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 significant 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, fixed and scanned with using the Agilent Microarray Scanner. Agilent Feature Extraction software (version 18.104.22.168) 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 identified through Fold Change filtering (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 identification, behavioral testing and pathological testing. Blood pressure identification 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.
Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs) were purchased from The Global Bioresource Center (ATCC). After the HUVECs recovery, cells were cultured in the 90% Dulbecco's modified Eagle's medium (DMEM, 12430054, Gibco, USA) supplemented with 10% of fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 µg/mL streptomycin at 37 ℃ in a 5% CO2-contained incubator under 95% saturation humidity. HUVECs was induced for 12 hour by 1.00µmol/L AngII (Sigma) to establish HICH cell model. For function analysis of miR-20a-5p or RBM24, HUVECs were seeded in a six-well plate until the confluence reached at 70–90%. And then miR-20a-5p mimics or inhibitor, pCDNA-RBM24 or RBM24 SiRNA were transiently transfected into HUVECs and cultured for 24h, respectively. And then the cells were used for further analysis. The miR-20a-5p mimics, inhibitor and NC sequences were synthesized by Shanghai GenePharma Co., Ltd. and sequences were listed as follows: miR-20a-5p mimics: 5’-UAAAGUGCUUAUAGUGCAGGUAGCUACCUGCACUAUAAGCACUUUA-3’; miR-20a-5p inhibitor: 5’-UAAAGUGCUGACAGUGCAGAU GCGAAGAGGTGACAGUGCAGA-3’; NC: 5’-GCACCGUCAAGGCUGAGAACUGGTGAAGACGCCAGUGGA-3’.
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 firefly luciferase relative to that of renilla luciferase.
RNA extraction and quantification
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 H2O. Thermal cycling parameters for the amplification 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-5p Forward: 5’-TAAAGTGCTTATAGTGCAGGTAG-3’, Reverse: 5’-GCAATGTAGAATCCGCTGGG-3’; U6 Forward: 5’-ACGCAAATTCGTGAAGCGTT-3’, Reverse: 5’-CAGTCCAACTAGCACAATTCG-3’; GAPDH Forward 5’-GCACCGTCAAGGCTGAGAAC-3, Reverse: 5’-TGGTGAAGACGCCAGTGGA-3’.
Total protein from HUVECs and brain tissues were extracted by using RIPA Lysis buffer. Protein abundances were determined using the BCA Protein Assay Kit (70-PQ0012, MultiSciences, China) following the manufacturer’s instructions. 20 ug proteins for each sample with protein loading buffer were boiled at 100℃ for 5 min, followed by separation in 10–12% SDS-PAGE electrophoresis and transferred onto PVDF membranes. The membranes were then blocked with 5% lipid-free milk/TBST buffer for 2 h at room temperature, incubated with anti-RBM24, anti-HIF-1α antibody (ab51608, 1:2000), anti-VEGF-A antibody (ab183100, 1:450) and anti-GAPDH (ab8245, 1:5000, abcam, Cambridge, UK) primary antibodies for 2 h at 4℃overnight, respectively. After being incubation with secondary antibodies anti-mouse IgG (ab205719, 1:20000, abcam, Cambridge, UK) or anti-rabbit IgG (ab6721, 1:20000, abcam, Cambridge, UK) for 1–2 h at room temperature, the immuno-complexes were finally detected by ECL after washing by TBST and analyzed using the Image-Pro Plus 6.0 software.
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. Briefly, HUVECs in were seeded in the 96-well plates, incubated with CCK-8 solution at 37°C for 24 h. Proliferation rates were finally 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 fixation, 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. Briefly, 1x106 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 fluorescence 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 fluorescence microscope (Olympus Corporation) and analyzed by image J.
The expression and distribution of HIF-α and VEGFR were also detected by immunofluorescence in vivo and in vitro. In vitro, cells were seeded onto 12 mm coverslip in 24 well plates and cultured until their confluence reached about 70–80% and then fixed 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 fluorescent microscope.
Brain tissues from different groups were obtained and fixed with 4% paraformaldehyde followed by embedded in paraffin. 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).
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 significance.