Long Noncoding RNA H19 Overexpression Protects against Hypoxic-Ischemic Brain Damage by Inhibiting miR-107 and Up-Regulating Vascular Endothelial Growth Factor

Long noncoding RNAs play critical roles in cellular homeostasis, and long RNA H19 (H19) is implicated several pathologic conditions. The putative role of the pathogenesis progression hypoxic-ischemic brain damage (HIBD) not yet understood. Therefore, a series vivo vitro experiments were designed to investigate potential neuronal apoptosis cognitive dysfunction HIBD. expression was decreased HIBD rat models established by partial occlusion carotid artery. bound miR-107, which also increased VEGF expression. overexpression reduced alleviated rats. up-regulation miR-107 reversed protective effects conferred H19. In addition, cell model oxygen-glucose deprivation cells used. neurons B-cell lymphoma-2 lymphoma-2–associated X, total cleaved caspase-3 expressions. Taken together, results showed that expresses at low level expression, resulted repressed dysfunction. Thus, may serve as molecular target for translational research therapy. Hypoxic-ischemic affects approximately 0.6% neonates 25% 30% newborns, with serious long-term consequences1Yin X. Meng F. Wang Y. Wei W. Li A. Chai Feng Z. Effect hyperbaric oxygen on neurological recovery neonatal rats following its underlying mechanism.Int J Clin Exp Pathol. 2013; 6: 66-75PubMed Google Scholar such epilepsy, mental retardation, cerebral palsy.2Wang Zhang J. Yang Dong L. Progesterone attenuates edema inhibiting matrix metalloproteinase-9 aquaporin-4.Exp Ther Med. 263-267Crossref PubMed Scopus (35) characterized apoptosis, dysfunction, hippocampus.3Li C. Mo Lei H. Fu R. Huang Luo S. Edaravone via suppression TRAIL signaling pathway.Int Biochem Cell Biol. 2018; 99: 169-177Crossref (14) Scholar,4Byeon J.H. Kim G.H. J.Y. Sun Eun B.L. Cognitive hippocampal induced injury prolonged febrile convulsions immature rats.J Korean Neurosurg Soc. 2015; 58: 22-29Crossref (12) At present, there notable scarcity effective therapeutic strategies HIBD.5van Velthoven C.T. Kavelaars van Bel Heijnen C.J. Mesenchymal stem treatment after improves behavioral outcome induces oligodendrocyte regeneration.Brain Behav Immun. 2010; 24: 387-393Crossref (178) An understanding events underpinning valuable advances alleviation (lncRNAs) have been neurodevelopment etiological HIBD.6Zhou M. Zou Y.G. Xue Y.Z. X.H. Gao H.W. Q. non-coding protects acute myocardial infarction through activating autophagy mice.Eur Rev Med Pharmacol Sci. 22: 5647-5651PubMed LncRNAs represent transcripts >200 nucleotides comprise largest portion transcriptome.7Kaur P. Liu Tan J.R. Lim K.Y. Sepramaniam Karolina D.S. Armugam Jeyaseelan K. Non-coding neuroprotectants against ischemic injury.Brain 3: 360-395Crossref (34) crucial embryogenesis development central nervous system.7Kaur are highly expressed reported regulate activities, including plasticity, development.8Zhao Qu Gan Lu Mu D. Microarray profiling co-expression network analysis lncRNAs mRNAs damage.Sci Rep. 5: 13850Crossref (60) LncRNA first identified lncRNA has multiple conditions.9Han C.L. Ge Y.P. Zhao X.M. K.L. Chen N. Hu J.G. F.G. contributes let-7b temporal lobe epilepsy.Cell Death Dis. 9: 617Crossref (56) be comprehensively described. Certain can bind miRNAs diminish bioavailability their mRNAs.10Paci Colombo T. Farina Computational identifies sponge interaction between messenger human breast cancer.BMC Syst 2014; 8: 83Crossref (173) small 22 bp length silence genes degrading or translation.11Moon J.M. Xu Giffard R.G. Inhibition microRNA-181 reduces forebrain ischemia-induced loss.J Cereb Blood Flow Metab. 33: 1976-1982Crossref (114) known modulators post-transcriptional gene silencing HIBD.12Zhou S.L. Pei M.H. microRNA-129-5p involved neuroprotective effect dexmedetomidine targeting COL3A1 Wnt/beta-catenin pathway Biochem. 120: 6908-6919Crossref Specifically, activation suppress thus promote cycle non–small-cell lung cancer,13Cui Yu Zhou c-Myc-activated downregulates promotes non-small cancer.Int 12400-12409PubMed suggesting binding site miR-107. documented vital traumatic neurodegenerative disease, Alzheimer disease.14Wang W.X. Wilfred B.R. Madathil S.K. Tang G. Dimayuga Stromberg A.J. Saatman K.E. Nelson P.T. regulates granulin/progranulin implications disease.Am 177: 334-345Abstract Full Text PDF (153) bioinformatics website Targetscan (http://www.targetscan.org/vert_71, last accessed December 14, 2018) adopted our study predict relationship HIBD, specific 3′-untranslated region (UTR) sequence, Moreover, evidence collected basis analyses, indicating vascular endothelial growth factor (VEGF) one Expression VEGF, trophic factor, increases system injury, promoting angiogenesis neurogenesis ischemia.15Thau-Zuchman O. Shohami E. Alexandrovich A.G. Leker R.R. Vascular injury.J 30: 1008-1016Crossref (143) regarded molecule injury.16Zhao Bao X.J. R.Z. G.L. Ma S.H. J.J. Y.J. W.B. Y.N. Kong Postacute ischemia transfer transferrin-targeted liposomes experimental stroke rats.Hum Gene Ther. 2011; 207-215Crossref (52) On literature analysis, we hypothesized H19, contribute histologic deficits. Animal protocols approved Ethics Committee Guizhou Provincial People's Hospital (approval number 201803005). All animal performed accordance Guide Care Use Laboratory Animals, published US NIH,17Committee Update AnimalsNational Research Council: Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref care taken minimize numbers suffering included animals. Neonatal male Sprague-Dawley (n = 25; age, 7 days; weight, 11.84 ± 1.46 g) this study. kept breastfed under controlled conditions an room 20°C 24°C temperature, 40% 70% relative humidity, >50-dB noise 12-hour light/dark cycle. A 15 used establish model. anesthetized i.p. injection 30 mg/kg 0.3% pentobarbital sodium solution. then placed onto operating table supine position. neck disinfected alcohol, 0.5-cm incision made along midline guidance dissecting microscope. anterior cervical muscles trachea separated expose triangle using glass needle. Carotid pulsations observed right side adjacent vagus nerve. artery separated, vessels ligated double limit blood flow. surgery lasted period <5 minutes nonsurgical injuries from operation. closed sutures. cage 1 2 hours surgery. When confirmed fully awake state, they sealed 50-mL hypoxic container 20°C. After exposure conditions, breastfed. remaining 10 subjected sham operation, all identical procedures conducted rats, except ligation (Table 1). fulfilled criteria considered successful models: negative geotaxis (ie, response when head down incline 40 45 degrees); bar holding; finger climbing ability climb four horizontally extended fingers examiner's palm inclined 60 score <3. Responses various tests graded 0 (no response) 5 (maximal optimal response).18Rice 3rd, J.E. Vannucci R.C. Brierley J.B. influence immaturity rat.Ann Neurol. 1981; 131-141Crossref (1858) successfully models.Table 1The Classification RatsGroupingRats experiment, nSuccessfully modeled nSham-operated rats1010HIBD rats1510HIBD, damage. Open new tab Morris water maze 3 days 100-cm–diameter round pool divided into separate regions (I, II, III, IV regions). hidden, 6-cm–diameter platform cm below surface IV. Pool (25°C) opaque adding appropriate amount milk, camera above center pool. Rat swimming traced automatic video recording system. trained once day days. facing wall time spent finding hidden recorded. Rats failed find within 90 seconds guided seconds. case, recorded Probe test 6 experiment. removed. entry point order I-II-III-IV. Time IV, where originally placed. test, euthanized solution (30 mg/kg). hippocampus harvested stored −80°C subsequent experiments.19Zhou Silencing MEG3 enhances protection mice miR-129-5p.J 2019; 7978-7988Crossref (15) prepared. injected adenovirus (Genomeditech, Shanghai, China) expressing 10), vectors carrying siRNA blank 10). Male carbon dioxide asphyxiation 6) 24 birth, bilateral cortex collected. meninges, cerebellum, hippocampus, medulla removed, tissues cut trypsinized 37°C minutes. suspension differential adhesion method unattached cultured 75-cm2 culture bottle coated l-polylysine 5% CO2 37°C. Half medium renewed every 85% 95% confluency, oscillated (150 × g; hours) remove supernatant. To simulate vitro, attached subcultured (OGD) Dulbecco's modified Eagle's replaced Hanks’ balanced salt without glucose. incubated chamber (mic-101; Billups-Rothenberg, Del Mar, CA; nitrogen CO2) normoxic/normoglycemic condition hours.20Guan Du Lv Yuan Oxygen-glucose preconditioning deprivation/reperfusion bone morphogenetic protein-7 mediated ERK, p38 Smad signalling pathways.Clin Physiol. 2016; 43: 125-134Crossref (3) Scholar,21Luan Ding Cui Jiang Zhu Lin Tian Neuroprotective salvianolate ischaemia-reperfusion signal pathway.Eur Pharmacol. 2020; 872: 172944Crossref 80% confluent primary infected (Genomeditech) plasmids, mimics, control (NC) plasmids. infection, 37°C, CO2, saturated humidity. hours, (Santa Cruz Biotechnology, Inc., Dallas, TX) containing 10% fetal bovine serum 72 further incubation.22Ju Cao B. Song M.Q. Z.B. Effects miR-223 colorectal cancer proliferation regulating FoxO3a/BIM.Eur 3771-3778PubMed transfection reagents purchased Shanghai GenePharma Co Ltd (Shanghai, China). experiment repeated three times. Total extracted TRIZOL (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), concentration determined. primer sequences listed Table (TaKaRa Biotechnology Co, Ltd, Liaoning, cDNA synthesized reverse transcription kit (D0401; Harbin HaiGene Harbin, Reverse antisense miRNA one-step (D1801; Ltd). Next, (1 μL) employed quantitative fluorescence, PCR coding (AQ101-02; Beijing TransGen Biotech RT-PCR (RT-qPCR) (ABI ViiA7; Da Guangzhou, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) U6 internal references. 2−ΔΔCt applied quantify expressions genes.23Ayuk S.M. Abrahamse Houreld N.N. photobiomodulation molecules diabetic wounded fibroblasts vitro.J Photochem Photobiol 161: 368-374Crossref (197) times.Table 2Primers Quantitative RT-PCRGenePrimer (5′-3′)LncRNA H19F: 5′-CAAGATGGGGGGGTCAGAC-3′R: 5′-GAGCCACTTCTGAACCTGCT-3′miR-107F: 5′-ATACCGCTCGAGTGCCATGTGTCCACTGAAT-3′R: 5′-ATACCGCTCGAGTTCCATGCCTCAACTCC-3′VEGFF: 5′-TGTGCGGGCTGCTGCAATGAT-3′R: 5′-TGTGCTGGCTTTGGTGAGGTTTGA-3′GAPDHF: 5′-GATGGTGAAGGTCGGTGTGA-3′R: 5′-TGAACTTGCCGTGGGTAGAG-3′U6F: 5′-GCCGCATACAGAGAAGATTA-3′R: 5′-AGTGCAGGGTCCGAGGTA-3′GAPDH, glyceraldehyde-3-phosphate dehydrogenase; F, forward; H19; R, reverse; factor. GAPDH, Western blot previously reported.24Pan Lewno M.T. Wu Highly dynamic changes activity regulation macroautophagy hearts proteotoxic stress.Front 10: 758Crossref (17) Scholar,25Zhang Pan Parajuli Rekhter M.D. Goldberg A.L. PDE1 inhibition facilitates proteasomal degradation misfolded proteins cardiac proteinopathy.Sci Adv. eaaw5870Crossref (28) protein radioimmunoprecipitation assay lysis (R0010; Ltd.) 4°C minutes, centrifuged 24,000 g each sample determined bicinchoninic acid (20201ES76; YeSen Equal amounts μg fractionated 8% 14% SDS-PAGE gel hour. transferred polyvinylidene difluoride membrane. membrane blocked nonfat milk antibodies (ab32152; 1:1000; Abcam, Cambridge, UK), (ab13847; 1:500; Abcam), (ab32499; 1:10,000; (Bcl-2; ab196495; Bcl-2–associated X (Bax; ab232479; GAPDH (ab8245; Abcam) overnight 4°C. washing Tris-buffered saline Tween 20 times (5 each), horseradish peroxidase–labeled goat anti-rabbit IgG (ab205718; 1:20,000; anti-rat (ab6789; 1:5000; temperature washed each) subsequently developed. Data semiquantitatively analyzed ImageJ software version 1.8.0 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). ratio gray value GAPDH. Target web-based biological prediction resource. predicted sites 3′-UTR dual-luciferase reporter essay. Dual-luciferase wild-type (WT) WT mutants (MUTs) constructed. two cotransfected mimics HEK293T cells. digested TransDetect Double-Luciferase Reporter Assay Kit (FR201-01; supernatant Luciferase detected Dual-Luciferase System (E1910; Promega Corp., Madison, WI). reaction reagent (100 added lysate (20 measure Firefly luciferase activity. II determine Renilla Firefly/Renilla luciferase. Immunofluorescence staining method.10Paci Scholar,26Pan Tong STI571 neurotoxic prion fragment-induced apoptosis.Neuropharmacology. 93: 191-198Crossref (25) Briefly, fixed 4% paraformaldehyde 3% albumin Antibodies Bcl-2 (ab32370; Bax (ab32503; microtubule-associated (ab5392; P-τ (EPR2731; 1:50; added, respectively, green red fluorescent (Interwiki, Baltimore, MD) hours. Thereafter, fixed, imaged fluorescence microscope (Olympus, Tokyo, Japan) DAPI nuclear-specific markers sequence probe hybridization. brief, cy5-labeled whereas dye–labeled nucleus stained DAPI. manufacturer’s instructions (Gene Pharma, images captured Zeiss LSM880 NLO (2 + BIG) confocal (Leica Microsystems, Mannheim, Germany). immunoprecipitation (Millipore, Bedford, MA). treated beads rinsing buffer six Purified RT-qPCR.27Guo Hao PVT1 modulates hepatocellular carcinoma recruiting EZH2.Cancer Int. 18: 98Crossref (42) WT-bio-LncRNA MUT-bio-LncRNA (50 nmol/L; biotin labeled) transfected 293T 48 phosphate-buffered saline, followed incubation (Ambion, Austin, 33,000 obtain Protein M-280 streptavidin magnetic (S3762; Sigma-Aldrich, St. Louis, MO) precoated RNase-free yeast tRNA (TRNABAK-RO; Sigma-Aldrich). Magnetic twice precooled buffer, low-salt high-salt buffer. TRIzol purify RNA, RT-qPCR.28Liu Liang Xing Yi LINC00852 adenocarcinoma spinal metastasis S100A9.J Cancer. 4139-4149Crossref (13) brains, euthanizing rinsed running water, dehydrated gradient cleared xylene, embedded. Paraffin-embedded sliced sections μm thick) dried overnight. dewaxing, 0.5% hematoxylin, differentiated 75% hydrochloric water. ammonia bluing solution, samples eosin, ethanol, mounted neutral resin, dewaxed protease K working (protease storage diluted 1:9) rupture membranes (consistent immunofluorescence staining). Reagent [terminal deoxynucleotidyl transferase (TdT)] (dUTP) mixed (2:29) terminal transferase-mediated dUTP nick-end labeling (11684817910; Roche Diagnostics GmbH, Germany) samples. wet box Randomly selected fields section Apoptosis index calculated positive values total. cresol violet Nissl differentiation immersed distilled terminate staining. ethanol gum. Morphologic optical microscope.29Shan Shang Dexmedetomidine developing neurotoxicity wrought sevoflurane: Drp1-Bax signaling.Drug Des Devel 12: 3617-3624Crossref (24) deep anesthesia, perfused supplemented 2% glutaraldehyde subcutaneously. cold 1% OsO4 osmium oxide Ultrathin (90 nm) uranyl acetate lead acetate. Synaptic vesicles post-tactile synapse density investigators blinded groups JEOL 200CX electron kV; JEOL, Japan).30Hongpaisan M.K. Alkon D.L. PKC epsilon prevents synaptic loss, Abeta elevation, deficits Alzheimer's disease transgenic mice.J Neurosci. 31: 630-643Crossref (154) Statistical analyses SPSS 21.0 (IBM Armonk, NY). means SD. data sets evaluated normal distribution homogeneity variance. Comparison unpaired t-test. Comparisons one-way variance, post hoc Tukey test. different points repeated-measures Differences statistical significance P < 0.05. functions less removed