Circular RNA Rap1b promotes Hoxa5 histone acetylation by recruiting Kat7 to inhibit neuronal apoptosis in acute ischemic stroke

doi:10.21203/rs.3.rs-1622225/v1

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Circular RNA Rap1b promotes Hoxa5 histone acetylation by recruiting Kat7 to inhibit neuronal apoptosis in acute ischemic stroke

https://doi.org/10.21203/rs.3.rs-1622225/v1

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Stroke is a common neurological disease and one of the major causes of permanent morbidity and disability worldwide. Although many drugs have shown significant neuroprotective effects in animal models, these results have not been confirmed in human clinical trials. Therefore, search for new treatments is urgent. In human patients and animal stroke models, acute ischemic primary neurons with blocked arterial blood supply die rapidly, while core and surrounding acute ischemic primary neurons that are infused by other arteries undergo delayed apoptosis to some extent. In the present study, we found that circRAP1B was downregulated in patients with acute ischemia stroke (AIS), and it was validated that circRap1b was downregulated in the hippocampus tissues and neurons HT22 cells of C57BL/6J mice treated with acute ischemia-hypoxia (AIS). Moreover, overexpression of circRAap1b inhibited the apoptosis of HT22-AIS cells. We attempted to prove that neuronal apoptosis inhibited by circRap1b/Hoxa5 in vitro might be ascribed to Kat7-induced acetylation of histones H3 Lysine 14 modification in the Hoxa5 promoter region. Promisingly, we hope that circRap1b and Hoxa5 will provide new treatment strategy for stroke.

circRNAs

epigenetics

stroke

ischemia

Stroke is a common neurological disease and one of the major causes of permanent morbidity and disability worldwide [1, 2]. The two main types of stroke are acute ischemic stroke (AIS) and hemorrhagic stroke. The currently FAD-approved treatment for AIS is intravenous injection of recombinant human tissue plasminogen activator within 3 hours after symptom onset, but due to the narrow time window of recombinant tissue plasminogen activator and the risk of intracranial hemorrhage and angioedema, only about 7% of patients meet the treatment conditions, and the efficacy of thrombolysis therapy for ischemic stroke is limited [3–7]. In human patients and animal stroke models, acute ischemic primary neurons with blocked arterial blood supply die rapidly, while core and surrounding acute ischemic primary neurons that are infused by other arteries undergo delayed apoptosis to some extent [8, 9]. Although many drugs have shown significant neuroprotective effects in animal models, these results have not been confirmed in human clinical trials[10]. Given the complexity of ischemic brain injury and its systemic consequences, cell therapy is more effective by targeting multiple mechanisms than monotherapy[11, 12].

Circular RNAs (circRNAs) are a new class of non-coding RNAs, a class of single-stranded, stable RNAs with a covalent bond between the 3' and 5' ends, forming a circular structure without polyadenylation tail and are not affected by RNA exonuclease [13–15]. Recent studies have shown that circRNAs are highly conserved and abundantly expressed in human cells [13, 16, 17]. Importantly, recent studies have found that the expression of circRNAs is tissue-specific and regulated by different biological processes, such as stroke, neuroinflammation and aging, cardiac hypertrophy and heart failure, and cell growth [18–23]. CircRNAs can regulate gene expression at the transcriptional and post-transcriptional levels through miRNA interactions [13]. Heart-related circRNAs can act as endogenous sponges for miR-223 [22], preventing heart failure and cardiac hypertrophy. CircRNA ciRS-7 regulates the pathophysiological process of Parkinson's disease through the adsorption function of miRNA-7 [24]. Recent studies have shown that the expression profiles of circRNAs in transient focal ischemia neurons (HT22) and mouse brains are significantly altered after hypoxia/reoxygenation [25–27].

High-throughput sequencing found that a large number of circRNAs were significantly differentially expressed in peripheral blood mononuclear cells of 28 patients with acute ischemic stroke, among which circRAP1B was significantly under-expressed. Rap1 is a small GTPase in the Ras family that combines extracellular stimuli with intracellular effectors and the resulting biological responses by cycling between inactive GDP and active GTP-bound states. Rap1a and Rap1b, two members of the Rap1 family, are encoded by different genes but share 95% of the same amino acid homology [28]. Members of this family regulate a variety of cellular processes, including cell adhesion, growth and differentiation. RAP1B is localized to the cell membrane and has been shown to regulate integrin-mediated cell signaling; it also plays a role in regulating platelet outside-in signaling. However, the functional role of circRAP1B has not been reported yet.

In vitro studies were conducted using HT22 neurons in the hippocampus of C57BL/6J mice, and after acute ischemia-hypoxia (AIS) treatment (No serum medium, O₂ concentration of 3%, 37°C incubator for 24 h) it was verified that the expression level of circRap1b was significantly downregulated, and the apoptosis rate of HT22 was significantly increased, and the expression level of neuronal apoptosis-related protein Fam3a was significantly decreased.

Further research found that overexpression of circRap1b led to the most significant increase in the expression of transcription factor Hoxa5 among the differentially expressed proteins. Transcription factors encoded by the hox gene family help determine the fate of neurons [29]. Specifically, Hoxa5 was recently shown to be involved in neuronal differentiation of autonomic functional nuclei in mouse brains [30] and the axonal growth of the posterior vagus motor neurons [31]. Hoxa5 is a crucial regulator of neuronal inflammation between sevoflurane and Gm5106 [32]. HOXA5 is also involved in neuroinflammation by interacting with the TGF signaling pathway [33]. Rosa Hernández et al. identified Hoxa5 as an important gene for neuronal differentiation of hASCs and thus a potential candidate gene for the development of cell therapy strategies in neurological diseases [34]. There are few other studies of Hoxa5 in neurological injury disease-stroke, so this study selected Hoxa5 as a candidate research object.

Lysine acetyltransferase 7 (KAT7), a member of the MYST domain family, plays a crucial role in transcription and DNA replication by acetylating histones H3 Lysine 14 (H3K14ac) and H4 Lysine 5, 8 and 12 (H4K5ac, H4K8ac, H4K12ac)[35, 36]. KAT7 is a driver of cellular senescence[37]. KAT is essential for the maintenance and self-renewal of adult hematopoietic stem cells [38]. KAT7 promotes efficient germination of tip cells during angiogenesis [39]. Overexpression of KAT7 attenuated cigarette extract-induced emphysema changes and lung cell apoptosis in mice with chronic obstructive pulmonary disease [40].

Family 3A with sequence similarity (FAM3A) is a mitochondrial protein that plays an important role in cell adaptation to stress and cell survival [41] and is involved in the regulation of neuronal apoptosis[42]. FAM3A has been reported to inhibit IL-1β-induced chondrocyte apoptosis by activating the pro-survival PI3K/Akt/mTOR pathway [41]. FAM3A can improve hypoxic-ischemic brain injury in neonatal rats [43]. In this study, it was found that there are 44 potential binding sites for HOXA5 in the promoter region − 3000 ~ 0bp of human FAM3A and four potential binding sites for Hoxa5 were found in the promoter region-3000 ~ 0bp of mouse Fam3a using the bioinformatics database JASPAR.

In the present study we attempted to prove that neuronal apoptosis inhibited by circRap1b/Hoxa5 in vitro might be ascribed to Kat7-induced H3K14ac modification in the Hoxa5 promoter region. Additionally, hope that circRap1b and Hoxa5 will provide new treatment strategy for stroke.

HT22 cell culture, AIS treatment

The mouse hippocampal neuronal cell line HT22 was purchased from Procell Life Science & Technology Co, Ltd (Wuhan, China). The cells were cultured in DMEM (HyClone, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in a cell humidified Calorstat with a condition of 37 °C, 5% CO₂ and 95% air. HT22 cells were treated to acute ischemia-hypoxia (AIS), in brief, the culture medium was replaced by glucose-free medium and cells were cultured for 24 h in a tri-gas incubator (SANYO, Osaka, Japan) with a humidified condition of at 37 °C 3% O₂ and 5% CO₂. As normal, the previous culture medium was replaced by fresh DMEM medium for 24 h in a humidified condition of 37 °C, 95% air and 5% CO₂.

Permanent MCAO (pMCAO) models and experimental design

pMCAO was processed according to a previous study [44] utilizing the adult male C57BL/6J mice of aged 12–14 weeks (weighing 24–30 g). For more details, see Supplemental Materials and Methods.

Microarray Analysis of Human circRNAs Expression Profile

Sample preparation, microarray hybridization and the expression profile of circRNAs analysis were carried out with the help of Kangchen Biotech (Shanghai, P.R. China) utilizing an Agilent chip platform.

Quantitative Real-Time PCR

The total RNA in cells was extracted by Trizol, and the purity and concentration of RNA were detected by NanoDrop 2000 ultra-micro spectrophotometer. The primers are listed in Table S1. For details, see Supplemental Materials and Methods.

Western Blot

Firstly, prepare the protein lysate according to the ratio of PMSF:RIPA=1:100; cells were added an equal volume of lysate to extract protein. For details, see Supplemental Materials and Methods.

FISH

To investigate circRap1b localization in HT22 cells, FISH assays were carried out with circRap1b probe (5′-CCTTGAACAAACTGTACAGT-3′) as previously described [45]

Cell Transfections

CircRap1b overexpression (circRap1b(+)) plasmids and its corresponding negative control plasmids were constructed using pCDH-CMV-MCS-EF1-Puro vector (Geneseed Biotech, Guangzhou, China); the plasmids of Hoxa5 and Fam3a overexpression (Hoxa5(+) and Fam3a(+))were synthesized and constructed (GenePharma, Shanghai, China); siRNA of Hoxa5, Fam3a and Kat7 (Hoxa5(-), Fam3a(-) and siKat7) plasmids and their corresponding negative control (NC) plasmids were constructed by GeneChem (Shanghai, China); For details, see Supplemental Materials and Methods.

RNA Pull-Down

The procedure of the assays was performed as previously described[45]. For details, see Supplemental Materials and Methods.

RIP Assays

The procedure of the assays was performed as previously described[45]. For details, see Supplemental Materials and Methods.

Flow Cytometry

The apoptosis of HT22 cells was assessed by ApoScreen Annexin 7AAD/PE kit (Southern Biotech, USA). For details, see Supplemental Materials and Methods.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) analysis

As previously described[42] that Neuronal apoptosis in the hippocampal CA1 region was assessed by terminal deoxynucleotide transferase (TdT) -mediated DUTP-biotin-notched-end labeling (TUNEL) according to the manufacturer's protocol of The One Step TUNEL Apoptosis Assay Kit (Beyotime, Jiangsu, China).

Reporter Vectors Construction and Luciferase Reporter Assay

As previously described[46], promoter region -3000~0bp of Fam3a was amplified by PCR and cloned into the pmirGLO of Dual Luciferase Expression Vector (Promega, Madison, WI, USA) to construct wild-type and mutation-type luciferase reporter vectors (Generay Biotech Co., Shanghai, China). For details, see Supplemental Materials and Methods.

ChIP Assay

The procedure of the assays was performed as previously described[45]. The primers were provided in Table S2-S3. For details, see Supplemental Materials and Methods.

ChIRP

The procedure of the assays was performed as previously described [47].The probes were provided in Table S4. For details, see Supplemental Materials and Methods.

Statistical Analysis

All data in this study were analyzed by GraphPad Prism v7.0 (GraphPad, CA, USA) and expressed as mean ± standard deviation (SD), and T-test or one-way analysis of variance (ANOVA) test was used between groups. If P<0.05, the difference was considered statistically significant.

CircRap1b is down-expressed in the hippocampus and neuronal cells of mice with acute ischemic stroke, and inhibits neuronal apoptosis

Microarray Analysis revealed a large number of significantly differentially expressed circRNAs in peripheral blood monocytes of 28 patients with acute ischemic stroke (AIS), among which circRAP1B was significantly under-expressed (Fig.1Sa) The acute ischemia model (mouse AIS and HT22-AIS) cells were constructed by using C57BL/6J mice and hippocampal neuron cells HT22 respectively for in vivo and in vitro studies. The apoptosis of hippocampal CA1 neurons in sham and AIS groups was detected by HE staining and TUNEL (Fig.1a). As the Fig.1a shown, HE staining showed that neurons in the hippocampal CA1 area appeared atrophy and nuclear pyknosis; TUNEL analysis showed that the apoptosis rate of acute ischemia (AIS) group was significantly increased (Fig.1b-c, the nuclei were blue, TUNEL staining positive cells (apoptotic cells) were shown in green, and the apoptotic rate = the number of TUNEL-positive neurons in the CA1 area/the total number of neurons in the CA1 area). And it was verified that the expression level of circRap1b was significantly decreased in mouse AIS hippocampus and HT22-AIS cells by quantitative real-time PCR (qRT-PCR) (Fig. 1d-e, h-i). Flow cytometry analysis of HT22-AIS cells apoptosis rate was significantly increased in the HT22-AIS cells treated with circRap1b plasmids and empty vector negative control (NC) (Fig. 1f-g), and circRap1b was shown to inhibit HT22-AIS cells apoptosis (Fig. 1j-k).

Hoxa5 is down-expressed in mouse acute ischemic hippocampus tissues and neurons HT22-AIS cells and inhibits their apoptosis

After overexpression of circRap1b in HT22-AIS cells, the mRNA expression profile of proteins was detected, and it was found that the expression of transcription factor Hoxa5 was the most significantly increased among the differentially expressed proteins (Fig. 1Sb). As shown in Fig. 2a-c, Hoxa5 mRNA and protein expression levels were significantly decreased in mouse AIS hippocampus tissues and neurons HT22-AIS cells by qRT-PCR and Western blot. Further, flow cytometry analysis showed that the apoptosis rate of HT22-AIS cells was significantly decreased after Hoxa5 overexpression, and the apoptosis rate of HT22-AIS cells was significantly increased after Hoxa5 knockdown (Fig. 2d-e).

CircRap1b induces H3K14ac modification in the Hoxa5 promoter region by recruiting the acetyltransferase Kat7, thereby promoting the transcription of Hoxa5

After overexpression of circRap1b in HT22-AIS cells, the Hoxa5 mRNA level increased (Fig. 3a). To further explore the mechanism by which circRap1b regulates Hoxa5 transcription, we first performed FISH assays to determine the localization of circRap1b in HT-22 cells. FISH results showed that circRap1b (green) was mainly present in the nucleus (Fig. 3b). Kat7 is often involved in transcriptional regulation, we hypothesized that Kat7 may take participate in the transcription regulation of circRap1b to Hoxa5. Furtherly, RNA pull-down assays were carried out, and the results showed that Kat7 was bound to circRap1b (Fig. 3c). Consistently, the results of Fig. 3d shown that the enrichment of U1 in anti-SNRNP70 group was significantly increased in RIP assays, which proved the effectiveness of this experimental operation; and the enrichment of circRap1b in the anti-Kat7 group was significantly increased, compared with the negative control IgG group and the negative RNA control GAPDH group (Fig. 3e), which further verified the binding of circRap1b to Kat7. Further, we divided the Hoxa5 promoter region -3000~0 bp into six segments (P1-P6), and designed primers (Table S5), used H3K14ac, H4K5ac and H4K12ac antibodies for chromatin immunoprecipitation (ChIP) assays, and it was found that the H3K14ac modification was present in promoter region -1000~0bp (P2 and P1) (Fig. 3f), but no H4K5ac and H4K12ac modification (Fig. S2c-d); further we used qRT-PCR to amplify the immunoprecipitated DNA fragments, the enrichment of H3K14ac in the P1 and P2 regions was detected, as shown in Fig. 3g, compared with the negative control IgG, the enrichment of H3K14ac was significantly increased, and the enrichment of H3K14ac in the P1 region was higher. To further prove that circRap1b and Kat7 jointly regulate H3K14ac modification in the Hoxa5 promoter region, we used circRap1b plasmids and its negative control circ-NC plasmids, small interfering RNA siKat7 plasmids and its negative control siNC plasmids to transiently transfect HT22-AIS. The HT22-AIS cell lines of circ-NC+siNC, circ+siNC, and circ+siKat7 were obtained, and the enrichment of H3K14ac in each group was detected by ChIP-PCR, as shown in Fig. 3h-i, compared with circ-NC+siNC, the H3K14ac enrichment of circ+siNC group was significantly increased, but compared with circ+siNC group , the enrichment of H3K14ac in the circ+siKat7 group was significantly decreased, and the above results indicated that knockdown of Kat7 could attenuate the effect of circRap1b on the increase of H3K14ac level in the Hoxa5 promoter region; similarly, the Hoxa5 mRNA and protein expression levels changed accordingly (Fig. 3j-k). We further demonstrated the interaction between H3K14ac and circRap1b using RNA pull-down (Fig. 3l). Next, we performed RNA purification isolated chromatin (ChIRP) assays, 20%–40% of circRap1b retrieval was obtained using tiling probes from the RNA fraction recovered, the promoter region 500–1000 bp of Hoxa5 was obtained 50% retrieval, and the promoter region 0-500bp was obtained 20% retrieval from the DNA fraction recovered (Fig.3m-n).

Hoxa5 regulates neuronal apoptosis by transcriptionally activating the expression of Fam3a

Fam3a is a neuronal apoptosis-related protein, as Fig.4a-c shown, it was found that Fam3a mRNA and protein expression levels were significantly decreased in mouse AIS hippocampus tissues and neurons HT22-AIS cells. Furtherly, the results of flow cytometry displayed, the apoptotic rate of HT22-AIS was significantly decreased after overexpression of Fam3a, and the apoptotic rate of HT22-AIS was significantly increased after Fam3a knockdown (Fig. 4d-e). Importantly, the Fam3a mRNA and protein were increased and decreased (Fig. 4f-h) following Hoxa5 overexpression and knockdown, respectively. Further, to explore the mechanism of Hoxa5 regulation of Fam3a transcription, there were four putative binding sites for Hoxa5 were predicted in the promoter region -3,000 to 0 bp of Fam3a using the bioinformatics database JASPAR, three of which were in the antisense strand, one of which were in the sense strand. The ChIP and luciferase reporter gene system assays were performed, and the results showed that Hoxa5 bound to the binding site in the -1000-500 bp of Fam3a promoter, and Hoxa5 could activate Fam3a transcription (Fig. 4i-j), and make the Fam3a expression increased.

CircRap1b regulates the expression of Fam3a through Hoxa5, and then regulates neuronal apoptosis

The expression of circRap1b and Hoxa5 in HT22-AIS model and their effects on apoptosis have been determined. To further clarify the mechanism of circRap1b and Hoxa5 co-regulating HT22-AIS apoptosis, circRap1b(+) and Hoxa5(-) and their negative control plasmids were co-transfected in HT22-AIS cell model, cell apoptosis rate was further detected by flow cytometry. The results showed that overexpression of circRap1b inhibited cell apoptosis rate, knocking down Hoxa5 promoted cell apoptosis, and knocking down Hoxa5 reversed the inhibitory effect circRap1b overexpression alone on cell apoptosis (Fig.5a-b). How did circRap1b and Hoxa5 affect cell apoptosis, and we detected their effects on the expression of neuronal apoptosis-related protein Fam3a. As shown, after overexpressing circRap1b, the protein expression level of Fam3a was raised (Fig. 5c-d); the level of protein expression of Hoxa5 was also raised after circRap1b overexpression (Fig. 5e-f), and as Fig. 5g-h shown that knockdown of Hoxa5 could reverse the up-regulation of Fam3a protein induced by circRap1b overexpression, considering that Hoxa5 could activate Fam3a expression in transcription level, in summary, circRap1b regulated the transcription and expression of Hoxa5 and then affected the expression of Fam3a, ultimately affecting cell apoptosis.

Inhibition of neuronal apoptosis by circRap1b and Hoxa5 in vivo

Given that circRap1b regulates Hoxa5 transcription by histone modification to regulate neuronal apoptosis, we hope to further explore its clinical transformation significance. Therefore, circRap1b(+) and Hoxa5(+) as well as in vivo transfection reagents were injected into the lateral ventricle of AIS mouse model. As Fig. 6a displayed HE staining sections of hippocampal CA1 region in each group (scale =50 or 10μm). The results of TUNEL assays showed the effect of circRap1b and Hoxa5 overexpression on AIS induced neuronal apoptosis in hippocampal CA1 region (scale =50μm) (Fig. 6b). Apoptosis rate = TUNEL-positive cells/total cells, Figure 6c showed neuronal apoptosis rate was significantly reduced in AIS + circRap1b(+) + Hoxa5(+)NC group, AIS + circRap1b(+)NC + Hoxa5(+) group and AIS + circRap1b(+) + Hoxa5(+) group, compared to AIS + circRap1b(+)NC + Hoxa5(+)NC group; neuronal apoptosis rate was significantly decreased of AIS + circRap1b(+) +Hoxa5(+) group when compared to AIS + circRap1b(+)+ Hoxa5(+)NC group; neuronal apoptosis rate was decreased in AIS + circRap1b(+) + Hoxa5(+) group compared with AIS + circRap1b(+)NC + Hoxa5(+) group. Figure 6d is an experimental schedule to explore the effects of circRap1b and Hoxa5 on AIS-induced apoptosis in vivo. Figure 6e is a schematic diagram of the mechanism of circRap1b / Hoxa5 / Fam3a axis regulatory neuronal apoptosis.

Studies have confirmed that circRNAs are involved in the regulation of the occurrence and progression of ischemic cerebrovascular diseases [18, 25, 27]. Circ DLGAP4 regulates the expression of HER1 by binding to miR-143, affecting endothelial cell dedifferentiation into mesenchymal cells, thereby reducing cerebral infarction size and blood-brain barrier injury. Previous studies showed that circRNAs were involved in regulating apoptosis in a variety of cells, including neurons. Circ_0000950 negatively regulated neuronal apoptosis via miR-103 during the development of Alzheimer's [48]. Also, it was found that circ_0000296 was downregulated in hippocampal tissues and neurons HT22 cells treated with Chronic cerebral ischemia-hypoxia (CCI), and overexpression of circ_0000296 significantly inhibited CCI-induced neuronal apoptosis. In present study, it was found that circRap1b was under-expressed in mouse hippocampal tissues and HT22-AIS cells treated with acute ischemia-hypoxia (AIS), and circRap1b overexpression significantly inhibited neuronal apoptosis AIS-induced. The expression of transcription factor Hoxa5 was most significantly increased among the proteins with differential expression caused by overexpression of circRap1b. Transcription factors encoded by the Hox gene family contribute to determine the fate of neurons [23]. Specifically, Hoxa5 has recently been shown to be involved in axonal growth of autonomously functioning nuclear cells [24] and post-vagal motor neurons [25] in the mouse brain. Rosa Hernandez et al. suggested that Hoxa5 is an important gene in the differentiation of hASCs neurons and is therefore a potential candidate gene for the exploration of cell therapy strategies for neurological disorders [34]. Our data confirmed that Hoxa5 was low-expressed in mouse hippocampal tissues and HT22-AIS cells treated with AIS. Overexpression of Hoxa5 significantly inhibited the apoptosis of neurons AIS-induced, and the apoptosis rate of HT22-AIS cells was significantly increased after knockdown of Hoxa5. Consistent with our study, HOXA5 in fibroblasts that overexpress hypertrophic scar or keloid origin attenuated cell proliferation, migration and collagen synthesis, while accelerated apoptosis [49]. Effectively silenced HOXA5 gene expression, induced Jurkat cell apoptosis and cell cycle arrest [50]. HOXA5 directly acted downstream of retinoic acid receptors and is involved in retinoic acid-induced apoptosis and growth inhibition [51]. Hoxa13 was down-regulated in CCI-induced hippocampal tissues and neuron cells HT22, and overexpression of Hoxa13 attenuated the apoptosis of CCI-induced hippocampal neuron HT22 [42]. Mouse Hoxa13 mutant inhibits urinary epithelial cell apoptosis by down-regulating Bmp7 expression [52].

Histone modifications mainly include methylation, acetylation, phosphorylation, ubiquitination and sumoylation [53]. Histone acetylation is dynamically regulated by histone acetyl transferase (HAT) and histone deacetylation enzyme (HDAC). Previous studies have shown that Kat7 tends to catalyze the acetylation of lysine 5 and lysine 12 of histone H4, and lysine 14 of histone H3 [54, 55]. It was found that overexpression of circFoxo3 inhibited oxygen-glucose deprivation (OGD) and induced autophagy, apoptosis, inflammation and injury of cardiomyocytes in vitro, and overexpression of circFoxo3 inhibited HMGB1 expression by reducing the enrichment of KAT7, H3K14ac and RNA Poly II in HMGB1 promoter and thus inhibiting autophagy and alleviating myocardial ischemia/reperfusion injury [56]. Overexpression of CDK11(P58) in mammalian cells can enhance the acetyltransferase activity of Kat7 to free histones. CDK11(P58) is a regulatory protein of Kat7, which interacts with Kat7 in vivo and in vitro and plays an important role in cell cycle progression and is closely related to apoptosis [57]. In this study, we found Hoxa5 mRNA and protein expression levels were significantly increased after circRap1b overexpression. CircRap1b was localized in the nucleus and interacted with Kat7 to co-exist in the promoter region of Hoxa5. Furthermore, H3K14ac enrichment was detected in the promoter region of Hoxa5, and it was mainly enriched in -1000 ~ 0bp (P2 and P1), while H4K5ac and H4K12ac were not detected. CircRap1b overexpression and Kat7 knockdown plasmid were co-transfected into HT22-AIS cells. The enrichment of H3K14ac and the changes of Hoxa5 mRNA and protein expression levels indicated that knocking down Kat7 could reverse the increase of circRap1b overexpression on the enrichment of H3K14ac and the increase of Hoxa5 mRNA and protein. Therefore, the data in this study supported that circRap1b recruited Kat7 to induce H3K14ac modification in the Hoxa5 promoter region to activate its transcription. Similar studies were as follows: circMRPS35 increased the H4K5ac level of FOXO1 and FOXO3a promoter region through the recruitment of KAT7, thereby inhibiting the progression of gastric cancer [58]. Similar to our results, lncRNA-MRCCAT1 mediated NPR3 transcription by binding EZH2 to induce H3K27me3 in the NPR3 promoter region [59]. the binding of EZH2 to the HIV-1 LTR promoter was separated by binding of MALAT1 to PRC2, resulting in the removal of H3K27me3 and reversing the epigenetic silencing of HIV-1 transcription[60].

Family with sequence similarity 3A (FAM3A) is a mitochondrial protein that plays an important role in cell adaptation to stress and cell survival and takes participate in the regulation of neuronal apoptosis[41, 42]. It was reported that FAM3A is an important regulator of ER-stress-induced neuronal HT22 cell death [61]. FAM3A protected HT22 cells from hydrogen peroxide-induced oxidative stress by activating the PI3K/Akt pathway[62]. The expression of FAM3A was down-regulated in hippocampal tissues and neuron cells induced by CCI, and overexpression of FAM3A could inhibit the apoptosis of hippocampal neurons induced by CCI [42]. In this study, we found that Fam3a was significantly underexpressed in AIS-induced hippocampal tissues and neuron cells HT22; overexpression of Fam3a significantly inhibited the apoptosis of HT22-AIS cells; knockdown of Fam3a significantly promoted the apoptosis of HT22-AIS cells. As reported that FAM3A inhibited interleukin IL-1β-induced chondrocyte apoptosis by activating the pro-survival PI3K/AKT/mTOR pathway[41]. FAM3A positively regulated post-ischemia angiogenesis in endothelial cells [63]. FAM3A could improve hypoxic-ischemic brain injury in neonatal mice[43]. In this study, according to the bioinformatics database JASPA 44 putative binding sites of HOXA5 were found in the promoter region − 3000 ~ 0bp of human FAM3A, and 4 putative binding sites of Hoxa5 were found in the promoter region − 3000 ~ 0bp of mouse Fam3a. Furthermore, ChIP and luciferase reporter gene system were used to verify that Hoxa5 can bind to the promoter region of Fam3a to promote its transcription and its protein expression, and thus regulating the apoptosis of hippocampal neurons. Reports that HOX family transcription factors play a positive role in transcriptional regulation were as follows: Hoxa13 activated the expression of FAM3A protein through transcription and inhibited CCI-induced hippocampal neuronal apoptosis[42]. Wu et al. found that Hoxa13 promotes transcriptional expression in gastric cancer by targeting the BMP7 promoter region[64]. Hoxa13 targeted the Aldh1a2 promoter and upregulated its expression to regulate limb morphogenesis[65].

To further clarify the mechanism of circRap1b and Hoxa5 co-regulating HT22-AIS apoptosis, we co-transfected circRap1b overexpression, siRNA of Hoxa5 and their negative control plasmids into HT22-AIS cell model and found that overexpression of circRap1b inhibited cell apoptosis rate and knockdown of Hoxa5 promoted apoptosis, and Hoxa5 knockdown could reverse the inhibitory effect of circRap1b overexpression alone on cell apoptosis. After overexpression of circRap1b, mRNA and protein expression levels of Hoxa5 and Fam3a were both upregulated, and knockdown of Hoxa5 could reverse the upregulation effect of overexpression of circRap1b on Fam3a protein, suggesting that circRap1b regulated the expression of Fam3a through Hoxa5 In summary, circRap1b acted as a modular scaffold to recruit KAT7 in the promoter regions of Hoxa5, which subsequently increased the level of H3K14ac modification to activate Hoxa5 transcription and expression and ultimately suppressed hippocampal neuronal apoptosis.

Finally, we verified in vivo the lowest apoptosis rate of hippocampal neurons after overexpression of circRap1b and Hoxa5. Currently, it has been reported that natural protective circRNAs can be overexpressed in vivo utilizing lentiviral or adenovirus vectors. They encode microcassettes with exons (s), as well as endogenous splicing donor and recipient sites, and inverted repeats of flanking introns that support RNA reverse folding[66]. For example, microinjection of circDLGAP4 lentivirus could significantly improve ischemic stroke prognosis[67], Therefore, the use of lentiviral or adenovirus vectors may be a feasible way to study circRap1b in vivo or in clinical trials. This study may provide a new idea for developing an ideal treatment for acute ischemic stroke and broadening the therapeutic target.

Ethical Approval and Consent to participate

Patients participating this study voluntarily signed informed consent, all experiments were approved by the Ethics Committee of People's Hospital affiliated to China Medical University.

Human and Animal Ethics

Animal experiments were received approval from the approved by the Ethics Committee of China Medical University.

Consent for publication

All authors reviewed the manuscript and gave consent for the publication of the manuscript in Translational Stroke Research.

Availability of supporting data

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This work is supported by Natural Science Foundation of Liaoning Province (2021-MS-061).

Authors' contributions

Study design: F.Z., L.Z. and L.Z., experiments implementation and data acquisition: F.Z., L.Z. and Y.L., data analysis: F.Z., L.Z., Y.L., X.D., Y.L., Y. L., S.G., S.Z., Y.X., Y.J., data interpretation: F.Z., L.Z. and L.Z., paper discussion and writing: F.Z., L.Z. and L.Z., manuscript drafting: F.Z., administrative and material support: L.Z., all authors reviewed the manuscript.

Acknowledgements

We thank all individuals who took part in this research and the biotechnology companies which provided helps.

Authors' information

Fangfang Zhang^1,#, Lin Zhao^1,#, Yu Lu¹, Xin Dong¹, Yanqi Liu¹, Yu Li¹, Shuang Guo¹, Siyuan Zheng¹, Ying Xiao¹, Yuzhu Jiang¹, Liang Zhang^1,*

1 Department of Rehabilitation Medicine, The People's Hospital of China Medical University (The People's Hospital of Liaoning Province), No.33, Wenyi Road, Shenhe District, Shenyang, 110016, Liaoning Province, PR China.

# Fangfang Zhang and Lin Zhao contributed equally to this work.

*Correspondence:

Liang Zhang, Department of Rehabilitation Medicine, The People's Hospital of China Medical University (The People's Hospital of Liaoning Province), No.33, Wenyi Road, Shenhe District, Shenyang, 110016, Liaoning Province, PR China. E-mail: [email protected]

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No competing interests reported.

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Circular RNA Rap1b promotes Hoxa5 histone acetylation by recruiting Kat7 to inhibit neuronal apoptosis in acute ischemic stroke

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