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Research Paper

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

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Pages 10512-10524 | Received 08 Sep 2021, Accepted 24 Oct 2021, Published online: 11 Dec 2021

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 [Citation1]. Nearly 85% of stroke cases might be attributed to cerebral ischemia [Citation2,Citation3]. 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 [Citation4]. 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 [Citation5]. Although numerous drugs are of neuroprotective capability, many of them fail to therapeutic effects in cerebral I/R (CI/R) treatment [Citation6]. 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 [Citation7]. Besides, DEX also exerts essential pharmacological effects on reducing apoptosis, diminishing inflammation, and relieving neuropathic pain [Citation8]. For example, Kang et al. revealed that DEX eliminated diabetes-induced neuropathic pain in mice by regulating P2X4 and NLRP3 expressions [Citation9]. Li et al. found that DEX reduced renal I/R-induced apoptosis via the α2 Adrenoceptor/PI3K/Akt signaling [Citation10]. He et al. demonstrated that DEX alleviated doxorubicin-mediated apoptosis and inflammation of myocardial cells [Citation11]. Recently, there is a heated topic on DEX-mediated neuroprotection in CI/R [Citation12–14]. 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 [Citation15–18]. To cite an instance, Zhang et al. disclosed circRNA CAMK4 aggravated CI/R injury by accelerating neuron cell death [Citation19]. Yang et al. revealed that circ_008018 exacerbated CI/R-induced neuronal cell apoptosis via regulating miR-99a [Citation20]. Liu et al. circ_002664 promoted CI/R-induced neuron cell apoptosis via regulating Herpud1 through interaction with miR-182-5p [Citation21]. 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 [Citation22], 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 [Citation23]. 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.

Materials and methods

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% CO2; 37°C) as per standard protocols. For DEX treatment, HT-22 cells were exposed to DEX (100 µM) for 24 h.

Establishment of hypoxia/reoxygenation (H/R) cell model

To construct an H/R cell model, HT-22 cells were cultivated in a low-oxygen atmosphere (94% N2 + 5% CO2 + 1% O2; 37°C) for 6 h. Thereafter, the HT-22 cells were transferred to fresh DMEM supplemented with 10% FBS and cultured in a regular incubator (95% O2 + 5% CO2; 37°C) for 6 h. Untreated HT-22 cells were used as the blank control group (Control group) [Citation24].

Cell transfection

Small interfering RNAs against circ-CDR1as (si-circ-CDR1as: 5′-UAAUGUGAGACGUCAUAGAAC-3′), TRAF3 (si-TRAF3: 5′-AUAGAGAAUAUAACCUGUCGA-3′), scramble control (si-NC: 5′-AUUGUAGAUAAUCAGCUUAAU-3′), pcDNA3.1 overexpression vector for circ-CDR1as (oe-circ-CDR1as), empty vector (Vector), miR-28-3p overexpression and inhibition plasmids (miR-28-3p mimics: 5′-UAGAUCACAGUCCUUUGUUAU-3′ and miR-28-3p inhibitor: 5′-AUCUAGUGUCAGGAAACAAAUA-3′), and corresponding negative controls (NC mimics: 5′-AUCUAGUCAGUCCUUUGUUUAU-3′ and NC inhibitor: 5′-AUCUAGUGUCAGGAAACAAAUA-3′) were provided by GenePharma (Shanghai, China) and transfected into HT-22 cells via Lipofectamine 2000 (Invitrogen, USA).

CCK-8 assay

CCK-8 assay was utilized for cell viability assessment. HT-22 cells were seeded into six-well plates (3 × 105 cells/well) and cultured for 24 h. Afterward, 10 μl CCK-8 reagent was added into each well. After incubation with CCK-8 reagent for 1 h, the absorbance (optical density) was measured with a microplate reader (Bio-Rad, USA) [Citation25].

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) [Citation26].

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 [Citation27].

RT-qPCR

Isolation of total RNA from HT-22 cells was performed via TRIzol (Invitrogen). Then, cDNA was generated with a reverse transcriptase kit (Takara, Japan) or Thermo Fisher’s K1622 kit (Thermo Fisher Scientific, USA). Afterward, qPCR was accomplished with SYBR-Green PCR Master Mix kit (Takara, China). Relative gene expression was evaluated by 2−ΔΔCt method, with GAPDH or U6 as the internal reference [Citation28]. The primers used were as follows: circ-CDR1as forward (F): 5′-GTGTCTCCAGTGTATCGGCG-3′ and reverse (R): 5′-TACTGGCACCACTGGAAACC-3′; TRAF3 F: 5′-CTTCCCGGGCTGTGATATTG-3′ and R: 5′-GGCTGTATCTGACCGCTAGG-3′; GAPDH F: 5′-ACCCACTCCTCCACCTTTGAC-3′ and R: 5′-TGTTGCTGTAGCCAAATTCGTT-3′; miR-28-3p F: 5′-CGCGCACTAGATTGTGAGCT-3′ and R: 5′-AGTGCAGGGTCCGAGGTATT-3′; U6 F: 5′-CTCGCTTCGGCAGCACATATACT-3′ and R: 5′-ACGCTTCACGAATTTGCGTGTC-3′.

Dual-luciferase assay

The 3′ UTR regions of TRAF3 and circ-CDR1as with binding or mutant sequences for miR-28-3p were cloned into pmirGLO luciferase vectors (Promega, USA) to synthesize wild-type plasmids (TRAF3-WT and circ-CDR1as-WT) or mutant-type plasmids (TRAF3-MUT and circ-CDR1as-MUT). The above plasmids were respectively transfected into HT-22 cells, together with NC mimics or miR-28-3p with Lipofectamine 2000 (Invitrogen). 48 h later, the luciferase activity was determined via the dual-luciferase reporter assay kit (Promega, USA) and normalized to Renilla luciferase activity.

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 ), 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 ()), 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 (). To sum up, DEX relieved H/R-induced apoptosis and inflammatory responses in HT-22 cells.

Figure 1. DEX relieves HT-22 cell dysfunction induced by H/R treatment. (a) Relative cell viability after H/R injury and DEX treatment was measured using CCK-8 assay. (b) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (c-e) TNF-α (d), IL-6 (e), and IL-1β (f) levels were measured by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 1. DEX relieves HT-22 cell dysfunction induced by H/R treatment. (a) Relative cell viability after H/R injury and DEX treatment was measured using CCK-8 assay. (b) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (c-e) TNF-α (d), IL-6 (e), and IL-1β (f) levels were measured by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001

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 down-regulated circ-CDR1as expression ()). 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 ()). 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 ()). 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. 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. (a) Circ-CDR1as expression in HT-22 cells after H/R injury and DEX treatment was detected via RT-qPCR. (b) Circ-CDR1as overexpression efficiency was evaluated by RT-qPCR. (c) Circ-CDR1as expression in Control group, H/R group, H/R+ DEX group, H/R+ DEX+Vector group, or H/R+ DEX+oe-circ-CDR1as group was evaluated by RT-qPCR. (d) cell viability was measured using CCK-8 assay. (e) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (f-h) TNF-α (g), IL-6 (h), and IL-1β (i) levels were measured by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 2. 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. (a) Circ-CDR1as expression in HT-22 cells after H/R injury and DEX treatment was detected via RT-qPCR. (b) Circ-CDR1as overexpression efficiency was evaluated by RT-qPCR. (c) Circ-CDR1as expression in Control group, H/R group, H/R+ DEX group, H/R+ DEX+Vector group, or H/R+ DEX+oe-circ-CDR1as group was evaluated by RT-qPCR. (d) cell viability was measured using CCK-8 assay. (e) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (f-h) TNF-α (g), IL-6 (h), and IL-1β (i) levels were measured by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001

MiR-28-3p directly binds to circ-CDR1as

StarBase predicted that circ-CDR1as contains a latent binding region for miR-28-3p ()). The dual-luciferase reporter assay was performed to verify the binding condition between miR-28-3p and circ-CDR1as. Firstly, miR-28-3p was overexpressed in HT-22 cells ()). The results showed that miR-28-3p upregulation inhibited the luciferase activity of circ-CDR1as-WT but almost had no effect on the luciferase activity of circ-CDR1as-MUT ()). RT-qPCR results revealed that miR-28-3p was lowly expressed in H/R-challenged HT-22 cells, while DEX upregulated miR-28-3p expression ()). Next, circ-CDR1as was knockdown in HT-22 cells ()). Furthermore, miR-28-3p expression was downregulated or overexpressed in HT-22 cells after circ-CDR1as overexpression or knockdown, respectively ()). These data showed that circ-CDR1as negatively regulated miR-28-3p expression.

Figure 3. MiR-28-3p directly binds to circ-CDR1as. (a) Binding site between circ-CDR1as and miR-28-3p. (b) MiR-28-3p overexpression efficiency was evaluated by RT-qPCR. (c) The binding relationship between circ-CDR1as and miR-28-3p was verified by dual-luciferase activity assay. (d) MiR-28-3p expression in HT-22 cells after H/R injury and DEX treatment was detected via RT-qPCR. (e) Circ-CDR1as knockdown efficiency was evaluated by RT-qPCR. (f) MiR-28-3p expression in HT-22 cells transfected with Vector, oe-circ-CDR1as, si-NC, or si-circ-CDR1as was detected by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 3. MiR-28-3p directly binds to circ-CDR1as. (a) Binding site between circ-CDR1as and miR-28-3p. (b) MiR-28-3p overexpression efficiency was evaluated by RT-qPCR. (c) The binding relationship between circ-CDR1as and miR-28-3p was verified by dual-luciferase activity assay. (d) MiR-28-3p expression in HT-22 cells after H/R injury and DEX treatment was detected via RT-qPCR. (e) Circ-CDR1as knockdown efficiency was evaluated by RT-qPCR. (f) MiR-28-3p expression in HT-22 cells transfected with Vector, oe-circ-CDR1as, si-NC, or si-circ-CDR1as was detected by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001

Circ-CDR1as expedited apoptosis and inflammatory responses of H/R-treated HT-22 cells via regulating miR-28-3p

To explore the regulating role of circ-CDR1as and miR-28-3p in H/R-induced HT-22 cell dysfunction, H/R-treated HT-22 cells were transfected with Vector, oe-circ-CDR1as, oe-circ-CDR1as+NC mimics, or oe-circ-CDR1as+miR-28-3p mimics, respectively. RT-qPCR assay revealed that miR-28-3p expression was decreased after circ-CDR1as overexpression and then increased after miR-28-3p addition ()). Next, we measured cell viability, apoptotic rate, and levels of pro-inflammatory cytokines in each group. It was revealed that circ-CDR1as upregulation significantly inhibited proliferation and increased apoptosis of H/R-treated HT-22 cells; while miR-28-3p upregulation partly reversed such phenomena (). In addition, the TNF-α, IL-6, and IL-1β levels in H/R-treated HT-22 cells were remarkably lifted after circ-CDR1as amplification and declined after miR-28-3p overexpression (). Taken together, circ-CDR1as exacerbated H/R-induced apoptosis and inflammatory responses via miR-28-3p in HT-22 cells.

Figure 4. Circ-CDR1as expedited apoptosis and inflammatory responses of H/R-treated HT-22 cells via regulating miR-28-3p. (a) HT-22 cells were subject to H/R treatment and then transfected with Vector, oe-circ-CDR1as, oe-circ-CDR1as+NC mimics, or oe-circ-CDR1as+miR-28-3p mimics, with untreated HT-22 cells as the Control group. MiR-28-3p expression in each group was evaluated by RT-qPCR. (b) cell viability was measured using CCK-8 assay. (c) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (d-f) TNF-α (e), IL-6 (f), and IL-1β (g) levels were measured by ELISA. *P < 0.05; **P < 0.01

Figure 4. Circ-CDR1as expedited apoptosis and inflammatory responses of H/R-treated HT-22 cells via regulating miR-28-3p. (a) HT-22 cells were subject to H/R treatment and then transfected with Vector, oe-circ-CDR1as, oe-circ-CDR1as+NC mimics, or oe-circ-CDR1as+miR-28-3p mimics, with untreated HT-22 cells as the Control group. MiR-28-3p expression in each group was evaluated by RT-qPCR. (b) cell viability was measured using CCK-8 assay. (c) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (d-f) TNF-α (e), IL-6 (f), and IL-1β (g) levels were measured by ELISA. *P < 0.05; **P < 0.01

MiR-28-3p targets TRAF3

Via 5 miRNA databases (microT, miRmap, PITA, PicTar, and TargetScan), 10 candidate downstream genes (MARCH6, RNF216, FAF2, ARF6, ZFP91, C15orf48, FUBP3, FAM168B, NR3C2, and TRAF3) were predicted for miR-28-3p ()). As Yao et al. disclosed that TRAF3 (tumor necrosis factor receptor-associated factor-3) expression was upregulated in neurons from hippocampus after cerebral ischemia [Citation29], TRAF3 was selected for subsequent experiments. The putative binding site between miR-28-3p and 3′UTR of TRAF3 was presented in ). Dual-luciferase activity assay manifested that miR-28-3p upregulation remarkably reduced the luciferase activity of TRAF3-WT, while the luciferase activity of TRAF3-MUT was nearly unchanged ()). RT-qPCR revealed that TRAF3 expression in HT-22 cells were significantly lifted after H/R and substantially declined after DEX treatment ()). Then, the efficiency of miR-28-3p inhibition was detected via RT-qPCR ()). TRAF3 expression were apparently decreased after miR-28-3p addition and distinctly increased after miR-28-3p inhibition; however, such phenomena were partly abrogated by circ-CDR1as amplification or circ-CDR1as silencing, respectively (). These results indicated that circ-CDR1as positively regulated TRAF3 expression in HT-22 cells via interaction with miR-28-3p, suggesting that circ-CDR1as exerting its effects via the miR-28-3p/TRAF3 pathway.

Figure 5. MiR-28-3p targets TRAF3. (a) Venn diagram of target mRNAs for miR-28-3p predicted by 5 databases (microT, miRmap, PITA, PicTar, and TargetScan). (b) Binding site between TRAF3 and miR-28-3p. (c) The binding relationship between TRAF3 and miR-28-3p was verified by dual-luciferase activity assay. (d) TRAF3 expression in HT-22 cells after H/R injury and DEX treatment were detected via RT-qPCR. (e) MiR-28-3p knockdown efficiency was evaluated by RT-qPCR. (f) TRAF3 expression in HT-22 cells transfected with NC mimics, miR-28-3p mimics, miR-28-3p mimics+Vector, or miR-28-3p mimics+oe-circ-CDR1as were detected by RT-qPC. (g) TRAF3 expression in HT-22 cells transfected with NC inhibitor, miR-28-3p inhibitor, miR-28-3p inhibitor+si-NC, or miR-28-3p inhibitor+si-circ-CDR1as were detected by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 5. MiR-28-3p targets TRAF3. (a) Venn diagram of target mRNAs for miR-28-3p predicted by 5 databases (microT, miRmap, PITA, PicTar, and TargetScan). (b) Binding site between TRAF3 and miR-28-3p. (c) The binding relationship between TRAF3 and miR-28-3p was verified by dual-luciferase activity assay. (d) TRAF3 expression in HT-22 cells after H/R injury and DEX treatment were detected via RT-qPCR. (e) MiR-28-3p knockdown efficiency was evaluated by RT-qPCR. (f) TRAF3 expression in HT-22 cells transfected with NC mimics, miR-28-3p mimics, miR-28-3p mimics+Vector, or miR-28-3p mimics+oe-circ-CDR1as were detected by RT-qPC. (g) TRAF3 expression in HT-22 cells transfected with NC inhibitor, miR-28-3p inhibitor, miR-28-3p inhibitor+si-NC, or miR-28-3p inhibitor+si-circ-CDR1as were detected by RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001

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 ()). As indicated by RT-qPCR, TRAF3 expression was significantly elevated after circ-CDR1as addition, but declined after TRAF3 depletion ()). 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. TRAF3 knockdown reverses the effect of circ-CDR1as overexpression on H/R-challenged HT-22 cells. (a) TRAF3 knockdown efficiency was evaluated by RT-qPCR. (b) HT-22 cells were subject to H/R treatment and then transfected with Vector, oe-circ-CDR1as, oe-circ-CDR1as+si-NC, and oe-circ-CDR1as+si-TRAF3, with untreated HT-22 cells as the Control group. TRAF3 mRNA and protein levels in HT-22 cells from each group were detected by RT-qPCR. (c) cell viability was measured using CCK-8 assay. (d) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (e-g) TNF-α (f), IL-6 (g), and IL-1β (h) levels were measured by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 6. TRAF3 knockdown reverses the effect of circ-CDR1as overexpression on H/R-challenged HT-22 cells. (a) TRAF3 knockdown efficiency was evaluated by RT-qPCR. (b) HT-22 cells were subject to H/R treatment and then transfected with Vector, oe-circ-CDR1as, oe-circ-CDR1as+si-NC, and oe-circ-CDR1as+si-TRAF3, with untreated HT-22 cells as the Control group. TRAF3 mRNA and protein levels in HT-22 cells from each group were detected by RT-qPCR. (c) cell viability was measured using CCK-8 assay. (d) Cell apoptosis rate after H/R injury and DEX treatment was measured by flow cytometry. (e-g) TNF-α (f), IL-6 (g), and IL-1β (h) levels were measured by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001

Discussion

DEX has not only effects on the hippocampus but also sensory processing, which involves the sensory cortex [Citation30]. Moreover, DEX significantly regulates the thalamus, the pulvinar nucleus in particular [Citation31], which plays a vital role in visual and auditory processing [Citation32,Citation33]. Moreover, accumulating evidence shows that DEX exerts significant neuroprotective effects in neurological diseases [Citation34–36]. In this work, the protective effect and regulatory mechanism of DEX on H/R-induced HT-22 cell dysfunction were further investigated. Our findings substantiated that DEX considerably attenuated H/R-induced apoptosis and inflammation in hippocampal neurons via the circ-CDR1as/miR-28-3p/TRAF3 cascade.

It has been widely recognized that circRNAs play crucial roles in the development and progression of neurological diseases, such as cerebral I/R [Citation37], Alzheimer’s disease (AD) [Citation38], Parkinson’s disease (PD) [Citation39], and Hirschsprung’s disease (HSCR) [Citation40]. 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 [Citation41]. Geng et al. revealed that circ-CDR1as contributed to apoptosis of hypoxia-treated mouse cardiac myocytes [Citation42]. Besides, Zhang et al. uncovered that circ-CDR1as also exacerbated inflammatory responses [Citation43]. 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) [Citation44]. Moreover, such a circRNA-miRNA-mRNA regulating network also exerts key effects in diverse neurological disorders, including cerebral I/R [Citation39,Citation45,Citation46]. 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 [Citation47]. Xing et al. disclosed that miR-374 targeted WNT5A to alleviate CI/R injury [Citation48]. Besides, Liu et al. revealed that miR-211 exerted protective effects on OGD/R-challenged PC12 cells via reducing cell apoptotic rate [Citation49]. MiR-28-3p is differently regulated in human diseases and deeply involved in the regulation of cellular functions [Citation50]. In addition, Fan et al. disclosed that miR-28-3p expression was negatively related to IL-1β level in colorectal cancer [Citation51]. 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 [Citation52]. Sun et al. disclosed that TRAF3 upregulation substantially enhanced the inflammatory responses of caerulein-induced AR42J cells [Citation53]. Liu et al. revealed that TRAF3 promoted apoptosis and inflammatory responses of oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells [Citation54]. Zhang et al. found that TRAF3 impaired the proliferation of MDA-MB-231 cells via inhibiting miR-29b-3p [Citation55]. Also, Liu et al. demonstrated that TRAF3 aggravated cardiac I/R-induced apoptosis and inflammation [Citation56]. 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.

Research highlights

1. DEX attenuated H/R-induced dysfunctions of HT-22 cells by regulating circ-CDR1as

2.circ-CDR1as upregulated TRAF3 expression by targeting miR-28-3p

3.circ-CDR1as promoted dysfunction of H/R-induced HT-22 cells via miR-28-3p/TRAF3 axis

Disclosure statement

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

Additional information

Funding

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

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