NcRNAs: A synergistically antiapoptosis therapeutic tool in Alzheimer's disease

Abstract Aims The aim of this review is to systematically summarize and analyze the noncoding RNAs (ncRNAs), especially microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), in the cell apoptosis among Alzheimer's disease (AD) in recent years to demonstrate their value in the diagnosis and treatment of AD. Methods We systematically summarized in vitro and in vivo studies focusing on the ncRNAs in the regulation of cell apoptosis among AD in PubMed, ScienceDirect, and Google Scholar. Results We discover three patterns of ncRNAs (including ‘miRNA‐mRNA’, ‘lncRNA‐miRNA‐mRNA’, and ‘circRNA‐miRNA‐mRNA’) form the ncRNA‐based regulatory networks in regulating cell apoptosis in AD. Conclusions This review provides a future diagnosis and treatment strategy for AD patients based on ncRNAs.

AD brains.Clarifying the mechanism of cell apoptosis is always an important work in diagnosing and treating AD, which cannot be ignored.
Numerous original articles reported that noncoding RNAs (ncRNAs), especially microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), participate in regulating cell apoptosis in AD in the past decade.NcRNAs have become an important research direction to explore cell apoptosis in AD.This review highlights the role of miRNAs, lncRNAs, and circRNAs in regulating cell apoptosis among AD.Three classic patterns, including 'miRNA-mRNA', 'lncRNA-miRNA-mRNA', and 'circRNA-miRNA-mRNA', are discovered.Furthermore, the potential ncRNA-based networks in regulating cell apoptosis among AD are built.Subsequently, this review proposes an ncRNA-based synergistically antiapoptosis strategy in investigating the diagnosis and treatment of AD patients.

| CELL AP OP TOS IS
Cell apoptosis was first described as chromatin condensation, nuclear membrane breakdown, cell shrinkage, and formation of small vesicular bodies near the cell surface (apoptotic bodies) in 1972. 10 is triggered by two principal pathways referred to as the intrinsic (or mitochondrial) pathway and extrinsic (or death receptor) pathway (Figure 1).

| The intrinsic pathway
The intrinsic pathway involves the regulation of mitochondrial outer membrane permeabilization (MOMP) in cell apoptosis by the BCl-2 family proteins, 11 which results in the release of Cytochrome c from the mitochondria into the cytosol. 12MOMP and release of Cytochrome c are critical requirements to trigger the formation of apoptosomes, which are considered the point of no return in apoptotic cell death. 135][16] Oligomerized BAX and BAK form macropores in the mitochondria membrane, causing MOMP. 14X and BAK are considered necessary for the execution of cell apoptosis via the intrinsic pathway. 14The release of Cytochrome c is inhibited by the antiapoptotic BCL-2 family members, such as BCL-2, BCL-XL, and BCL-W. 11,17These antiapoptotic proteins contain two BCL-2 homology (BH3) domains, which constitute a binding trench that sequesters BAX and BAK. 18The affinity and expression level of BCL-2 family members ultimately determine the sensitivity or resistance to cell apoptosis. 18[21] The mature apoptosome cleaves and activates Pro-Caspase-9, initiating the caspase-processing cascade (activating Pro-Caspase-3 and Pro-Caspase-7). 22,23I G U R E 1 Apoptosis is triggered by the intrinsic and extrinsic pathways.The intrinsic pathway is activated by BAK and BAX.BAK/BAX oligomers form pores, leading to the release of Cytochrome c into the cytosol.Activation of BAK/BAX is promoted by BID or inhibited by BCL-2/BCL-XL/BCL-W.Cytochrome c binds to Apaf-1, which recruits Pro-Caspase-9, forming the apoptosome.In the apoptosome, Pro-Caspase-9 is activated by autoproteolytic cleavage, initiating the caspase-processing cascade.The extrinsic pathway is activated by engagement of membrane receptors.These proteins induce DISC involving TRADD, FADD, Caspase-8, and Caspase-10.Initiator Caspase-8/10 activation is negatively regulated by c-FLIP.Once active, executioner caspases (Caspase-3/6/7) bring about apoptosis.
Subsequently, the complete 'lncRNA-miRNA-mRNA' axes will be verified in regulating cell apoptosis in the AD cellular models.
We need to solve the mystery left behind.
Aβ 42 triggered a significant downregulation in circHDAC9 and a striking upregulation in miR-142-5p in human neuronal cells. 77The direct interaction between circHDAC9 and miR-142-5p was confirmed by dual-luciferase reporter, RIP, and RNA pull-down assays. 77rcHDAC9-miR-142-5p axis inhibited cell apoptosis in Aβ 42 -treated human neuronal cells. 77The expression level of circ_0000950 was not changed in PC12 cells and rat primary cerebral cortex neurons. 78wever, overexpression of circ_0000950 aggravated Aβ 42 -induced cell apoptosis in PC12 cells and rat primary cerebral cortex neurons. 78Compensation experiment and dual-luciferase reporter assay further validated the sponging effect of circ_0000950 on miR-103 as well as the mechanism of circ_0000950-miR-103 axis on promoting cell apoptosis. 78The above studies require further research to identify the direct target mRNAs of 'lncRNA-miRNA' axes.Then, the complete 'lncRNA-miRNA-mRNA' axes will be verified in regulating cell apoptosis in the AD cellular model.

| AB NORMALLY E XPRE SS ED N CRNA S IN AD ANIMAL MODEL S IN REG UL ATING CELL AP OP TOS IS IN AD
Transgene (such as APP/PS1, 5xFAD, and SAMP8) and microinjection (such as Aβ 42 , Aβ 25-35 , and Aβ 40 ) are often used to construct AD animal models. 79Many studies have shown that numerous abnormally expressed ncRNAs in AD animal models are deeply involved in regulating cell apoptosis in AD.Therefore, it is necessary to summarize and analyze it.
Interestingly, the relationship between miR-34a and cell apoptosis in AD required special attention.Wang et al. and Li et al. showed that the expression level of miR-34a is increased in APPswe/PSΔE9 mice and PS-2 mutant (N141I) transgenic mice. 87,89BCL-2 and SIRT1 were both the targets of miR-34a. 87,89MiR-34a promoted cell apoptosis by negatively regulating the expression of BCL-2 and SIRT1. 87,89nversely, Modi et al. showed that miR-34a expression increases significantly until 6 months, followed by a decrease, which was significantly lower than in the WT (wild-type) animals aged more than 12 months in APP/PS1 transgenic mice. 88Cyclin D1 was a direct target of miR-34a. 88MiR-34a inhibited cell apoptosis by negatively regulating the expression of Cyclin D1. 88 We noticed that Wang et al. used 3-month-old and 6-month-old transgenic mice in their study, 87 and Li et al. used 9-month-old transgenic mice in their study. 89And Modi et al. found that miR-34a expression increases significantly until 6-month-old, followed by a decrease more than 12-month-old, in the transgenic mice. 88Therefore, whether miR-34a promotes or inhibits cell apoptosis in AD may be related to the age of the transgenic mice.
The expression of lncRNA EBF3-AS was upregulated in hippocampus of APP/PS1 mice. 126EBF3-AS promoted cell apoptosis in Aβ 25-35 -induced SH-SY5Y cells and might be a new therapeutic target for treatment of AD. 126 In addition, downregulated lncRNA MEG3 was detected in the hippocampus tissues of AD rats (microinjection of Aβ 25-35 ). 127Upregulation of MEG3 inhibited cell apoptosis in hippocampal neurons in AD rats. 127EBF3-AS and MEG3 are closely related to cell apoptosis in AD.However, whether they regulate cell apoptosis through 'lncRNA-miRNA-mRNA' axis has not been explored.We need to solve the mystery left behind.
Interestingly, lncRNA n336694 and miR-106b were both upregulated in APP/PS1 double transgenic mice. 128Dual-luciferase reporter assay showed that n336694 directly targets the 3′-UTR of miR-106b. 128Overexpression of n336694 obviously increased the expression of miR-106b. 128Subsequently, transfecting miR-106b mimics promoted cell apoptosis in SH-SY5Y cells. 128Moreover, bioinformatics analysis revealed that lncRNA SOX21-AS1 was upregulated in AD. 129 SOX21-AS1 directly targeted the 3′-UTR of FZD3/5. 129Silencing of SOX21-AS1 could act to alleviate neuronal apoptosis in AD mice through the upregulation of FZD3/5 and subsequent activation of the Wnt signaling pathway. 129Similarly, the expression of lncRNA XIST was increased in AD mice. 130RIP validated the combination of XIST and EZH2. 130XIST promoted cell apoptosis by negatively regulating EZH2 in AD. 130 Conversely, the expression of lncRNA WT1-AS was decreased in the brain tissues of Aβ 25-35induced mice. 131WT1-AS overexpression inhibited WT1 expression, and WT1 could directly target the promoter region of miR-375 to promote its expression. 131MiR-375 could bind the 3′-UTR of SIX4, and miR-375-SIX4 axis promoted cell apoptosis in SH-SY5Y cells. 131ese studies show that the abnormally expressed lncRNAs in the AD animal models can regulate cell apoptosis in AD by these nonclassic patterns.These nonclassic patterns are independent on the 'lncRNA-miRNA-mRNA' axis described previously.

| AB NORMALLY E XPRE SS ED N CRNA S IN AD PATIENTS IN REG UL ATING CELL AP OP TOS IS IN AD
No matter what role ncRNAs play on cell apoptosis in cellular AD models and AD animal models, we should first verify whether the related ncRNAs are abnormally expressed in AD patients.In other words, if the expression of these ncRNAs does not change in AD patients, it would be a devastating blow to relevant research.Many studies have shown that numerous ncRNAs, which are abnormally expressed in AD patients, are deeply involved in regulating cell apoptosis in AD in recent years.Therefore, it is necessary to summarize and analyze it.

| miRNA-mRNA
MiR-212 downregulated in human plasma and brains with AD. 132,133 PTEN and FOXO3a were direct targets of miR-212, 132 and PDCD4 was also a direct target of miR-212. 133MiR-212-PTEN/FOXO3 axes 132 and miR-212-PDCD4 axis 133 reduced neuronal apoptosis and contributed to AD neuroprotection.Additionally, a decreased expression of miR-129 was detected in serum of AD patients. 134MiR-129 directly targeted YAP1 and disrupted its interaction with JAG1, leading to a decline in hippocampal neuron apoptosis and attenuated cognitive impairment in Aβ 42 -injected mice. 134In other words, miR-129-YAP1 axis might be a new therapeutic target for AD treatment. 134Moreover, the expression of miR-539-5p was significantly downregulated in human cerebrospinal fluid (CSF) with AD, upregulation of miR-539-5p inhibited cell apoptosis in APP/PS1 transgenic mice. 135Dual-luciferase reporter assay showed that APP, CAV1, and GSK-3β are direct targets of miR-539-5p. 135MiR-539-5p was negatively correlated with the expression of APP, CAV1, and GSK-3β, and miR-539-5p-APP/CAV1/ GSK-3β axes might be a novel pathologic mechanism in regulating cell apoptosis in APP/PS1 transgenic mice. 135Conversely, miR-26b significantly elevated in the defined pathological areas of human postmortem brains, starting from early stages of AD (Braak III). 136Rb1 was a direct target of miR-26b. 136MiR-26b-Rb1 axis promoted cell apoptosis and inhibited cell viability in human primary cortical neurons. 136Similarly, miR-425-5p was upregulated in human postmortem brain with AD. 137 HSPB8 was directly targeted by miR-425-5p. 137MiR-425-5p-HSPB8 axis induced cell apoptosis and promoted Tau phosphorylation in AD, and might act as a new therapeutic target for AD treatment. 137 addition, miR-485-3p was overexpressed in the brain tissues, CSF, and plasma of patients with AD. 138 CD36 was a direct target of miR-485-3p. 138MiR-485-3p-CD36 axis weakened the phagocytosis of Aβ in vitro and in vivo and might promote cell apoptosis in AD. 138 Table S3 summarizes all the abnormally expressed and cell apoptosis-related miRNAs in AD patients.  These RNAs can form 'miRNA-mRNA' axis in regulating cell apoptosis in AD.
Interestingly, miR-132 downregulated in human AD brains. 132EN and FOXO3a were both the direct targets of miR-132. 132MiR-132-PTEN/FOXO3 axes reduced neuronal apoptosis and contributed to AD neuroprotection. 132However, Liu et al. showed that the expression of miR-132 is significantly higher in human AD brains. 139al-luciferase reporter assay verified that GTDC-1 is a direct target of miR-132. 139MiR-132-GTDC-1 axis promoted cell apoptosis in human primary cortical neurons. 139Similarly, the expression level of miR-125b-5p was increased in the CSF samples of AD patients 140 but decreased in the serum samples of AD patients. 141Jin et al.

confirmed that miR-125b-5p promotes cell apoptosis in Neuro2a
APPswe/Δ9 cells. 140Li et al. confirmed that BACE1 acts as a direct target of miR-125b-5p, and miR-125b-5p-BACE1 axis inhibited cell apoptosis in Aβ 25-35 -treated MCN and N2a cells. 141These studies showed that the true expression and function of miR-132 and miR-125b-5p need to be further confirmed in AD.

| LncRNA-miRNA-mRNA
LncRNA ATB was overexpressed in the CSF and serum of AD patients. 161ATB negatively regulated the expression of miR-200, and suppression of ATB alleviated Aβ 25-35 -induced cell apoptosis by regulation of miR-200 in PC12 cells. 161ZNF217 was a direct target of miR-200. 161ATB might protect PC12 cells against Aβ 25-35 -induced cell apoptosis via regulating miR-200/ZNF217 axis, and ATB-miR-200-ZNF217 axis may provide a new insight for preventing AD. 161 Similarly, lncRNA RMRP was upregulated in serum of AD patients. 162al-luciferase reporter assay showed that RMRP acts as a sponge of miR-3142, and miR-3142 directly targets TRIB3. 162RMRP-miR-3142-TRIB3 axis promoted cell apoptosis in Aβ 42 -inudced SH-SY5Y cells. 162These above lncRNAs abnormally express in AD patients, and the 'lncRNA-miRNA-mRNA' axis is also a classic pattern in regulating cell apoptosis in AD.
LncRNA RP11-59J16.2 was upregulated in serum of AD patients. 163Interestingly, dual-luciferase reporter assay verified that RP11-59J16.2 can directly interact with 3′-UTR of MCM2 and further downregulated the expression of MCM2. 163RP11-59J16.2 aggravated Aβ 42 -induced cell apoptosis by negatively regulating the expression of MCM2 in SH-SY5Y cells. 163This indicates that ln-cRNAs can also directly target mRNA and regulate cell apoptosis in AD.In other words, 'lncRNA-mRNA' axis may be another direction of exploration in regulating cell apoptosis in AD.

| THE N CRNA-BA S ED ANTIAP OP TOS IS REG UL ATORY NE T WORK S IN AD
All the abovementioned studies are related to the abnormally expressed ncRNAs in cellular AD models, AD animal models, and AD patients, and their function in regulating cell apoptosis in AD.
Therefore, we can build the potential and broader ncRNA-based antiapoptosis regulatory networks based on 'miRNA' or 'mRNA' as the core, respectively.Figure 2A   strategies for the diagnosis and treatment of cell apoptosis in AD will also become more and more perfect.

| DISCUSS ION
At present, studies on the roles of ncRNAs in regulating cell apoptosis of AD and their clinical relevance are still in the development stage.0][171][172][173] The identification of whether these ncRNAs participate in regulating cell apoptosis in AD will be our important work.The verification of the functions of the above ncRNAs will greatly expand the breadth of ncRNAs in regulating cell apoptosis in AD.
Many studies demonstrated that numerous cell apoptosisrelated and abnormally expressed ncRNAs in AD also participate in regulating Aβ accumulation, Tau phosphorylation, inflammation, oxidative stress, and so on.For example, downregulation of miR-98-5p decreased SNX6-dependent levels of Aβ 40 , Aβ 42 , BACE1, soluble amyloid precursor protein β (sAPPβ), and membraneassociated APP β-carboxyl terminal fragment (βCTF) in SK-N-SH cells. 54In other words, the cell apoptosis-related miR-98-5p played a critical role in accumulation of Aβ in AD. 54 Overexpression of miR-132 induced neuronal apoptosis by increasing BAX and decreasing BCL-2 and also upregulated phosphorylation of Tau. 136Sequencespecific inhibition of miR-132 and miR-212 induced cell apoptosis in cultured primary neurons, whereas their overexpression was neuroprotective against oxidative stress. 132Circ_0000950 promoted neuron apoptosis, suppressed neurite outgrowth, and elevated inflammatory cytokine levels through directly sponging miR-103 in AD. 139 These studies imply that when we develop the antiapoptosis strategies based on ncRNAs, we may gain additional benefits in against Aβ accumulation, Tau phosphorylation, inflammation, oxidative stress, and so on.Concurrently, we should be aware that we may find a large number of ncRNAs involved in the regulation of cell apoptosis in the studies focusing on ncRNAs in regulating other AD-related pathological changes.It will help us amplify the ncRNAbased cell apoptosis regulatory network in AD and establish a more perfect antiapoptosis strategy in AD.
In future, as more ncRNAs in cell apoptosis regulatory of AD are identified, a more complete ncRNA-based regulatory network will be built.Based on this gradually improved network, we will have a more complete understanding of ncRNAs in cell apoptosis regulatory of AD.This will help us formulate ncRNA-based antiapoptosis synergistic strategies and improve the role of ncRNAs in the diagnosis and treatment of AD.
In addition, three suggestions were proposed for future research on the role of ncRNAs in cell apoptosis regulatory among AD.First,

Jin et al. found that miR-125b-5p induced cell apoptosis in Neuro2a
APPswe/Δ9 cells, 140 but Li et al. 141 showed that miR-125b-5p inhibited cell apoptosis in Aβ 25-35 -induced primary mouse cortical neurons and Neuro2a cells.In these cases, more studies are needed to confirm the real role of such ncRNAs.Second, future studies can focus on whether ncRNAs that function in other AD pathological changes affect the cell apoptosis in AD.Third, future studies can focus on lncRNAs and circRNAs in regulating cell apoptosis among AD.After all, there are fewer studies on lncRNAs and circRNAs comparing with miRNAs so far.Moreover, Cai et al. 69 showed that lncRNA RP11-543N12.1 and miR-324-3p are both upregulated in Aβ 25-35 -induced SH-SY5Y cells, and RP11-543N12.1 directly targets miR-324-3p.Unusually, overexpressed RP11-543N12.1 upregulated the expression of miR-324-3p, and RP11-543N12.1 and miR-324-3p both promoted cell apoptosis in Aβ 25-35 -induced SH-SY5Y cells. 69Additionally, Huang et al. found that lncRNA n336694 and miR-106b are overexpressed in APP/PS1 mice brain tissues, and miR-106b is a direct target of n336694. 128conventionally, overexpressed n336694 upregulated the expression of miR-106b, and n336694 and miR-106b both promoted cell apoptosis in SH-SY5Y cells. 128The two articles show that lncRNAs may act as a stabilizer of miRNAs and upregulate its expression.Otherwise, bioinformatics analysis revealed that lncRNA SOX21-AS1 was upregulated in AD. 129 SOX21-AS1 directly targeted the 3′-UTR of FZD3/5. 129Silencing of SOX21-AS1 alleviated neuronal apoptosis in AD mice through the upregulation of FZD3/5. 129Similarly, the expression of lncRNA XIST was increased in AD mice. 130RIP validated the combination of XIST and EZH2. 130XIST promoted cell apoptosis by negatively regulating EZH2 in AD. 130 The two articles show that 'lncRNA-mRNA' axis can also regulate cell apoptosis in AD.These ncRNA-based nonclassic patterns in regulating cell apoptosis in AD are an important supplements for our networks described previously.It will help us understand better ncRNAs in regulating cell apoptosis in AD.

| CON CLUS IONS
SH-SY5Y cell line, PC12 cell line, SK-N-SH cell line, and primary neurons are often used in in vitro studies in AD.The establishment of in vitro models among AD highly depends on the toxicity of Aβ, such as Aβ 42 , Aβ 25-35 , and Aβ 40 .Many studies have shown that ncRNAs are deeply involved in the regulation of cell apoptosis among AD in recent years, especially in in vitro studies.Therefore, it is necessary to summarize and analyze ncRNAs in regulating cell apoptosis in AD cellular models.
Figure 2B shows an ncRNA-based antiapoptosis regulatory network constructed with BACE1 as the core.Downregulation of lncRNA NEAT1 and circ-AXL, and upregulation of miR-124, miR-328, miR-200a-3p, miR-340, miR-19b-3p, miR-125b-5p, miR-34a-5p, miR-29c-3p, and miR-16 should exert synergistic antiapoptosis effects in AD (Figure 2B).Tables S4 and S5 summarize other ncRNAs that synergistically inhibit cell apoptosis in AD with miRNAs or mRNAs as the cores.Although these ncRNAs cannot form a broad regulatory network as miR-107 and BACE1, they also deserve our attention nonetheless.More importantly, based on the three core regulatory axes, 'miRNA-mRNA', 'lncRNA-miRNA-mRNA', and 'circRNA-miRNA-mRNA', this | 7 of 13 LI et al. review constructs a schematic diagram of a broader regulatory network that helps us understand better the mechanism by which ncRNAs involved in cell apoptosis of AD (Figure 3).As the studies on the cell apoptosis regulatory in AD by ncRNAs become more and more abundant, the ncRNA-based cell apoptosis regulatory network map will become more and more complete, and our ncRNA-based F I G U R E 2 The potential synergistically antiapoptosis ncRNA-based regulatory networks exist in AD. (A) Based on miR-107, the network is constructed in regulating cell apoptosis in AD and the simple antiapoptotic strategy is proposed.(B) Based on BACE1, the network is constructed in regulating cell apoptosis in AD and the simple antiapoptotic strategy is proposed.Red arrows (' ' and ' ') present for upregulation and downregulation, respectively.F I G U R E 3 The potential synergistically ncRNA-based apoptosis regulatory mechanism exists in AD.First, three independent regulatory axes in left, 'miRNA-mRNA', 'lncRNA-miRNA-mRNA', and 'circRNA-miRNA-mRNA', regulate cell apoptosis in AD.Then, intertwined ncRNAbased regulatory network in right regulates cell apoptosis in AD.
This review summarizes for the first time that the mechanisms which ncRNAs regulate cell apoptosis in AD.We propose to build the regulatory network based on the three classical regulatory patterns in helping us understand better ncRNAs in regulating cell apoptosis in AD.Subsequently, the network facilitates the construction of ncRNA-based synergistically diagnostic and therapeutic strategies for cell apoptosis in AD.Finally, we must be aware that such ncRNAbased network can also be constructed in different diseases.It will improve our understanding of ncRNAs in regulating the occurrence and development of various diseases, and help formulate a more effective and reasonable ncRNA-based diagnosis and treatment strategies.AUTH O R CO NTR I B UTI O N S Liangxian Li contributed to conceptualization, methodology, software, data curation, and original draft preparation.Liangxian Li and Mingyue Jin contributed to visualization and investigation.Jie Tan and Bo Xiao contributed to supervision, writing, reviewing, and editing.All authors read and approved the final manuscript document.