Analyses of the Competing Endogenous RNA Network in Rats with Middle Cerebral Artery Occlusion Based on lncRNA, miRNA, and mRNA Expression

Summary

This study used bioinformatics analyses and experimental validation to systematically investigate the regulatory roles and underlying mechanisms of long noncoding RNAs (lncRNAs) functioning as competitive endogenous RNAs (ceRNAs) in ischemic stroke.

Abstract

This study aims to investigate the regulatory roles and underlying mechanisms of lncRNAs acting as ceRNAs in ischemic stroke. Based on the ceRNA hypothesis, lncRNAs, miRNAs, and mRNAs were identified as components of a regulatory network involved in stroke. Key lncRNAs from the resulting subnetwork were selected for detailed analysis. Functional enrichment analysis using Gene Ontology and pathway mapping through the Kyoto Encyclopedia of Genes and Genomes revealed critical interactions within the lncRNA-associated ceRNA network. Key pathways, including calcium signaling, gap junction signaling, and neuroactive ligand receptor interaction, were further validated using Western blot analysis. The constructed ceRNA network comprised 334 lncRNAs, miRNAs, and mRNAs, with functional enrichment analysis predicting their biological roles. Three lncRNAs with high degree centrality were selected to construct a representative ceRNA subnetwork. Western blot analysis revealed that, compared to the sham group, the expression levels of key proteins -- CaMKII, calmodulin, CX36, PKC, CX43, GRIA3, GABRA6, and NPY1R were significantly downregulated in the model group, while the expression of CaN was significantly upregulated (P < 0.05). These findings suggest that lncRNAs are significantly involved in stroke pathogenesis. In conclusion, lncRNAs acting as ceRNAs serve critical regulatory roles in the pathogenesis of ischemic stroke.

Introduction

Ischemic stroke, a prevalent neurological disorder, leads to permanent brain damage, long-term disability, or death^1,2,3,4. MicroRNAs (miRNAs or miR), long noncoding RNAs (lncRNAs), and messenger RNAs (mRNA) form RNA-mediated regulatory networks^5,6,7,8. These molecules regulate cellular functions through various complex mechanisms^9,10are increasingly recogn....

Protocol

The animal experiments were approved by the Experimental Ethics Committee of Anhui University of Chinese Medicine (license number: AHUCM-rats-2024006) and conducted in accordance with institutional guidelines and JoVE's animal use standards. Forty male SPF-grade SD rats (220 g ± 30 g) were used in this study. The reagents and equipment used are listed in the Table of Materials.

1. Experimental animals

1. House the animals in the Animal Experimental Center under standard experimental conditions. Maintain a 12 h light/dark cycle, change bedding daily, and provide free access to food and wate....

Results

The success rate verification experimental results of each group of rat models are shown in Figure 1. After modeling, neurological function scoring was performed on the rats (Figure 1A). The model group exhibited severe neurological deficits, and those with scores ranging from 1 to 3 were included in subsequent experiments. Figure 1B and C show the TTC staining results, with red representing normal brain tissue and white indicating .......

Discussion

To investigate the molecular mechanisms underlying stroke pathogenesis and identify potential diagnostic and therapeutic targets, RNA-Seq and small RNA-Seq (sRNA-Seq) analyses were conducted on brain tissues from rats following MCAO. This study primarily aims to investigate the complex regulatory networks involving lncRNAs, miRNAs, and mRNAs. A comprehensive analysis of the RNA-Seq and sRNA-Seq datasets from MCAO-induced rat brain tissues was conducted to elucidate the lncRNA-miRNA-mRNA regulatory network. An extensive n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% (w/v) TTC staining solution	Beijing Kangle Clone Biotechnology Co., Ltd.	20230805	TTC staining
4% (w/v) paraformaldehyde solution	Beyotime	P0099	Fixed brain tissue
40 male SPF-grade SD Rats	Hagzhou Ziyuan Laboratory Animal Breeding Co.,Ltd	SCXK [zhe] 2024-0004	laboratory animal
5% skimmed milk powder	Scientific Phygene	PH1519	WB
agarose electrophoresis system	Beijing Liuyi Biotechnology Co., Ltd.	DYCP-44P	WB
automatic exposure instrument	Shanghai Peiqing Technology Co., Ltd.	JS-M6P	WB
Automatic exposure meter	Shanghai Peiqing Technology Co., LTD	JS-M6P	WB
Automatic ice maker	Changshu City Xueke Electric Appliance Co., LTD	IMS-20	WB
Calmodulin	Affinity	49B2443	WB
CamKII	Affinity	14G0796	WB
centrifuge	Haimen Qilinbeier Instrument Manufacturing Co., Ltd.	LX 300	WB
clusterProfiler package	Bioconductor	clusterProfiler_4.17	enrichment analyses
CX36	ZENBIO	N24AP24	WB
CX43	ZENBIO	M08NO01	WB
ECL ultra-sensitive chemiluminescence kit	biosharp	BL520B	WB
Electric thermostatic air drying oven	Shanghai Sanfa Scientific Instrument Co., LTD	DHG-9070	WB
Electrophoresis apparatus	Shanghai Tianeng Technology Co., LTD. (Tanon)	EPS300	WB
electrophoresis apparatus	Shanghai Tanon Science & Technology Co., Ltd.	EPS300	WB
electrophoresis tank	Shanghai Tanon Science & Technology Co., Ltd.	VE-180	WB
GABRA6	Affinity	46V5371	WB
Goat Anti-mouse IgG	Zs-BIO	142637	WB
Goat Anti-Rabbit IgG	Zs-BIO	139931	WB
GRIA3	Affinity	0C33051	WB
High speed refrigerated centrifuge	Anhui Jiawen instrument equipment Co., LTD	JW-3021HR	WB
HiSeq 2500 system	illumina	/	RNA high-throughput sequencing
ImageJ software	National Institutes of Health	ImageJ 1.54k	TTC staining
Magnetic heating agitator	Changzhou city and instrument factory	JJ-79-1	WB
Micropipette	Germany Eppendorf	/	WB
Normal temperature micro centrifuge	Haimen Qi Limber Instrument Manufacturing Co., LTD. LX300	LX300	WB
NPY1R	Affinity	2D38248	WB
pipette	eppendorf	0.5-10ul	WB
PKC	Affinity	17E3745	WB
pre-stained protein Marker	biosharp	BL712C	WB
PVDF membrane	Millipore	IPVH00010	WB
Ribo-Zero rRNA Removal kit	Illumina, San Diego, California, USA	/	RNA extraction
RIPA lysate	Biosharp	BL504A	WB
Sodium pentobarbital	Beijing Think-Far Technology Co., Ltd.	MERCK	anesthetic
transfer membrane instrument	Shanghai Tanon Science & Technology Co., Ltd.	VE-186	WB
Transmembrane apparatus	Shanghai Tianeng Technology Co., LTD. (Tanon)	VE-186	WB
β-actin	Zs-BIO	19AW0505	WB