miR-20a-5p/RBM24 Axis Alleviated Hypertensive Intracerebral Hemorrhage via Regulating HIF1α/VEGFA Signaling Pathway

Background: Hypertensive intracerebral hemorrhage presented high incidence and high mortality owing to its dicult to diagnose. However, the molecular mechanism of HICH remains unclear. Therefore, this study aims to investigate the key miRNAs and the mechanism of the key miRNAs in HICH. Methods: miRNAs chip was used to explore the differentially expressed miRNAs in HICH patients. In vitro and in vivo HICH models were established by Ang-II. Cell Counting Kit-8 (CCK8), ow cytometry, transwell assay and tube formation analysis were used to detect cell proliferation, apoptosis, cell migration and tube formation, respectively. Hematoxylin-eosin staining was used to evaluate the intracerebral hemorrhage in vivo HICH model. The regulatory mechanism of miR-20a-5p in HICH was conrmed by dual luciferase reporter assay, immunouorescence, qRT-PCR, western blot and rescue experiments. Results: miR-20a-5p showed the most downregulated in HICH patients compared with healthy individuals and signicantly associated with clinicopathological characteristics of HICH. Upregulation of miR-20a-5p promoted cell proliferation, migration and tube formation while inhibited apoptosis in vitro and ameliorated the development of HICH in vivo. RBM24 is a direct target of miR-20a-5p and silencing RBM24 could partially recovery the development of HICH caused by miR-20a-5p inhibition both in vivo and in vitro. miR-20a-5p regulated the development of HICH depending on HIF1α/VEGFA pathway. Conclusion: Our results demonstrated that miR-20a-5p/RBM24 axis regulated hypertensive intracerebral hemorrhage via regulating HIF1α/VEGFA signaling pathway, in support of further investigation into miR-20a-5p therapies for HICH treatment. The proliferation rates of cultured HUVECs with different treatments were measured by the Cell Counting 8 kit (#C0038; Beyotime) following the manufacturer’s instruction. Briey, HUVECs in were seeded in the 96-well plates, incubated with CCK-8 solution at 37°C for 24 h. Proliferation rates were nally evaluated by using microplate reader at 450 nm. For migration, Transwell chamber systems of 24-well plate with 8 µm wells were performed for cell migration assays. In brief, HUVECs were adjusted using the serum-free RPMI-1640 medium and 200 µL of cell suspension were added into the upper chambers. The cells that transferred to the lower chamber containing 10% FBS-supplemented DMEM (Invitrogen) incubation 24h at 37 ℃ were subject to 4% paraformaldehyde xation, 0.2% Triton X-100 treatment, and 0.05% crystal violet staining. For tube formation, HUVECs were plated in the plates coated by Matrigel (300 µL/well) at 3× 104 cells/well. The formation of vessels-like tube structures and migration were observed by using the inverted microscope (XDS-800D, Shanghai Caikang Optical Co. Ltd., China) and quantitated with Image J software. suggesting the effects of miR-20a-5p on the HIF-1α/VEGF-A pathway is dependent of RBM24. Taken together, our results demonstrate miR-20a-5p/RBM24 axis regulated hypertensive intracerebral hemorrhage via regulating HIF1α/VEGFA signaling pathway, in support of further investigation into miR-20a-5p therapies for HICH treatment.


Introduction
Hypertensive cerebral hemorrhage (HICH) refers to the spontaneous hemorrhage in the brain parenchyma caused by hypertension or sudden increase in blood pressure [1,2]. With the development of population aging, the incidence of HICH in our country is increasing year by year. So far, China has become one of the countries with high incidence of HICH in the world [3]. HICH has the characteristics of high disability and fatality rate, which brings a heavy burden to individuals, families and society. Although there are a variety of treatments in the pre-clinical eld, many sequelae still occur, which affect the patient's ability of daily living and greatly reduce the patient's quality of life [4]. Therefore, it is of great signi cance to nd effective molecular markers and study its molecular mechanism to provide more effective clinical treatments for HICH.
In recent years, with the development of medical imaging technology and the improvement of neurosurgery treatment, the disability and fatality rate of HICH has decreased, but the pathogenesis and the reasons for the high incidence of HICH are still not fully understood. The pathogenesis of HICH is relatively complex, involving multiple factors such as genetics, environment, infection, immunity, among which hypertension is the most important cause [5,6]. Because intracranial arteries have the characteristics of less middle muscle cells and outer connective tissue, and lack of outer elastic layer, long-term hypertension can cause hyalinosis, brinoid necrosis, and even the formation of microaneurysms or dissecting arteries. This will lead to the blood vessels rupture and cause bleeding when the blood pressure suddenly rises [7,8]. With the rapid development of the advanced study in HICH, several molecular markers have been investigated on the incidence of HICH. Such as the COL1A2 rs42524 polymorphism was associated with the development of hypertensive intracerebral hemorrhage, particularly in conjunction with tobacco use and alcohol consumption in a Chinese population [9].
Recently, Liu et al., demonstrated that NIHSS score and high-density lipoprotein cholesterol level were prominently higher in HICH patients with CG and GG genotypes of ET-1 gene polymorphism rs1920453 than those in patients with CC genotype, suggesting that Rs1920453 in the promoter region of ET-1 gene was correlated with the occurrence of HICH [10]. However, there is still a lack of real targets for the diagnosis and treatment of HICH.
MicroRNAs (microRNAs, miRNAs) are endogenous non-coding single-stranded small RNAs with a length of 19-22 nucleotides, which are widely present in the biological world [11]. miRNAs regulate the expression of target genes by fully or partially complementary binding to the 3'UTR region of target mRNA [12] and participate in different physiological and pathological processes [13,14]. Increasing numbers of studies have demonstrated that miRNAs play crucial roles in intracerebral hemorrhage by regulating various kinds of progresses [15]. For example, lncRNA Mtss1 promoted in ammatory responses and secondary brain injury after intracerebral hemorrhage by targeting miR-709 in mice [16].
However, it remains far from enough to discover key miRNAs in HICH, nor the mechanism. Therefore, in the present study, we intend to investigate the key miRNAs in the HICH and the mechanism of the miRNA involved in the development of HICH.

Clinical tissue collection
The human peripheral blood was collected from 60 HICH patients and healthy individuals who were admitted into the PuKou Branch Hospital of Jiangsu Province Hospital between 2017 and 2018. The diagnosis of HICH relies on CT imaging diagnosis and a history of hypertension. According to the results of CT examination and the bleeding site, HICH consists of basal ganglia, ventricular, thalamic, brainstem and cerebellar hemorrhage. The criteria that need to be excluded, including trauma, brain tumor, cerebral infarction, vascular malformation and secondary cerebral hemorrhage caused by other reasons. There was no signi cant difference between the two groups of patients'age, gender and other basic data after statistical analysis (P > 0.05). The experimental procedures were approved by the Ethics Committee of the PuKou Branch Hospital of Jiangsu Province Hospital, and written consents were obtained from each subject in advance.

RNA extraction and miRNA chip
The peripheral blood was collected from the HICH patients to perform miRNA chip, the healthy tissues act as control (N = 3). RNeasy Mini Kit (74104, QIAGEN) was used to isolate the RNA and RNA quantity and quality were measured by NanoDrop. Sample labeling and array hybridization were performed according to the Agilent miRNA Microarray Systemwith miRNA Complete Labeling and Hyb Kit protocol (Agilent Technology). After The hybridized arrays were washed, xed and scanned with using the Agilent Microarray Scanner. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v14.9 software package (Agilent Technologies). Differentially expressed miRNAs were identi ed through Fold Change ltering (Fold Change > 2.0, FDR < 1.0). R scripts was used to perform Hierarchical Clustering.

Hypertensive intracerebral hemorrhage model
Eight months old C57BL/6 mice were purchased from Charles River Laboratories (Beijing) to establish HICH model. The mice housed at room temperature (20-25°C) with a constant humidity (55 ± 5%) with free access to food and water at a regular 12/12-h light/dark cycle. The C57BL/6 mice were randomly divided into experimental and normal groups (n = 20). The experimental group was given angiotensin II (100mg/kg/day) in subcutaneously embedded micro-osmotic pump. Meanwhile, the mice were feed L-NAME L-NAME (100mg/kg/day) to construct a mouse model of hypertensive cerebral hemorrhage. The mice were fed normally for 18 weeks. Without act as control group. The HICH model was evaluated by blood pressure identi cation, behavioral testing and pathological testing. Blood pressure identi cation was measured by BP2000 sphygmomanometer; Behavioral tests were performed on the mice three times a day, including morning, middle and evening. When the contralateral forelimb stretched, hovered, trembling, or other motor dysfunction, it was regarded as a behavioral sign of cerebral hemorrhage in mice. The experiments involved animals were performed with the approval from the institutional animal care and the Ethics committee of the PuKou Branch Hospital of Jiangsu Province Hospital. For the function analysis of miR-20a-5p, 20 µg miR-20a-5p mimics or inhibitors were injected into mice via tail vein. For the function analysis of RBM24, lentivirus harboring the full length of RBM24 and sh-RNA sequence of RBM24 were injected into mice via tail vein, respectively.

Luciferase assay
Fragments of RBM24 3'untranslated regions (UTR) including wild type (wt) or mutant (mt) miR-20a-5p binding sites were respectively inserted into the 3'-UTR of the luciferase reporter gene vector pmiGLO. The RBM24-wt or RBM24-mut pmiGLO vector were delivered into HEK293T cells by Lipofectamine® 3000 (Thermo Fisher) in the presence of miR-20a-5p mimic or inhibitor. The luciferase activity was determined using dual-luciferase reporter assay system kit (E1910, Promega, USA) according to the manufacturer's instruction. The luciferase activity was presented as re y luciferase relative to that of renilla luciferase.

RNA extraction and quanti cation
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instruction. cDNA was synthesized from mRNA by using a HiScript II One Step RT-PCR Kit (Dye Plus) (P612-01, Vazyme, Nanjing, China) and miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (MR101-01, Vazyme, Nanjing, China) following the instructions which used for detecting the expression of RBM24, CTD, OPCML, HIF-α, VEGFA and miR-20a-5p following the instructions provided by the manufacturer's instruction. Real time quantitative PCR (qRT-qPCR) was performed with the ABI 7500 instrument (ABI, USA) RT-qPCR reaction mixture of volume 20 µL contained 9 µL of SYBR Mix, 0.5 µL of each primer (10 µM), 2 µL of the cDNA template, and 8 µL of RNase free H 2 O. Thermal cycling parameters for the ampli cation were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min. The miR-20a-5p level was normalized to U6 and the target mRNA level to GAPDH. Results were calculated by using 2 −△△CT method. The primers used in the present study was list as follows: miR-20a-

Cell proliferation, migration and tube formation analysis
The proliferation rates of cultured HUVECs with different treatments were measured by the Cell Counting 8 kit (#C0038; Beyotime) following the manufacturer's instruction. Brie y, HUVECs in were seeded in the 96-well plates, incubated with CCK-8 solution at 37°C for 24 h. Proliferation rates were nally evaluated by using microplate reader at 450 nm. For migration, Transwell chamber systems of 24-well plate with 8 µm wells were performed for cell migration assays. In brief, HUVECs were adjusted using the serum-free RPMI-1640 medium and 200 µL of cell suspension were added into the upper chambers. The cells that transferred to the lower chamber containing 10% FBS-supplemented DMEM (Invitrogen) incubation 24h at 37℃were subject to 4% paraformaldehyde xation, 0.2% Triton X-100 treatment, and 0.05% crystal violet staining. For tube formation, HUVECs were plated in the plates coated by Matrigel (300 µL/well) at 3× 104 cells/well. The formation of vessels-like tube structures and migration were observed by using the inverted microscope (XDS-800D, Shanghai Caikang Optical Co. Ltd., China) and quantitated with Image J software.
Apoptosis analysis by TUNEL Apoptosis analysis was performed by TUNEL analysis using a TUNEL detection kit (cat. no. KGA702; Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Brie y, 1x10 6 cells were permeabilized with 0.1% Triton X-100 for 30 min and incubated in 50 mM TUNEL reaction mixture for 2 h at room temperature. In vivo apoptosis analysis, brain tissues slides were stained with 10 µl hematoxylin (cat. no. ab220365; Abcam) for 3 min at room temperature for nuclear staining. Images were captured using XSP-36 inverted uorescence microscope (Boshida Optical Co., Ltd.). Each experiment was performed three times. Slides were counterstained with DAPI for 10 min at room temperature for nuclear staining. Images were captured using a uorescence microscope (Olympus Corporation) and analyzed by image J.

Immuno uorescence staining
The expression and distribution of HIF-α and VEGFR were also detected by immuno uorescence in vivo and in vitro. In vitro, cells were seeded onto 12 mm coverslip in 24 well plates and cultured until their con uence reached about 70-80% and then xed with 4% paraformaldehyde for 30 min at room temperature. The cells and the slides were blocked with 10% goat serum for 15 min followed by incubation with RBM24 (1:200, Abcam), HIFa (1:200, Abcam) or VEGFR primary antibodies overnight at 4ºC. The cells or slides were incubation with TRITC-conjugated or FITC-conjugated secondary antibody (Thermo Fisher, 1:200) for 1 hour at 37℃ in the dark after washing with PBS and then counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, 0.1µg/ml) for 5 min. Images were taken using a uorescent microscope.
Brain tissues from different groups were obtained and xed with 4% paraformaldehyde followed by embedded in para n. Brain tissues were sliced into 4um sections for hematoxylin-eosin (HE) staining. Finally, the sections were rinsed and differentiated with 1% glacial acetic acid, and dehydrated in two tanks of absolute ethanol and dehydrate and sealed the slides with neutral gum. The tissue slices were taken by a microscope (Olympus, Japan).

Statistical analysis
All the statistical analyses in the present study were completed with SPSS 21.0 software (IBM, Armonk, NY, USA). Data are shown as the mean ± standard deviation with at least three independent experiments. Statistical comparisons were performed using unpaired t test between two groups and one-way analysis of variance (ANOVA) were used to compare more than two groups. Correlation of measurements was yielded with Pearson's correlation analysis. P < 0.05 was as a level of statistical signi cance.

MicroRNA microarray pro ling and differentially expressed miRNAs
Among the 2549 miRNAs tested using the Agilent 8 × 60 K miRNA-array platform, a total of 2134 miRNAs were found (Supplementary Table 1). Of them, a total of 715 miRNAs were found to be differentially expressed with 394 upregulation and 321 downregulation in the HICH patients compared to healthy individuals (Supplementary Table 2). And miR-20a-5p showed the most downregulated in HICH patients compared with healthy individuals (Fig. 1A). In order to con rm the results, the expression of miR-20a-5p were veri ed in HICH patients (n = 20) were by qRT-PCR. As shown in Fig. 1B, the expression of miR-20a-5p was signi cantly in HICH patients compared with healthy individuals. Therefore, miR-20a-5p was selected for further analysis.
miR-20a-5p is signi cantly associated with clinicopathological characteristics of HICH We have demonstrated that miR-20a-5p was signi cantly in HICH patients compared with healthy individuals. In order to know whether miR-20a-5p participate in HICH, the correlation between miR-20a-5p expression and the clinicopathological characteristics of HICH was investigated. According to the expression of miR-20a-5p, 33 patients were divided into miR-20a-5p high expression group (n = 17) and low expression group (n = 16). The results showed the expression of miR-20a-5p was signi cantly correlated with higher cerebral hematoma volume, higher NIHSS index score and lower BI index score (Table 1). In order to investigate the function of miR-20a-5p in HICH, the HICH model was established in HUVECs transfection with miR-20a-5p mimics or inhibitor. As shown in Fig. 2A-C, cell proliferation, migration and tube formation were signi cantly inhibited in miR-20a-5p inhibitor group while promoted in miR-20a-5p mimics group. Furthermore, apoptosis was dramatically increased in miR-20a-5p inhibitor group while decreased in miR-20a-5p mimics group (Fig. 2D). In addition, we found that the contents of HCy (homocysteine), ATII (angtiotensin ) and cTn I (cardiac troponin ) were signi cantly elevated in miR-20a-5p inhibitor group compared with miRNA-NC group while overexpression of miR-20a-5p decreased the contents of HCy, ATII and cTn I (Fig. 2E). These ndings demonstrated that miR-195 plays an important role in cell proliferation, migration and tube formation of HUVECs.

Overexpression of miR-20a-5p inhibited HICH development in vivo
We have demonstrated that miR-20a-5p could regulate the development of HUVECs in vitro. Therefore, HICH model was constructed to con rm whether miR-20a-5p participated in the regulation of HICH in vivo. The blood pressure of the mice in the experimental group gradually increased and nally stabilized at about 180 mmHg while the blood pressure of the mice in the control group was stable at about 100 mm Hg (Fig. 3A). HE staining results showed that large bleeding area was observed in HICH rats (Fig. 3B), demonstrating the HICH was successfully established. Further analysis showed that the blood pressure was elevated when downregulation of miR-20a-5p while declined when overexpressing miR-20a-5p (Fig. 3C). The bleeding area was also sharply reduced in miR-20a-5p mimics group while increased in miR-20a-5p inhibitor group when compared with NC group (Fig. 3D). In addition, the contents of HCy, ATII and cTn I were signi cantly decreased in miR-20a-5p mimics group compared with miRNA-NC group while miR-20a-5p silencing increased the contents of HCy, ATII and cTn I (Fig. 3E).
RBM24 is a direct target of miR-20a-5p In order to elucidate the underlying mechanism of miR-20a-5p involved in, the targets of miR-20a-5p were predicted by Targetscan7.2 (Supplementary Table 3). The expression of CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A (CTD), small phosphatase-like opioid binding protein/cell adhesion molecule-like (OPCML) and RNA binding motif protein 24 (RBM24) which showed the highest cumulative weighted context + + score was measured by qRT-PCR and western blot. As shown in Fig. 4A and B, RBM24 showed the highest fold change and selected for further analysis. To con rm the relationship between miR-20a-5p and RBM24, a luciferase activity assay was conducted. As shown in Fig. 4C, the luciferase activity was signi cantly decreased in RBM24 WT group compared with control group while no obvious changes observed in RBM24 Mutant group compared with control group. In addition, the expression of RBM24 is signi cantly upregulated in HICH compared with healthy individuals. Further analysis showed that the expression of RBM24 is upregulated in miR-20a-5p inhibitor group while miR-20a-5p mimics could signi cantly attenuate the expression of RBM24 both in vitro and in vivo HICH models ( Fig. 4D and E). These results indicated that RBM24 is a direct target of miR-20a-5p by binding its 3'UTR.

miR-20a-5p regulated the development of HICH by downregulation of RBM24
In order to know the function of RBM24 in the development of HICH, gain and loss function of RBM24 was performed in vivo and in vitro. In vitro experiments, we found that upregulation of RBM24 could inhibited cell proliferation, migration and tube formation while it could be promoted when downregulation of RBM24 (Fig. 5A-C). In addition, it showed that cell apoptosis was dramatically promoted in RBM24 overexpression group while inhibited in RBM24 suppression group (Fig. 5D). In vivo experiments, the blood pressure was elevated when upregulation of RBM24 while declined in suppressing RBM24 group (Fig. 5E). The bleeding area was also sharply increased in RBM24 overexpression group while reduced in RBM24 downregulation group compared with NC group (Fig. 5F). Furthermore, the contents of HCy, ATII and cTn I was signi cantly elevated in RBM24 upregulation group compared with miRNA-NC group while RBM24 suppressing decreased the contents of HCy, ATII and cTn I (Fig. 5G and H). In addition, the rescue experiment revealed that overexpression of RBM24 reversed the inhibitory effects of miR-20a-5p overexpression on the proliferation, migration, tube formation and apoptosis of HUVECs (Fig. 5I-L).
miR-20a-5p regulated the development of HICH depending on HIF1α/VEGFA pathway It has been demonstrated that the HIF1α/VEGFA axis has been documented to be of great importance to angiogenesis. Therefore, we speculated that might by affecting HIF1α/VEGFA signaling pathways. To clarify whether HIF1α/VEGFA signaling pathway participated in the development of HICH depended on the miR-20a-5p-RBM24 axis, the expression of HIF1α and VEGFA was measured by qRT-PCR, western blot and immuno uorescence staining. The results showed that the expression of HIF1α/VEGFA was signi cantly downregulated in HICH cells (Fig. 6A-C) and rats (Fig. 6D-F) compared with control group. Further analysis showed that overexpression of RBM24 could elevated the expression of HIF1α/VEGFA in HICH cells or rats (Fig. 6A-F), suggesting the effects of miR-20a-5p on the HIF-1α/VEGF-A pathway is dependent of RBM24.

Discussion
Surgical treatment alone cannot achieve satisfactory results to HICH due to the deep location of bleeding site in brain tissue. In view of the characteristics of rapid onset, high disability rate and high fatality rate, it brings a huge burden to individuals, families and society. Therefore, it is urgent to explore the effective biomarkers and in-depth study of their possible role in the pathogenesis, targeted therapy and prognosis of HICH. In the present study, we investigated the differentially expressed miRNAs in HICH. Of them, miR-20a-5p showed the most downregulated in HICH patients compared with healthy individuals and signi cantly associated with clinicopathological characteristics of HICH. Further analysis demonstrated that miR-20a-5p/RBM24 axis regulated hypertensive intracerebral hemorrhage via regulating HIF1α/VEGFA signaling pathway, in support of further investigation into miR-20a-5p therapies for HICH treatment. These results are of great signi cance for the diagnosis and treatment of HICH by miR-20a-5p.
Developmental defects in the middle layer of the arterial wall, arteriosclerosis and hypertension are three important factors for cerebral hemorrhage, among which hypertension is an important independent risk factor for cerebral hemorrhage [19]. Although the pathogenesis of HICH is not yet complete, it is currently believed that hypertension is an important basis for its pathogenesis, that is, hypertension induces mechanical stress changes to act on endothelial cells (ECs), causing a series of pathological changes, resulting in vascular wall damage [20]. However, the molecular mechanism of HICH is less studied.
miRNA played an important role in a series of key biological processes, including cell proliferation, differentiation and metabolism [21]. Numbers of miRNAs have been revealed play crucial roles in intracerebral hemorrhage, such as miR-124, miR-340-5p and miR-26a-5p [22][23][24]. However, there was less studies focus on HICH regulated by miRNAs. In the present study, XX differentially expressed miRNAs in HICH patients were found by miRNAs chip. Of them, miR-20a-5p showed the most downregulated in HICH patients compared with healthy individuals and signi cantly associated with clinicopathological characteristics of HICH, demonstrating that miR-20a-5p played important roles in HICH. In fact, miR-20a-5p inhibited epithelial to mesenchymal transition and invasion of endometrial cancer cells by targeting STAT3 [25]. Recently, it demonstrated that MicroRNA-20a-5p suppressed tumor angiogenesis of nonsmall cell lung cancer through RRM2-mediated PI3K/Akt signaling pathway [26]. In the present study, we found that downregulation of miR-20a-5p promoted cell proliferation, migration and tube formation while inhibited apoptosis in vitro and ameliorated the development of HICH in vivo. These results demonstrated that miR-20a-5p regulated the development of HICH by regulating the angiogenesis.
miRNA could inhibit the mRNA expression by binding the 3'UTR of its targets in various kinds of diseases.
In the present study, we found that RBM24 is a direct target of miR-20a-5p. It has been demonstrated that RBM24 played important roles in many progresses of various diseases, such as cell alternative splicing, lung cancer progression and skeletal muscle regeneration [27,28]. In addition, it also proved that RBM24 could regulated by miRNAs. For example, MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cell [29]; The restoration of RBM24 expression suppressed NPC cellular proliferation, migration and invasion and impeded metastatic colonization in mouse models by upregulating miR-25 [30]. However, the function of RBM24 remains unclear in HICH. In the present study, we showed that overexpression of RBM24 inhibited the cell proliferation, migration and tube formation while promoted apoptosis in vitro and ameliorated the development of HICH in vivo. Further analysis showed that silencing RBM24 could partially recovery the development of HICH caused by miR-20a-5p inhibition both in vivo and in vitro. These results demonstrated that miR-20a-5p regulated the development of HICH via regulating the expression of RBM24.
The HIF1α/VEGFA axis has been documented to be of great importance to angiogenesis in multiple diseases [31][32][33], as well as in intracerebral hemorrhage. Such as HIF-1α gene can promote the proliferation, migration and differentiation of endogenous neural stem cells after ICH, thereby contributing to neurofunctional recovery after ICH [34]. It also proved that HIF-1α and downstream pathways could regulate neuronal injury after intracerebral hemorrhage in diabetes [35]. Recently, Cui et al., demonstrated that thrombin reduced HIF-1α degradation and initiated angiogenesis by increasing miR-24, which targets PHD1 after ICH, suggesting HIF-1α might involve in the regulation of HICH [17]. In the present study, we demonstrated that the expression of HIF1α/VEGFA was signi cantly downregulated in HICH cells and rats compared with control group. Further analysis showed that overexpression of RBM24 could inhibit the expression of HIF1α/VEGFA while siRNA knockdown of RBM24 reversed the changes, suggesting the effects of miR-20a-5p on the HIF-1α/VEGF-A pathway is dependent of RBM24.
In summary, we found that miR-20a-5p showed the most downregulated in HICH patients compared with healthy individuals and signi cantly associated with clinicopathological characteristics of HICH. Further analysis showed that miR-20a-5p/RBM24 axis regulated hypertensive intracerebral hemorrhage via regulating HIF1α/VEGFA signaling pathway. These ndings will provide solid foundation to prevent the occurrence and development of HICH.

Declarations
Ethics approval and consent to participate    The expression levels of RMB24 protein after treatment with miR-20a-5p mimics, miR-20a-5p inhibitor or miR NC in vitro and in vivo, respectively. GAPDH act as control. **P<0.05, All data are expressed as the mean ± SD.  and VEGFA was measured by qRT-PCR, western blot and immuno uorescence staining after RBM24 overexpression or suppression in HICH rats, respectively GAPDH act as control for qRT-PCR and western