported in various pathological conditions, including malignancies, cardiovascular disorders, and metabolic diseases^7,8,9,10,11. Nevertheless, its biological significance in neurological diseases remains incompletely understood. Previous studies have shown that FGD5-AS1 participates in glioma and glioblastoma progression^12,13and contributes to neuronal injury under oxygen-glucose deprivation/reperfusion conditions¹⁴. These observations suggest that FGD5-AS1 may play a regulatory role in neural injury; however, its contribution to brain damage following ICH has not been fully elucidated.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by interacting with complementary sequences within target messenger RNAs¹⁵. Studies have shown that miRNAs also play important roles in ICH. Specifically, miR-143-3p promotes the transendothelial migration of neutrophils following acute brain injury by targeting ATP6V1A¹⁶. miR-195-5p exerts a protective effect against ICH-induced brain injury by inhibiting the expression of MMP-9 and MMP-2¹⁷. MiR-93-5p has also been identified as a therapeutic target to improve outcomes following ICH¹⁸. Although studies have confirmed the abnormal expression of FGD5-AS1 and miR-93-5p in ICH and their involvement in the progression of neurological dysfunction following ICH, previous research has primarily focused on their individual biological effects. The competitive regulatory relationship between FGD5-AS1 and miR-93-5p as endogenous RNAs has not yet been clearly established.

Protocol

1. Experimental animals

This research was approved by the Animal Ethics Committee of Ganzhou People’s Hospital (Approval No. 20230789) and was conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. SPF-grade female SD rats (6–8 weeks old, 180–230 g) were used in this study. The rats were housed with free access to food and water under controlled conditions of 22 °C ± 3 °C and a 12 h light/dark cycle. The animals were allowed to acclimate for 7 days before the experiment. The chemicals, reagents, and equipment used in the protocol are listed in the Table of Materials.

2. Establishment of the in vivo ICH model

To generate the experimental intracerebral hemorrhage (ICH) model, 0.23 U of type VII collagenase was dissolved in 1 µL of sterile physiological saline. Rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg), after which the collagenase solution was stereotactically infused into the striatum at 0.5 µL/min (0.2 mm behind the frontal point, 3.0 mm lateral to the midline, and 6 mm deep into the skull surface). Animals in the control group underwent the same procedure but received an equal volume of physiological saline instead of collagenase. A schematic overview of the experimental design is provided in Supplementary Figure 1.

Supplementary Figure 1: Animal experiment design diagram. Rats were randomly divided into two groups: one group used 0.23U type VII collagenase to construct an ICH model; One group used physiological saline as the control group. The model group rats were randomly divided into two batches for analyzing the role of FGD5-AS1 in ICH and the synergistic effect of FGD5-AS1/miR-93-5p axis. One hour after ICH induction, the transfection reagent was injected into the rat brain. Evaluate the neurological function of rats using mNSS on days 0, 1, 3, and 7; Collect brain tissue after inducing ICH for 7 days.Please click here to download this file.

The rats were randomly divided into two groups. One group was used to analyze the role of FGD5-AS1 in ICH and was further divided into four subgroups: Control, ICH, ICH + lentivirus (LV)-shNC, and ICH + LV-shFGD5-AS1. The other group was used to analyze the synergistic effects of the FGD5-AS1/miR-93-5p axis and was divided into five subgroups: ICH, ICH + LV-shNC, ICH + LV-shFGD5-AS1, ICH + LV-shFGD5-AS1 + antagomir NC, and ICH + LV-shFGD5-AS1 + miR-93-5p antagomir. Each group included 12 rats. 1 hour after ICH induction, the transfection reagent was injected into the rat brain at a dose of 2 mg/kg.

3. Cell culture and processing

Brain microvascular endothelial cells (BMECs) serve as a key model for studying the blood-brain barrier, mechanisms underlying neurological disorders, and drug delivery²⁰. An in vitro ICH model was established using oxygen-glucose deprivation (OGD)/hemin treatment in BMECs³. BMECs were cultured in glucose- and serum-free DMEM under a gas mixture of 5% CO² and 95% N₂ for 10 min. The cells were then treated with hemin (10µM) and transferred to an anaerobic chamber containing 5% CO₂ and 95% N₂ for 2 h. OGD was terminated by returning the cells to normal culture conditions. The control group was treated with PBS under the same conditions.

Cells were transfected according to the manufacturer’s instructions. Cells were transfected with 20 nM FGD5-AS1 siRNA (si-FGD5-AS1), negative control siRNA (si-NC), miR-93-5p inhibitor, or blank control inhibitor (NC inhibitor).

4. Modified neurological severity score test

After establishment of the in vivo ICH model, neurological function was evaluated using the modified Neurological Severity Score (mNSS) on days 0, 1, 3, and 7. Scores were quantified based on four domains: motor function, sensory function, balance, and reflex function. Scores of 1–4 indicated mild impairment, 5–9 indicated moderate impairment, and 10–14 indicated severe impairment.

5. Sample collection

7 days after ICH induction, rats were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium, followed by euthanasia through cervical dislocation and collection of brain tissue.

6. Assessment of cerebral edema

Cerebral edema was evaluated using the dry-wet weight method¹⁹. The brain was weighed immediately after extraction to obtain the wet weight. The tissue was then dried in a 100 °C oven for 24 h and weighed again to obtain the dry weight. Tissue water content was calculated as follows:

[(wet weight - dry weight) / wet weight] × 100%

7. Real-time quantitative PCR

Total RNA was isolated from tissue and cell samples using an RNA extraction reagent according to the manufacturer’s protocol. Complementary DNA was subsequently synthesized using a reverse transcription kit. Quantitative PCR analysis was then performed using a real-time PCR platform, with GAPDH and U6 as endogenous reference genes. The amplification protocol consisted of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 60 s. Relative gene expression levels were calculated using the 2^−ΔΔCt method. Primer sequences used in this study are provided in Supplementary Table 1.

Supplementary Table 1: The primer sequences and related reagent sequences used in this study. The sequences involved in real-time qPCR experiments and the relevant sequences of the reagents used in vitro experiments.Please click here to download this file.

8. Cell proliferation assay

Transfected cells were seeded into 96-well plates. After culturing for 0, 24, 48, and 72 h, 10 µL of CCK-8 reagent was added to each well. The cells were incubated at 37 °C for 2 h, and the optical density was measured at 450 nm using a microplate reader.

9. Dual-luciferase reporter assay

Wild-type (WT) and mutant (MUT) 3′-UTRs of FGD5-AS1 containing the miR-93-5p binding site were cloned into the pGL3 vector. The vector was co-transfected with a miR-93-5p mimic using a transfection reagent. Relative luciferase activity was measured using a dual-luciferase reporter system.

10. Enzyme-linked immunosorbent assay

Brain tissue samples (20–50 mg) were homogenized in ice-cold RIPA lysis buffer at a tissue-to-buffer ratio of 1:9. Following homogenization, the samples were centrifuged at 12,000 × g for 20 min at 4 °C, and the resulting supernatants were collected for analysis. BMECs from each experimental group were harvested and centrifuged at 12,000 × g for 10 min at 4 °C, after which the supernatants were retained. The concentrations of MDA, IL-1β, IL-6, and TNF-α were subsequently determined using the corresponding assay kits in accordance with the manufacturers’ instructions.

11. Statistical analysis

All data are presented as the mean ± SD. Data were visualized using statistical software. Each experiment was repeated at least three times. At-test was used to compare two groups, and one-way analysis of variance followed by Tukey’s post hoc test was used to compare more than two groups. A value of P < 0.05 was considered statistically significant.

Results

Effect of FGD5-AS1 on ICH rats

FGD5-AS1 expression was significantly higher in the ICH group than in the control group (P < 0.0001; Figure 1A). Following transfection with LV-shFGD5-AS1, FGD5-AS1 expression was markedly reduced compared with that in the LV-shNC group (Figure 1B). Assessment of cerebral edema revealed more severe brain edema in ICH rats than in control rats, whereas silencing FGD5-AS1 significantly ...

Discussion

Brain damage following intracerebral hemorrhage results not only from the initial bleeding event but also from a series of secondary pathological responses that evolve over time²¹. While the primary hematoma causes direct mechanical disruption of surrounding tissue, subsequent injury is mediated by processes such as inflammatory activation, oxidative damage, and programmed cell death. These secondary mechanisms substantially contribute to neurological deterioration and unfavorable clinical outcome...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antagomir NC	GenePharma, China	B04007	Negative control antagomir
BMECs	ATCC, USA	CRL-8270	Human brain microvascular endothelial cells
Cell Counting Kit-8 (CCK-8)	Beyotime, China	C0038	Cell viability assay kit
DMEM medium	Gibco, USA	12430054	Glucose-free, serum-free medium
Dual-Glo Luciferase Assay System	Promega, USA	E2920	Luciferase activity detection
Electronic balance	Sartorius	Secura224-1CN	Precision balance for tissue weighing
GAPDH and U6 primers	Sangon Biotech	Custom primer synthesis service	Internal reference primers
Graphpad Prism 8.0	GraphPad Software, USA	——	Statistical analysis software
Hemin	MCE, China	HY-19424	Hemin reagent for cell injury induction
IL-1 beta ELISA kits	Abcam, UK	ab197742	Inflammatory cytokine quantification
IL-6 ELISA kits	Abcam, UK	ab222503	Inflammatory cytokine quantification
LightCycler 96 PCR system	Roche,Switzerland	5815916001	Real-time PCR instrument
Lipofectamine 3000	Invitrogen, USA	L3000001	Transfection reagent
MDA assay kit	Beyotime, China	S0131	Lipid peroxidation assay
Microplate reader	BioTek	Synergy H1	OD450 absorbance measurement
miR-93-5p antagomir	GenePharma, China	B05001	miR-93-5p inhibitor for in vivo experiments
PBS	Solarbio, China	P1020	Phosphate-buffered saline
Pentobarbital sodium	Sigma, Merck KGaA	11715	Anesthetic agent
pGL3-Promoter	NovoPro, China	V011541	Luciferase reporter vector
PrimeScript RT Mix kit	Takara, Japan	RR037A	Reverse transcription kit
RIPA	Beyotime, China	P0013B	Protein/tissue lysis buffer
ShFGD5-AS1 lentivirus	GenePharma, China	D02001	FGD5-AS1 knockdown vector
SPF-grade female SD rat	Vital River	——	6–8 weeks old, SPF grade
SYBR Green Realtime PCR Master Mix	Takara, Japan	RR420A	Quantitative PCR reagent
TNF-α ELISA kits	Abcam, UK	ab208348	Inflammatory cytokine quantification
TRIzol reagent	Invitrogen, USA	15596026	Total RNA extraction reagent
Type VII collagenase	Sigma, Merck KGaA	C0773	From Clostridium histolyticum; ICH induction