Hsa

Background: Circular RNAs (circRNAs) exhibited important roles in Alzheimer ’ s disease (AD). Here, we focused on the dysregulation of hsa_circ_0049472 (circ_0049472) and potential functions in SK-N-SH cells with amyloid-beta peptide (A β ) treatment in AD. Methods: RNA expression was detected by real-time quantitative PCR. Cell viability and proliferation were measured by MTS and Edu assays. Flow cytometry was used for apoptosis detection, and cell inflammation was assessed using enzyme-linked immunosorbent assay. Target interaction was validated by dual-luciferase reporter assay and RNA immunoprecipitation assay. Protein expression and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) pathway were examined by Immunoblotting. Results: A β treatment inhibited cell viability and proliferation of SK-N-SH cells, but enhanced apoptosis rate, apoptosis protein levels (Bcl2-associated X protein and cleaved-caspase-3) and inflammatory cytokines (interleukin (cid:0) 6, IL-1 β , tumor necrosis factor-α ). Then, circ_0049472 expression was shown to be upregulated in response to A β stimulation and knockdown of circ_0049472 has ameliorated A β -induced cell injury. Circ_0049472 was identified as a sponge for miR-22 – 3p, and miR-22 – 3p inhibition reversed the regulation of circ_0049472 knockdown in A β -treated cells. Furthermore, ZNF217 acted as a target of miR-22 – 3p and circ_0049472 could regulate ZNF217 expression via binding to miR-22 – 3p. Overexpression of miR-22 – 3p abated A β -induced apoptosis and inflammation via downregulating ZNF217. Furthermore, A β reduced proteins levels of p-PI3K and p-AKT, and this inhibition of PI3K-AKT pathway was restored by the regulation of circ_0049472/miR-22 – 3p/ZNF217 axis. Conclusion: Circ_0049472 was involved in A β -induced neural injury by regulating miR-22 – 3p/ZNF217 axis to affect PI3K-AKT pathway. This study has discovered an innovative mechanism for AD.


Introduction
Alzheimer's disease (AD) is the most common form of senile dementia, a progressive neurodegenerative disorder that affects memory, language, cognition, and movement (Tromp et al., 2015;Silva et al., 2019).Extracellular plaques of amyloid-beta peptide (Aβ) have been previously addressed as a remarkable pathophysiological cause in patient's brain (Silva et al., 2019).However, the notion of the neurobiology of non-protein-coding RNAs (ncRNAs) has currently emerged in the context of AD pathogenesis, such as gene expression, Aβ production and deposit, inflammation, and neuronal survival (Lauretti et al., 2021).
Circular RNA (circRNA) is generally a kind of single-stranded ncRNA molecules with highly stable circular structures that can affect the expression of many protein-coding genes via several mechanisms including functioning as microRNA (miRNA) sponge in fine-tuning the expression of messenger RNA (mRNA) (Huang et al., 2020), resulting in a phenomenon known as the competing endogenous RNA (ceRNA) network (Moreno-Garcia et al., 2020).Furthermore, ceRNA networks are emerging biomarkers in neurodegenerative diseases like AD (Moreno-Garcia et al., 2020;Zhang et al., 2020).Differentially expressed circRNAs have been profiled in different biofluids, including cerebrospinal fluid (Li et al., 2020) and blood (Liu et al., 2020;Zacharias et al., 2020) of patients affected by AD.Hsa_circ_0049472 (circ_0049472) is a upregulated circRNA in microarray analysis of AD blood plasma (Liu et al., 2020).However, there is currently no report about circ_0049472 role in AD as well as any other human disorders.
MiRNA is a category of small ncRNA molecules efficient at posttranscriptionally regulating gene expression and activity.Circulating miRNAs are effective testing tools for the early diagnosis, staging, and progress monitoring of AD (Siedlecki-Wullich et al., 2021).MiRNA (miR)-22-3p, an evolutionarily-conserved gene primitively cloned from HeLa cells (Lagos-Quintana et al., 2001), is a novel regulator of cellular events, immune responses, and the associated diseases (Wang et al., 2017a(Wang et al., , 2017b)).MiR-22-3p expression fluctuates with disease progression of AD patients (Dong et al., 2021;Guo et al., 2017), thus serving as a common component in the miRNA-panel as blood-based mini-invasive diagnostic biomarker for AD.Given that many miRNAs role has been well-elaborated in AD pathology and progression (Samadian et al., 2021;Silvestro et al., 2019), it is valuable and compelling to decode the possible engagement of miR-22-3p in neural injury and neurological disorders.

Neuronal cells and Aβ stimulation
SK-N-SH cells (HTB-11; ATCC, Manassas, VA, USA) were cultured in E'EMEM (no L-Glutamine or phenol red) (51200087; Gibco, Grand Island, NY, USA) with 10 % FBS (Gibco).The solid Aβ25-35 (Aβ; HY-P0128; MedChemExpress, Shanghai, China) was dissolved in anhydrous DMSO (MedChemExpress) to obtain a 5 mM stock solution.It was diluted to the required concentration in serum-free EMEM with vortexing, and the diluted Aβ was maintained at 37 • C for 24 h before use to obtain its aggregated form.SK-N-SH cells were administrated with Aβ for 24 h to mimic activated neurotoxicity in AD pathogenesis.Cell culture and Aβ treatment were held with 5 % CO 2 at 37 • C.

MTS and EdU assays
Aβ toxicity was measured through Quick MTS Cell Proliferation Assay Kit (Biovision, San Francisco, CA, USA) and Yefluor 594 EdU Imaging Kit (Yeasen).In brief, cells were repeated in at least 3 paralleled wells in 96-well plates, and cells reached 80 % confluence were stained with (v:v) 10 % WST-1/ECS solution or 10 mM EdU in the fresh EMEM for 4 h.Then, MTS-stained cells were dissolved in 100 µL DMSO and absorbance measurement was administrated on microplate reader at 570 nm; EdU-incorporated cells were fixed, permeated and reacted with Yefluor 594 Azide, followed with DAPI counterstaining in accordance with the product manual.Relative cell viability was determined based on the percentage of absorbance with normalization to control group, and relative EdU positive cells were confirmed by the ratio of EdU signal to DAPI signal.

Total RNA isolation and RNase R assay
Total RNA was isolated through RNeasy Mini Kit (Qiagen), and isolated RNAs were quantified based on spectrophotometric method.Then, an aliquot of total RNA (5 μg) was incubated with 10 U RNase R (Sigma-Aldrich) for 0.5 h for the digestion of linear RNAs, and on the other hand incubated with mock solution without RNase R for the control group.

Real-time quantitative PCR (qPCR)
RNA samples were suffered from reverse transcription PCR based on HiScript® III 1st Strand cDNA Synthesis Kit (+gDNA wiper; Vazyme, Nanjing, China) and miRNA 1st Strand cDNA Synthesis Kit (Vazyme).Subsequently, AceQ Universal SYBR qPCR Master Mix (Vazyme) and miRNA Universal SYBR qPCR Master Mix (Vazyme) were employed for the quantification of cDNA products.The qPCR reaction was proceeded on Stratagene MX4000 Mutiplex Quantitative PCR System (Cedar Creek, Texas, USA), and PCR products were analyzed by cycle threshold (CT) method.GAPDH and U6 were utilized as the internal controls for RNA normalization.The sequence of qPCR primers was shown in Table 1.

Dual-luciferase reporter assay
According to starBase v2.0 algorithm, the miR-22-3p complementary sites in circ_0049472 and 3'untranslated region of ZNF217 (transcript ID ENST00000371471) were intended to be mutated.First of all, the intact sequence of circ_0049472 and ZNF217 3'UTR was respectively inserted into pGL4 vector (Promega, Madison, WI, USA) for constructing the recombinant plasmids: WT-circ_0049472, WT-ZNF217 3'UTR, mutant type of circ_0049472 (MUT-circ_0049472), and MUT-ZNF217 3'UTR.MUT vectors were generated based on WT vectors with the help of Hieff Mut™ Multi Site-Directed Mutagenesis Kit (Yeasen).After co-transfection of recombinant vectors and miR-22-3p/NC mimics for 36 h, SK-N-SH cells were obtained for determination of luciferase activity via Dual Glo Luciferase Reporter Assay System (Promega).

RNA immunoprecipitation (RNA-IP)
An RIP Kit (Geneseed, Guangzhou, China) was utilized to analyze the binding specificity between circ_0049472 or ZNF217 and miR-22-3p.Briefly, Protein A+G Beads were pre-treated with 1× Buffer A and Buffer D and linked with anti-Ago2 or anti-IgG.On the other side, cells were lysed in the 1× Buffer A pre-supplemented with 1 % proteinase inhibitor and 1 % RNase R inhibitor, and the supernatant after centrifuging at 14,000 g was added with 100 μL beads at 4 • C. Next, the precipitated mixture was co-cultured with protease K to decompose the protein content, and facilitated the isolation of RNA complex in RNeasy Mini Kit (Qiagen).Eventually, RNA enrichment was determined by qPCR.

Statistical analysis
Student's t-test was employed for two groups' comparison, and analysis of variance (ANOVA) followed with Turkey's test was used for comparison among more than two groups.Data of three independent experiments were expressed as mean ± SD, and P value for data comparison was deemed to be significant when it was less than 0.05.GraphPad Prism 5.0 (La Jolla, CA, USA) was employed to analyze data and image processing.

Circ_0049472 upregulation was associated with Aβ-stimulated neuronal injury
Circ_0049472 was highly expressed in AD patients compared normal controls (Supplementary Figure 1).As shown in Fig. 2A, circ_0049472 was gradually upregulated after Aβ treatment with the dose.Besides, not like linear GAPDH mRNA, circ_0049472 was stably expressed under RNase R digestion (Fig. 2B).By introducing siRNA, circ_0049472 level was inhibited in Aβ-treated cells (Fig. 2C), which resulted in a gain of cell viability and EdU positive rate (Figs.2D and 2E).Moreover, si-circ_0049472 transfection reduced apoptosis (Fig. 2F-2H) and secretions of pro-inflammatory cytokines (Fig. 2I-2K) under Aβ accumulation.Collectively, it proposed an inhibitive role of circ_0049472 silence in Aβstimulated neuronal injury.

Aβ-inactivated PI3K-AKT pathway was restrained by circ_0049472-miR-22-3p-ZNF217 axis
Immunoblotting data revealed that p-PI3K/PI3K and p-AKT/AKT protein levels were lowered after the administration of Aβ toxicant than control group, whereas this expression change was distinctively activated by introducing si-circ_0049472, and then this activation could be abrogated by the presence of anti-miR-22-3p or ectopic ZNF217 (Fig. 7).Moreover, flow cytometry showed that LY294002 (the inhibitor of PI3K-AKT pathway) reversed si-circ_0049472-indudced reduction of cell apoptosis rate in Aβ-treated cells (Supplementary Figure 2).These data concluded that interfering circ_0049472 could activate PI3K-AKT signaling pathway to regulate AD progression in Aβ-stimulated SK-N-SH cells.

Discussion
A previous microarray analysis screened out top 10 upregulated and top 10 downregulated circRNAs in the blood plasma samples of AD patients than age-matched controls (Liu et al., 2020); however, the researchers further focused on analyzing and confirming the downregulated circRNAs but let alone the upregulated ones.Here, we selected circ_0049472 for validation.Circ_0049472 is a circRNA generated from exons 4-18 from PRKCSH mRNA that codes beta-subunit of glucosidase II (Janssen et al., 2010;Kawaai et al., 2009).Aβ25-35 has been used to induce neurotoxicity in SK-N-SH cells for establishing AD cell model (Zhang et al., 2021;Gu et al., 2020).In this study, Aβ25-35 treatment was shown to reduce cell viability, proliferation but enhance apoptosis and inflammation in SK-N-SH cells, which demonstrated that Aβ25-35 induced significant cell neurotoxicity.We found that increased circ_0049472 was a contributing factor in Aβ aggregates-elicited damage in neurons, and its upregulation was dependent on Aβ dose, suggesting circ_0049472 as a potential indicator of the degree of Aβ deposits and even a biomarker for the diagnosis, staging, and progress of AD.RNA interference experiment revealed that inhibiting circ_0049472 could avoid Aβ-stimulated cytotoxicity, apoptosis and inflammatory factors release, implicating that targeting circ_0049472 might be a promising therapy for AD.Accidently, the circRNA derived from PRKCSH exons 2-3 also participated in inflammatory response and its silencing suppressed TNF-α-activated astrocytes inflammation by regulating CC chemokine CCL2 via miR-488 (Chen et al., 2022).Nevertheless, these emerging data documented a biological role of circPRKCSHs in the inflammatory response, even though this was not completely understood yet.
Besides, we noticed that miR-22-3p was sensitive to Aβ treatment and downregulated with the dose of Aβ.Previously, miR-22-3p was dysregulated in AD patients' serum and serum exosomes (Dong et al., 2021;Guo et al., 2017;Lu et al., 2021).Serum exosomal miR-22-3p was independently associated with AD and presented a certain diagnostic capability to distinguish AD from healthy subjects (Dong et al., 2021).Besides, serum miR-22-3p downregulation was also indicated to cause mild cognitive impairment of AD (Guo et al., 2017); more importantly, serum miR-22-3p could confer the best sensitivity and specificity among the 9 miRNAs-signature (Guo et al., 2017).Except for that, miR-146a and miR-181a upregulations in the blood were later published to involve in the progression of mild AD conversion within 2 years (Ansari et al., 2019).Here, functional experiment using its mimic showed that miR-22-3p overexpression went against to neuronal cell toxicity, apoptosis and inflammation under Aβ stimulation.As early documented, miR-22-3p was neuroprotective in multiple in vitro models for neurodegeneration partially through inhibiting caspase activation, improving cell viability and targeting pro-apoptotic genes (Jovicic et al., 2013).Similarly, we observed that apoptotic proteins Bax and cleaved-caspase-3 were consistently attenuated by restoring miR-22-3p via targeting and inhibiting oncogene ZNF217.Except for regulating ZNF217 node, miR-22-3p was predicted to fine-tune the expression of numerous network nodes of the senescence-accelerated mice affected with AD (Cheng et al., 2013).
Analogical to the existing literatures, our outcomes considered ZNF217 upregulation as the underlying molecular mechanism of Aβinduced apoptosis, inflammation and cell proliferation impairment in human neuronal cells via ceRNA networks (Wang et al., 2018;Gao et al., 2020;Wu et al., 2021).Furthermore, the promotion of oxidative stress and the reduction of mitochondrial membrane potential were coupled with ZNF217 (Wang et al., 2018).Therefore, Bax and cleaved-caspase-3 levels herein were highly enhanced by Aβ, implicating that Aβ induced a mitochondrial apoptotic pathway.

Table 1
The sequence of qPCR primers.