Circular RNA cerebellar degeneration-related protein 1 antisense RNA (Circ-CDR1as) downregulation induced by dexmedetomidine treatment protects hippocampal neurons against hypoxia/reoxygenation injury through the microRNA-28-3p (miR-28-3p)/tumor necrosis factor receptor-associated factor-3 (TRAF3) axis

ABSTRACT Cerebral ischemia/reperfusion (CI/R) injury results in serious brain tissue damage, thereby leading to long-term disability and mortality. It has been reported that dexmedetomidine (DEX) exerted neuroprotective effects in CI/R injury. Herein, we intended to investigate whether and how circular RNA (circRNA) cerebellar degeneration-related protein 1 antisense RNA (circ-CDR1as) was involved in the DEX-mediated protection on hippocampal neurons. In our work, the mouse hippocampal neuronal cells (HT-22) were used to construct a hypoxia/reperfusion (H/R) model for CI/R injury. Cell proliferation and apoptosis were evaluated by CCK-8 and flow cytometry. Gene expressions were detected by RT-qPCR. Levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) were measured by ELISA. The association between miR-28-3p and circ-CDR1as or TRAF3 was verified by dual-luciferase assay. The results indicated that DEX alleviated HT-22 cell dysfunction induced by H/R treatment. In addition, circ-CDR1as was downregulated after DEX treatment and reversed the effects of DEX on the proliferation, apoptosis, and inflammatory responses of H/R-treated HT-22 cells. Circ-CDR1as positively regulated TRAF3 expression via interaction with miR-28-3p in HT-22 cells. Circ-CDR1as aggravated H/R-treated HT-22 cell dysfunction through targeting miR-28-3p. Furthermore, TRAF3 inhibition partly abolished the effect of circ-CDR1as overexpression on cellular activities of H/R-treated HT-22 cells. To sum up, our findings, for the first time, demonstrated that DEX exerted neuroprotective effects on hippocampal neurons against H/R treatment via the circ-CDR1as/miR-28-3p/TRAF3 regulatory network, providing novel therapeutic targets for DEX administration in CI/R treatment.


Introduction
Stroke, an acute cerebrovascular disorder, is a leading cause of long-term disability and mortality in adults worldwide [1]. Nearly 85% of stroke cases might be attributed to cerebral ischemia [2,3]. At present, the most effective method for ischemia stroke treatment in clinical practice is thrombolytic therapy through which blood supply to brain tissues can be timely restored, thereby ameliorating ischemic stroke-induced brain injury [4]. However, sudden resumption of blood supply after cerebral ischemia may induce a succession of pathological reactions, such as aggravated apoptosis and inflammatory responses in neurons, and even cause secondary injury to local brain tissues, which is known as cerebral ischemia/reperfusion (I/R) injury [5]. Although numerous drugs are of neuroprotective capability, many of them fail to therapeutic effects in cerebral I/R (CI/R) treatment [6]. Hence, it is of great significance to find new therapeutic approaches for CI/R therapy. Dexmedetomidine (DEX), an activator of Alpha2-adrenoceptor, is a widely applied anesthetic drug for sympathetic activity depression, analgesia, and sedation in clinical anesthesia, without causing respiratory depression [7]. Besides, DEX also exerts essential pharmacological effects on reducing apoptosis, diminishing inflammation, and relieving neuropathic pain [8]. For example, Kang et al. revealed that DEX eliminated diabetesinduced neuropathic pain in mice by regulating P2X4 and NLRP3 expressions [9]. Li et al. found that DEX reduced renal I/R-induced apoptosis via the α 2 Adrenoceptor/PI3K/Akt signaling [10]. He et al. demonstrated that DEX alleviated doxorubicin-mediated apoptosis and inflammation of myocardial cells [11]. Recently, there is a heated topic on DEX-mediated neuroprotection in CI/R [12][13][14]. However, the pharmacological action of DEX in CI/R still needs further investigation. Hence, a deeper understanding of the underlying mechanisms of DEX in CI/R is imperative for the better improvement of DEX application in CI/R treatment.
Circular RNAs (circRNAs), a group of newly identified RNAs with covalent closed-loop structures, play vital roles in several diseases, including CI/R [15][16][17][18]. To cite an instance, Zhang et al. disclosed circRNA CAMK4 aggravated CI/R injury by accelerating neuron cell death [19]. Yang et al. revealed that circ_008018 exacerbated CI/R-induced neuronal cell apoptosis via regulating miR-99a [20]. Liu et al. circ_002664 promoted CI/R-induced neuron cell apoptosis via regulating Herpud1 through interaction with miR-182-5p [21]. As reported in a study by Quan et al., circRNA cerebellar degeneration-related protein 1 antisense (circ-CDR1as) was highly expressed in PD and cause cell damage in vitro [22], indicating its promoting role in neurological disorder. Nevertheless, the functions of circ-CDR1as in CI/R remains poorly understood.
In this study, a hypoxia/reoxygenation (H/ R)-induced neuronal cell model serves as an effective tool for the research on the cellular dysfunction caused by CI/R injury [23]. It was hypothesized that DEX exerted protective effects on H/R-induced hippocampal neuron cells via regulating circ-CDR1as. Herein, we for the first time explored the specific role of circ-CDR1as/miR-28-3p/TRAF3 competing endogenous RNA (ceRNA) network in the DEX-mediated protection against H/R-induced hippocampal neuronal dysfunction, thereby providing novel molecular targets for CI/R treatment with DEX.

Cell culture and DEX treatment
Mouse hippocampal neuronal cells (HT-22) purchased from BeNa Culture Collection (Beijing, China) were cultured in DMEM supplemented with 10% FBS in an incubator (5% CO 2 ; 37°C) as per standard protocols. For DEX treatment, HT-22 cells were exposed to DEX (100 µM) for 24 h.

Flow cytometry
Flow cytometry was applied for the analysis of HT-22 cell apoptosis via the Annexin V-FITC Apoptosis Detection Kit (eBioscience, USA). HT-22 cells in each group were centrifuged at 400 × g for 5 min, rinsed 3 times with PBS, and then incubated with 5 μL Annexin V-FITC reagent and 10 μL PI reagent (Sigma-Aldrich, USA) for 15 min at room temperature in darkness. The apoptotic HT-22 cells were analyzed with a flow cytometer (Beckman Coulter, China) [26].

ELISA
In order to determine TNF-α, IL-6, and IL-1β levels in HT-22 cells, ELISA assays were performed with corresponding ELISA assay kits (Mlbio, China) according to the standard protocol [27].

Statistical analysis
The data were expressed as mean ± standard deviation (SD). Each experiment was conducted three times. Student's t test or one-way ANOVA was applied to accomplish difference comparison between two or multiple groups. Statistical analysis was performed using GraphPad Prism 6.0. A difference with P < 0.05 was deemed statistically significant.

Results
In this work, we intended to investigate the role and molecular mechanism of DEX in H/ R-challenged hippocampal neuronal dysfunction. Our results demonstrated that DEX exerted neuroprotective effects on hippocampal neurons against H/R treatment via the circ-CDR1as/miR-28-3p/TRAF3 regulatory network, providing novel therapeutic targets for DEX administration in CI/ R treatment.

DEX relieves HT-22 cell dysfunction induced by H/ R treatment
To discover the possible effects of DEX treatment on H/R-induced HT-22 cells, we treated HT-22 cells with DEX after H/R treatment. As shown in Figure 1(a), HT-22 cell viability prominently dropped after H/R treatment, while DEX treatment everted an ameliorative effect on cell viability. In addition, it was discovered that HT-22 cell apoptosis rate was significantly increased after H/R treatment, while DEX led to an opposite result (Figure 1(b)), indicating that DEX reduced cell apoptosis. Moreover, the TNF-α, IL-6, and IL-1β levels were increased after H/R treatment, whereas DEX remarkably reversed such a phenomenon ( Figure 1(c-e)). To sum up, DEX relieved H/ R-induced apoptosis and inflammatory responses in HT-22 cells.

Circ-CDR1as is down-regulated after DEX treatment and reverses the effects of DEX on H/ R-treated HT-22 cell proliferation, apoptosis, and inflammation
It was found that circ-CDR1as expression was markedly increased in HT-22 cells subject to H/R treatment; on the contrary, DEX led to downregulated circ-CDR1as expression (Figure 2(a)). To further discover the potential functions of circ-CDR1as in DEX-mediated neuroprotection against H/R treatment, cell viability, apoptosis, and inflammatory responses of HT-22 cells were detected in each group. Firstly, circ-CDR1as was overexpressed in HT-22 cells (Figure 2(b)). As indicated by RT-qPCR assay, circ-CDR1as expression was evidently up-regulated by H/R, remarkably decreased by DEX, and increased again by circ-CDR1as overexpression (Figure 2(c)). Functional assays exhibited that circ-CDR1as upregulation reversed the effect of DEX on cellular processes of HT-22 cells, as indicated by decreased cell viability, elevated apoptotic rate, as well as increased inflammatory responses (Figure 2(d-h)).

TRAF3 knockdown reverses the effect of circ-CDR1as overexpression on H/R-challenged HT-22 cells
To further probe the role of TRAF3 in H/ R-mediated damage to HT-22 cells, H/R-induced HT-22 cells were transfected with Vector, oe-circ-CDR1as, oe-circ-CDR1as+si-NC, and oe-circ-CDR1as+si-TRAF3, respectively. First of all, TRAF3 was knocked down in H/R-induced HT-22 cells (Figure 6(a)). As indicated by RT-qPCR, TRAF3 expression was significantly elevated after circ-CDR1as addition, but declined after TRAF3 depletion (Figure 6(b)). Functional assays exhibited that TRAF3 knockdown partly neutralized the effects of circ-CDR1as overexpression on viability, apoptosis, and inflammatory responses of HT-22 cells subject to H/R treatment ( Figure 6(c-g)).
Circ-CDR1as, also known as ciRS-7, is deeply involved in the biological processes of cells, including cell viability and apoptosis. For example, Mao et al. found that circ-CDR1as inhibited the proliferation of bone microvascular endothelial cells by regulating FIH-1 via interaction with miR-135b [41]. Geng et al. revealed that circ-CDR1as contributed to apoptosis of hypoxia-treated mouse cardiac myocytes [42]. Besides, Zhang et al. uncovered that circ-CDR1as also exacerbated inflammatory responses [43]. In this study, we found that DEX exerted protective effects on hippocampal neuronal cells against H/R treatment, which was closely related to the downregulation of circ-CDR1as expression. However, circ-CDR1as overexpression could markedly weaken the protective effect of DEX on H/R-induced apoptosis and inflammation in HT-22 cells. Hence, DEX could attenuate H/ R-induced dysfunctions in hippocampal neurons via down-regulating circ-CDR1as.
Emerging evidence has demonstrated that circRNAs can post-transcriptionally regulate the transcription and translation of messenger RNAs (mRNAs) as endogenous competitive RNAs for specific microRNAs (miRNAs) [44]. Moreover, such a circRNA-miRNA-mRNA regulating network also exerts key effects in diverse neurological disorders, including cerebral I/R [39,45,46]. Interestingly, our data demonstrated that circ-CDR1as sponged miR-28-3p as a ceRNA and miR-28-3p targeted TRAF3. Previous studies demonstrated that miRNAs also play important roles in CI/R-induced nerve damage. For instance, a report from Ma et al. showed that miRNA-589 protected against CI/R-induced inflammatory responses in primary cortical neurons via mediating TRAF6 [47]. Xing et al. disclosed that miR-374 targeted WNT5A to alleviate CI/R injury [48]. Besides, Liu et al. revealed that miR-211 exerted protective effects on OGD/R-challenged PC12 cells via reducing cell apoptotic rate [49]. MiR-28-3p is differently regulated in human diseases and deeply involved in the regulation of cellular functions [50]. In addition, Fan et al. disclosed that miR-28-3p expression was negatively related to IL-1β level in colorectal cancer [51]. In the present study, we found that miR-28-3p was downregulated in H/ R-induced HT-22 cells, while DEX increased miR-28-3p expression. Further experiments revealed that miR-28-3p could partially abolish the effects of circ-CDR1as overexpression on H/R-induced HT-22 cells by enhancing proliferation, reducing apoptosis, and impairing the secretion of pro-inflammatory cytokines. Therefore, circ-CDR1as aggravated cellular dysfunction of H/R-induced HT-22 cells via interaction with miR-28-3p.
As a member of the TRAF adaptor protein family, TRAF3 exerts critical functions in regulating cellular activities in multiple diseases [52]. Sun et al. disclosed that TRAF3 upregulation substantially enhanced the inflammatory responses of caeruleininduced AR42J cells [53]. Liu et al. revealed that TRAF3 promoted apoptosis and inflammatory responses of oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells [54]. Zhang et al. found that TRAF3 impaired the proliferation of MDA-MB-231 cells via inhibiting miR-29b-3p [55]. Also, Liu et al. demonstrated that TRAF3 aggravated cardiac I/R-induced apoptosis and inflammation [56]. Consistent with the above findings, TRAF3 expression was significantly increased in HT-22 cells after H/R treatment and remarkably after DEX administration. Rescue experiments showed that the promoting effects of circ-CDR1as upregulation on cellular dysfunction of H/R-treated HT-22 cells were partly abrogated by TRAF3 silencing. Taken together, circ-CDR1as upregulated TRAF3 expression, thereby promoting apoptosis and inflammatory responses in H/R-treated HT-22 cells.

Conclusion
In summary, this work demonstrated that DEX exerted neuroprotective effects against H/ R-induced HT-22 cell dysfunction through regulating the circ-CDR1as/miR-28-3p/TRAF3 cascade. This study explored the neuroprotective effects and potential mechanisms of DEX in hippocampal neuron damage induced by cerebral I/ R, providing a theoretical basis and certain targets for DEX application in cerebral I/R. In the future, in vivo experiments should be performed to further confirm the role of circ-CDR1as/miR-28-3p/TRAF3 axis in CI/R injury.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.