Circular RNA expression profile of Alzheimer’s disease and its clinical significance as biomarkers for the disease risk and progression

doi:10.1016/j.biocel.2020.105747

The International Journal of Biochemistry & Cell Biology

Volume 123, June 2020, 105747

https://doi.org/10.1016/j.biocel.2020.105747 Get rights and content

Abstract

Objective

To investigate circular RNA (circRNA) expression profile via microarray, and further assess the potential of candidate circRNAs as biomarkers in Alzheimer’s disease (AD).

Methods

CircRNA expression profile in cerebrospinal fluid from 8 AD patients and 8 control (Ctrl) subjects was assessed by microarray. Subsequently, 10 candidate circRNAs from microarray were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in cerebrospinal fluid from 80 AD patients and 40 Ctrl subjects.

Results

By microarray, 112 circRNAs were upregulated and 51 circRNAs were downregulated in AD patients compared with Ctrl subjects, and these circRNAs were enriched in AD related pathways such as neurotrophin signaling pathway, natural killer cell mediated cytotoxicity and cholinergic synapse. By RT-qPCR, circ-LPAR1, circ-AXL and circ-GPHN were increased, whereas circ-PCCA, circ-HAUS4, circ-KIF18B and circ-TTC39C were decreased in AD patients compared with Ctrl subjects, and these circRNAs were disclosed to predict AD risk by receiver operating characteristics curve analysis. Further forward-stepwise multivariate logistic regression revealed that circ-AXL, circ-GPHN, circ-ITPR3, circ-PCCA and cic-TTC39C were independent predictive factors for AD risk. Besides, in AD patients, circ-AXL and circ-GPHN negatively correlated, while circ-PCCA and circ-HAUS4 positively correlated with mini–mental state examination score; Circ-AXL negatively correlated, while circ-PCCA, circ-HAUS4 and circ-KIF18B positively correlated with Aβ42; Circ-AXL and circ-GPHN positively correlated, whereas circ-HAUS4 negatively correlated with t-tau; Circ-AXL positively correlated with p-tau.

Conclusion

Our study provides an overview of circRNA expression profile in AD, and identifies that circ-AXL, circ-GPHN and circ-PCCA hold clinical implications for guiding disease management in AD patients.

Introduction

Alzheimer’s disease (AD) is the main cause of dementia in elderly population, which is marked by the accumulation of neurotoxic amyloid plaques, hyperphosphorylated tau tangles, neuroinflammation and extensive neuronal cell death in brain [[1], [2], [3]]. It is estimated to affect 40.0 million individuals (mostly older than 60 years) worldwide [3]. Generally, AD patients experience a long asymptomatic preclinical phase (more than 20 years) before the clinical onset of typical symptoms [4]. As the disease manifests, AD patients begin to suffer from devasting, persistent and progressive memory loss combined with cognitive decline and loss of independent care, which poses a great emotional and financial burden to both families and society [3]. Despite decades of tremendous pharmaceutical industry efforts to develop drug treatments for halting the progression of AD symptoms or curing AD, there is still no effective drug treatment available yet [5]. Therefore, it is of particular importance to investigate potential biomarkers for predicting disease risk, guiding management and decision-making of AD.

Circular RNAs (CircRNAs) is a class of endogenous noncoding RNAs, and are generated from back-splicing events and defined by covalently closed circular configuration without 5’ end caps and 3’ polyadenylated tails [6]. CircRNAs have been misinterpreted as by-products of aberrant splicing without any function for decades [7]. In recent years, a growing number of studies have focused on circRNAs and gradually uncover that a select number of circRNAs (such as circRNA Cdr1as and circ_000950) function as microRNAs (miRNAs) sponges to disturb the downstream signaling pathways of these miRNAs and participate in the pathogenesis of neurodegenerative diseases [[8], [9], [10]]. Notably, with the advent and application of microarrays coupled with novel bioinformatic analysis, our understanding about circRNA expression profile has been built and extended in various diseases [[11], [12], [13]]. Regarding AD, just presently two studies based on the mouse model, which analyze the circRNA expression profile of AD mice and disclose that a number of circRNAs are involved in the pathogenesis of AD [14,15]. For instance, 85 circRNAs are dysregulated (45 upregulated and 40 downregulated) in 10-month-old senescence accelerated mice P8 (SAMP8) compared with age-matched senescence-accelerated mouse resistant R1 (SAMR1), and mmu_circRNA_017963 is validated to be positively correlated with the progression of AD [14]. Another study illustrates that 10 circRNAs are differentially expressed (4 upregulated and 6 downregulated) in Panax notoginseng saponins (PNS)-treated SAMP8 compared with PNS-untreated SAMP8, and mmu_circRNA_013636 as well as mmu_circRNA_012180 participate in AD-associated signaling pathways [15]. However, in clinical studies, there is currently no report about the circRNA expression profile in AD patients.

In the current study, initially, we characterized the circRNA expression profile of 8 AD patients and 8 control (Ctrl) subjects by microarray analysis. Subsequently, we further used reverse transcription quantitative polymerase chain reaction (RT-qPCR) to validate 10 candidate circRNAs (top 5 upregulated and top 5 downregulated from microarray analysis) in 80 AD patients and 40 Ctrl subjects. Lastly, we investigated the correlation of 10 candidate circRNAs with disease risk and disease severity of AD. Through these explorations, we aimed to evaluate the potential of circRNA expression profile in predicting disease risk and assisting management of AD in clinical practice.

Section snippets

Participants

Eighty AD patients and forty Ctrl subjects were consecutively recruited in People’s Hospital of Zhengzhou University. The inclusion criteria of AD patients were: (1) newly diagnosed as AD according to the NIA-AA criteria [16], (2) presenting with decreased amyloid β-42 (Aβ42) (<550 pg/mL), increased total tau (t-tau) (≥350 pg/mL) and increased phosphorylated tau (p-tau) (≥70 pg/mL) in cerebrospinal fluid (CSF); (3) age≥40 years. And the patients who complicated with brain injuries (such as

Comparison of clinical features between 8 AD patients and 8 Ctrl subjects

For microarray analysis, 8 AD patients and 8 Ctrl subjects were included. No difference of mean age (P = 0.727), gender (P = 0.282) or mean education duration (P = 0.177) was observed between AD patients and Ctrl subjects. As for the mean MMSE score, it was decreased in AD patients compared with Ctrl subjects (P < 0.001). Besides, the mean Aβ42 level (P < 0.001) was lower, whereas the mean t-tau level (P < 0.001), mean p-tau level (P = 0.001) and mean 8-OHG level (P = 0.001) were higher in AD

Discussion

In the present study, a microarray analysis of circRNA expression profile in 8 AD patients and 8 Ctrl subjects disclosed that circRNA expression profile could differentiate AD patients from Ctrl subjects, and dysregulated circRNAs were enriched in multiple pathophysiology processes of AD, especially regarding neuron cell death, neuroinflammation and neurodegeneration pathways. Further validation by RT-qPCR in 80 AD patients and 40 Ctrl subjects found that circ-LPAR1, circ-AXL, circ-GPHN,

Conclusion

In conclusion, the present study provides a comprehensive overview of dysregulated circRNAs in AD, and identifies that circ-AXL, circ-GPHN and circ-PCCA may hold the clinical value for predicting disease risk and progression of AD. These findings may provide valuable insights for supporting patient care, guiding management and decision making of AD in clinics.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

This study was supported by Beijing Medical and Health Foundation (No. B17773) and The 23456 Talent Project of Henan Provincial People’s Hospital.

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Circular RNAs (circRNAs) is a fascinating covalently closed circular non-coding RNA that is abundantly present in the transcriptome of eukaryotic cells. Its versatile nature allows it to participate in a multitude of pathological and physiological processes within the organism. One of its crucial functions is acting as a microRNA sponge, modulating protein transcription levels, and forming interactions with essential RNA-binding proteins. Remarkably, circRNAs demonstrates a specific enrichment in various vital areas of the brain, including the cortex, hippocampus, white matter, and photoreceptor neurons, particularly in aging organisms. This intriguing characteristic has led scientists to explore its potential as a significant biological marker of neurodegeneration, offering promising insights into neurodegenerative diseases like Alzheimer's disease (AD). In AD, there has been an interesting observation of elevated levels of circRNAs in both peripheral blood and synaptic terminals of affected individuals. This intriguing finding raises the possibility that circRNAs may have a central role in the initiation and progression of AD. Notably, different categories of circRNAs, including HDAC9, HOMER1, Cwc27, Tulp4, and PTK2, have been implicated in driving the pathological changes associated with AD through diverse mechanisms. For instance, these circRNAs have been demonstrated to contribute to the accumulation of beta-amyloid, which is a hallmark characteristic of AD. Additionally, these circRNAs contribute to the excessive phosphorylation of tau protein, a phenomenon associated with neurofibrillary tangles, further exacerbating the disease. Moreover, they are involved in aggravating neuroinflammation, which is known to play a critical role in AD's pathogenesis. Lastly, these circRNAs can cause mitochondrial dysfunction, disrupting cellular energy production and leading to cognitive impairment. As researchers delve deeper into the intricate workings of circRNAs, they hope to unlock its full potential as a diagnostic tool and therapeutic target for neurodegenerative disorders, paving the way for innovative treatments and better management of such devastating conditions.
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So far, the search for a cure for Alzheimer Disease (AD) has been unsuccessful. The only approved drugs attenuate some symptoms, but do not halt the progress of this disease, which affects 50 million people worldwide and will increase its incidence in the coming decades. Such scenario demands new therapeutic approaches to fight against this devastating dementia. In recent years, multi-omics research and the analysis of differential epigenetic marks in AD subjects have contributed to our understanding of AD; however, the impact of epigenetic research is yet to be seen. This review integrates the most recent data on pathological processes and epigenetic changes relevant for aging and AD, as well as current therapies targeting epigenetic machinery in clinical trials. Evidence shows that epigenetic modifications play a key role in gene expression, which could provide multi-target preventative and therapeutic approaches in AD. Both novel and repurposed drugs are employed in AD clinical trials due to their epigenetic effects, as well as increasing number of natural compounds. Given the reversible nature of epigenetic modifications and the complexity of gene-environment interactions, the combination of epigenetic-based therapies with environmental strategies and drugs with multiple targets might be needed to properly help AD patients.
An insight into the TAM system in Alzheimer's disease
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The TAM receptors may help delay the progression of Alzheimer’s disease (AD). AD is the most common neurodegenerative disease associated with human aging. The TAM receptors, derived from the first letter of its three constituents -Tyro3, Axl, and Mertk, are associated with immune responses, cellular differentiation and migration, and clearance of apoptotic cells and debris, with the two canonical ligands, Growth Arrest Specific 6 (Gas6) and ProS1. Several kinds of research have indicated the participation of the TAM system in AD pathology. Also, the TAMs regulate multiple features of microglia, the significant sensors of disorder in the central nervous system (CNS).
In this review, we describe the biology of the TAM receptors and ligands in the CNS. Then, we discuss the relationship between the TAM system and AD, specially focusing on its functional expression in the microglia. Finally, we also summarize some agents that could interfere with the TAM signaling pathways and discuss potential difficulties and strategies for drug development.
CircUBXN7 suppresses cell proliferation and facilitates cell apoptosis in lipopolysaccharide-induced cell injury by sponging miR-622 and regulating the IL6ST/JAK1/STAT3 axis
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Acute respiratory distress syndrome (ARDS) is a common and serious respiratory illness with substantial morbidity and mortality. Circular RNAs have been demonstrated to participate in various diseases processes. However, the biological function and mechanism of most circular RNAs have not been elucidated in ARDS. In this study, we found that circUBXN7 was significantly increased in lipopolysaccharide (LPS)-induced A549 and Beas-2B cell injury. Inhibition of circUBXN7 significantly promoted cell proliferation and reduced cell apoptosis, while overexpression of circUBXN7 suppressed cell proliferation and accelerated cell apoptosis in LPS-induced A549 and Beas-2B cells. CircUBXN7 acted as a sponge for miR-622, and miR-622 rescued the effect of circUBXN7 on cell proliferation and apoptosis. We also found that IL6ST was a target gene of miR-622, and the expression of IL6ST was indirectly regulated by circUBXN7. Furthermore, western blotting indicated that the JAK1/STAT3 signaling pathway was involved in the circUBXN7/miR-622/IL6ST axis in LPS-induced A549 and Beas-2B cell injury. Overall, our study suggested that circUBXN7 suppressed cell proliferation and facilitated cell apoptosis by sponging miR-622 and regulating IL6ST, to activate the JAK1/STAT3 signaling pathway in LPS-induced A549 and Beas-2B cell injury. CircUBXN7 might therefore be a potential biomarker for ARDS, and dysregulation of circUBXN7 may be involved in the pathogenesis of ARDS.
Circ_0002945 functions as a competing endogenous RNA to promote Aβ<inf>25-35</inf>-induced endoplasmic reticulum stress and apoptosis in SK-N-SH cells and human primary neurons
2022, Brain Research
Citation Excerpt :
Moreover, the circHDAC9/miR-138/sirtuin-1 ceRNA network exerts crucial activity in synaptic function and Aβ production during AD (Lu et al., 2019). Circ_0002945, produced by the back-splicing of exons of AXL receptor tyrosine kinase (AXL), is upregulated in the cerebrospinal fluid of AD patients, and it may be a potential biomarker for the risk prediction and detection of AD (Li et al., 2020). However, it remains poorly understood whether circ_0002945 can impact AD pathogenesis.
Circular RNAs (circRNAs) have been shown to function as competing endogenous RNAs (ceRNAs) to participate in the pathogenesis of Alzheimer’s disease (AD). In this study, we set to identify the activity and mechanism of circ_0002945 in AD pathogenesis.
The levels of circ_0002945, microRNA (miR)-431-5p and TNF alpha induced protein 1 (TNFAIP1) were measured by quantitative real-time PCR (qRT-PCR) or western blot. The levels of cleaved caspase-12, glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP), the markers of cell endoplasmic reticulum stress, were gauged by western blot. Cell viability and apoptosis abilities were evaluated by MTT assay and flow cytometry, respectively. The direct relationship between miR-431-5p and circ_0002945 or TNFAIP1 was verified by the dual-luciferase reporter or RNA immunoprecipitation (RIP) assay.
Our data showed that circ_0002945 was overexpressed in AD serum and amyloid beta (Aβ)_25-35-stimulated SK-N-SH cells and human primary neurons (HPNs). Inhibition of circ_0002945 attenuated Aβ_25-35-induced cell apoptosis and endoplasmic reticulum stress. MiR-431-5p was regulated by circ_0002945 and it was responsible for the circ_0002945 function. Moreover, TNFAIP1 was a miR-431-5p target and circ_0002945 functioned as a ceRNA to control TNFAIP1 expression via miR-431-5p competition. Furthermore, miR-431-5p-mediated suppression of TNFAIP1 ameliorated Aβ_25-35-induced cell apoptosis and endoplasmic reticulum stress.
Our findings establish circ_0002945 as a crucial regulator of Aβ-induced neuron apoptosis and endoplasmic reticulum stress and uncover a novel circ_0002945/miR-431-5p/TNFAIP1 ceRNA network for the Aβ pathogenic pathway in AD.
Circular RNA AXL increases neuron injury and inflammation through targeting microRNA-328 mediated BACE1 in Alzheimer's disease
2022, Neuroscience Letters
Citation Excerpt :
The data showed that circ-AXL promoted neuron injury and inflammation in cellular AD models. The possible explanations might be that: (1) circ- AXL might serve as a competing endogenous RNA to target miR-328, consequently regulating BACE1 expression, which was involved in AD progression [15,16]; (2) our previous study discovered that circ-AXL might potentially target other microRNAs playing a crucial role in AD, such as miR-1233-5p, etc. [5,17,18]. The dysregulation of miRNA leads to neurodegenerative disorders, such as Parkinson’s disease, Huntington's disease, and AD [19–22].
Our previous study observed that circular RNA AXL (circ-AXL, access number circ_0002945) was closely correlated with disease risk and severity of Alzheimer’s Disease (AD) by microarray and RT-qPCR validation. Then this current study aimed to further investigate the effect of circ-AXL on regulating neuron injury and inflammation in cellular AD models and its underlying molecular mechanism.
SK-N-SH and SK-SY5Y cell lines were treated by amyloid β to construct cellular AD models. Then control or circ-AXL overexpression, control or circ-AXL knock-down, microRNA-328 (miR-328) knock-down with or without circ-AXL knock-down, as well as BACE1 overexpression with or without miR-328 overexpression plasmids were transfected into cellular AD models. Furthermore, neuron injury and inflammation were detected.
Circ-AXL overexpression increased apoptosis rate and declined neurite outgrowth, as well as elevated inflammatory cytokines in cellular AD models; but circ-AXL knockdown exhibited opposite effects. Additionally, circ-AXL negatively regulated miR-328 but positively modulated BACE1; besides, miR-328 negatively regulated BACE1; further luciferase reporter gene assay presented that circ-AXL directly bound miR-328, and miR-328 directly bound BACE1. Furthermore, miR-328 overexpression decreased apoptosis rate, elevated neurite outgrowth, and declined inflammatory cytokines in cellular AD models; but miR-328 knockdown presented opposite effects. Notably, miR-328 knockdown attenuated the effect of circ-AXL knockdown on cellular AD models. Moreover, BACE1 overexpression aggravated neuron injury and inflammation, as well as attenuated the effect of miR-328 overexpression on these functions in cellular AD models.
Circ-AXL may serve as a potential treatment target via miR-328 mediated BACE1 in AD.

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Circular RNA expression profile of Alzheimer’s disease and its clinical significance as biomarkers for the disease risk and progression

Abstract

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Participants

Comparison of clinical features between 8 AD patients and 8 Ctrl subjects

Discussion

Conclusion

Declaration of Competing Interest

Acknowledgements

Lancet

Pregnancy Hypertens

Trends Neurosci

Comput Struct Biotechnol J

Alzheimers Dement

Neurosci Lett

J Biol Chem

FEBS Lett

The road to restoring neural circuits for the treatment of Alzheimer’s disease

Nature

The new genetic landscape of Alzheimer’s disease: from amyloid cascade to genetically driven synaptic failure hypothesis?

Acta Neuropathol

Clinical and biomarker changes in dominantly inherited Alzheimer’s disease

N Engl J Med

Update on Alzheimer’s Disease Therapy and Prevention Strategies

Annu Rev Med

Identifying circRNA-associated-ceRNA networks in the hippocampus of Abeta1-42-induced Alzheimer’s disease-like rats using microarray analysis

Aging (Albany NY)

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function

Science

Circular RNA circ_0000950 promotes neuron apoptosis, suppresses neurite outgrowth and elevates inflammatory cytokines levels via directly sponging miR-103 in Alzheimer's disease

Cell Cycle

CircRNA expression profiles and function prediction in peripheral blood mononuclear cells of patients with acute ischemic stroke

J Cell Physiol

Novel circular RNA expression profile of uveal melanoma revealed by microarray

Chin J Cancer Res