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Genome-wide profiling of gene expression under electro-acupuncture pretreatment on myocardial I/R injury in rats

ABSTRACT: Purpose:Acupuncture exerts cardioprotective effect on myocardial I/R injury. In order to elucidate the potential mechanisms, RNA-seq by next generation sequencing was used to identify the rat genome-wide alterations by EA at Neiguan acupoint pretreatment in this study Methods: Adult male Sprague Dawley rats (250-300g) were divided into four groups: Sham operation(SO), I/R, electro-acupuncture at Neiguan pretreatment (EA) and electro-acupuncture at non-acupoint (NA) groups and myocardium mRNA profiles of rats in each group were generated by deep sequencing,using Illumina Hiseq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Results: Using an optimized data analysis workflow, we mapped about 20~43 million sequence reads per sample to the mouse genome (UCSC RN4) and identified 26190 22895 25160 22852 transcripts in the SO, I/R, EA and NA rats with TopHat and Cufflinks workflow respectively. Approximately 32% and 22% of the transcripts showed differential expression between the SO and I/R, I/R and EA , I/R and NA, respectively with a |log2 fold change |≥1. Conclusions: Our results for the first time generated genome-wide gene expression profiles both in the rat I/R model and in acupuncture treatment by high throughput sequencing. The optimized data analysis workflows reported here should provide a framework for acupuncture investigations of expression profiles.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE61840 | GEO | 2016/03/20

SECONDARY ACCESSION(S): PRJNA262523

REPOSITORIES: GEO

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Project description:Background and Objective: Cerebral infarction (CI) is a severe neurological disorder caused by localized disruption of blood supply to brain tissue, leading to high rates of disability and mortality. Circular RNAs (circRNAs), highly expressed in the brain, have been identified as potential therapeutic targets for CI. Although acupuncture has been shown to promote neurological recovery, the precise molecular mechanisms, particularly the role of circRNAs in regulating angiogenesis, remain insufficiently understood. This study is the first to demonstrate that electroacupuncture (EA) promotes angiogenesis by regulating the circRNA_011971/miR-196c-5p/ARL13B axis, enhancing the proliferation and migration of brain microvascular endothelial cells (BMECs), and accelerating angiogenesis. These findings provide new insights into the molecular mechanisms of acupuncture treatment for CI and identify potential therapeutic targets. Methods: Male Wistar rats were used to establish a cerebral infarction model via middle cerebral artery occlusion (MCAO). The rats were divided into a model group, an EA treatment group, and a control group. Immediately after MCAO induction, the EA group received acupuncture at the GV26 acupoint. Neurological deficits were assessed using the modified Neurological Severity Score (mNSS), and infarct volumes were measured using TTC staining. Immunofluorescence double staining was performed to detect angiogenesis markers CD31 and Ki67 in the peri-infarct area. CircRNA expression profiles in brain tissue were obtained through circRNA microarray analysis, and key circRNAs were validated via qRT-PCR and fluorescence in situ hybridization (FISH). In BMECs, circRNA_011971 knockdown was achieved using shRNA, and its biological functions were assessed through CCK8, wound healing, and transwell assays. The expression levels of Arl13b, VEGFA, and VEGFR2 were measured using qRT-PCR and Western blot after circRNA_011971 knockdown or overexpression. Molecular interactions among circRNA_011971, miR-196c-5p, and ARL13B were explored using dual-luciferase reporter assays, RNA immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP). Results: Compared to the untreated group, the EA group exhibited significantly smaller infarct volumes, suggesting immediate EA treatment at GV26 following infarction can effectively limit infarct progression. Immunofluorescence analysis revealed a marked increase in the co-expression of CD31 and Ki67 in the peri-infarct region of the EA-treated rats, indicating enhanced angiogenesis. CircRNA microarray analysis showed that circRNA_011971 was significantly upregulated following ischemia and downregulated after EA treatment. In BMECs, circRNA_011971 knockdown inhibited cell proliferation, migration, and invasion, whereas its overexpression promoted these processes. Further mechanistic studies revealed that EA downregulated rno_circ_011971 expression, lifting its inhibition on miR-196c-5p and activating the ARL13B/VEGF signaling pathway, thereby significantly promoting angiogenesis. This study discover a molecular mechanism by which EA regulates post-CI angiogenesis through the circRNA/miRNA axis for the first time, offering new potential targets for acupuncture therapy. Conclusion: EA improves neurological function, reduces infarct size, and promotes angiogenesis, potentially through regulating of VEGFR via the rno_circ_011971/miR-196c-5p/ARL13B axis.

Project description:Objective: To investigate the mechanisms depression-related manifestations ameliorated by electroacupuncture on genome-wide transcriptomes of the frontal cortex at the molecular level, we evaluated unbiased genome-wide RNA sequencing of depression-related rats suffer from maternal separation (MS). Methods: Rats were randomly divided into control, MS, electro-acupuncture treatment(EA) group . We demonstrated depression-like behavior deficiencies in a sucrose preference test and a forced swimming test in a rat model with neonatal maternal separation. Repeated EA treatment at the acupoints Baihui (GV20) and Yintang (GV29) during the adult period was shown to be remarkably attenuated above behavioral deficits. Sequencing was performed using Hiseq Xten (Illumina, USA). Results: Based on the data from RNA-seq analysis, for mRNA, 48 genes were significantly and differentially expressed in MS rats relative to control rats, with 39 up-regulated genes and 9 down-regulated genes. 40 genes in the EA rats relative to the MS rats show differential expression, with 21 genes up-regulated and 18 genes down-regulated. For lncRNA, 11 genes increased significantly in the MS rats relative and 4 genes decreased relative to controls. 31 genes increased in the EA rats relative to the MS rats while 1 gene decreased. For circRNA, 20 genes increased significantly in the MS rats relative to control rats and 17 genes decreased. 18 genes increased in the EA rats relative to the MS rats while 28 genes decreased. Using ceRNA, we narrowed the targets of mRNA from 31 to 17 genes with a circRNA_miRNA_mRNA network, and narrowed the targets of mRNA from 31 to 8 genes with an lncRNA_miRNA_mRNA network, pointing the way for further studies to verify the genes and describe their function. Conclusions: Our investigation demonstrates the first analysis of the frontal cortex genome-wide transcriptomes in depression rats under maternal separation by RNA-seq technology. These results suggest that EA at GV20 and GV29 ameliorates depression-related manifestations by regulating the expression of multiple genes.

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Genome-wide profiling of gene expression under electro-acupuncture pretreatment on myocardial I/R injury in rats

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