LncRNA Snhg1 Alleviates Neuronal Apoptosis During Cerebral Ischemic Stroke By Targeting Bcl2

BackgroundStroke is the leading cause of death and disability in China currently,with ischemic stroke(Ischemic stroke,IS)accounting for 69.6%-70.8%of stroke patients in China.Currently,the common clinical treatment for IS is pharlogical thrombolysis,intravascular thrombotomy,and stent placement to maintain blood supply.According to the China Acute Ischemic Stroke Treatment Guideline(2021 Edition),there are strict time windows and contraindications for the use of these methods.For example,Recombinant Human Tissue Plasminogen Activator for Injection(rt-PA)is only effective within 4-6 hours after IS onset.A large number of patients fail to benefit from revascularization therapy because of missing treatment windows or because of contraindication restrictions,resulting in post-stroke neuronal loss and neurological deficits that are difficult to salvage.Apoptosis is one of the main mechanisms of neuronal death in the acute phase after IS.Current studies have shown that interfering with the expression of apoptosis-related proteins in the early stage of apoptosis can effectively alleviate apoptosis.Thus,inhibition of post-stroke neuronal apoptosis is one of the important strategies in the treatment of IS.The changes of inhibitory factors in the apoptotic signaling in early IS and whether upregulation of inhibitory factors can ameliorate neurological deficits in IS animals became the focus of this topic.B-cell lymphoma-2(Bcl2)is an important member of neuronal apoptotic signaling that competitively binds to the mitochondrial permeability transition pore(PTP)and inhibits the release of cytochrome c(Cyt-C),thus exerting an inhibitory effect on apoptosis.However,the changes in neuronal Bcl2 levels after IS and the regulatory mechanisms are not fully understood.In this study,we investigated the changes of long non-coding RNA(LncRNA)in the early stage of IS,and explored the LncRNAs regulating the expression of Bcl2 and elucidated the regulatory mechanisms.Methods1.Mice model of IS.3-month-old C57BL/6 male mice were selected for middle cerebral artery occlusion(MCAO)for 60 minutes to induce IS.Sham-operated(Sham)animals were operated on with the wire embolus placed in the common carotid artery only.The intraoperative cerebral blood flow was measured using Doppler flowmetry,and the postoperative neurological deficits were detected by performing the animal’s neurological function score.The successful IS mouse model was defined as the blood flow on the ischemic side decreased to 30%of the normal level,and the neurological function score was higher than 3 points.The brain slices were subjected to Fluoro-Jade C(F-J C)staining and Triphenyl tetrazolium chloride(TTC)staining to detect brain tissue damage,apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining and caspase3 activity test.2.Cell model.N2a cells were cultured to mimic ischemic-hypoxic neurons by glyoxylation and glucose deprivation(OGD).To investigate the role of down-regulated Snhgl in cells,plasmids of down-regulated Snhgl(sh-Snhgl#1)and control plasmids(shCon)were transfected in N2a cells.To investigate the role of up-regulated Snhgl in cells,plasmids of up-regulated Snhg1(Snhg1)and control plasmids(Con)were transfected in N2a cells.cell viability was detected by CCK-8 kit and apoptosis was detected by TUNEL staining.3.Transcriptome study.Ischemic tissues from mice were sequenced at 24 hours poststroke and validated by quantitative reverse transcription polymerase chain reaction(RTqPCR)at 0,2,6,12 and 24 hours post-IS.Sequencing revealed that LncRNA Small Nucleolar RNA Host Genesl(Snhgl)was significantly upregulated after IS.Snhgl expression was further examined by qPCR at 7 days after IS.Cellular localization of LncRNA Snhgl was examined by Fluorescence in situ hybridization(FISH)and immunofluorescence(IF).4.Brain stereotaxic injection.To study the role of down-regulated Snhgl in IS,AdenoAssociated Virus(shSnhgl AAV)with down-regulated Snhg1 expression and control virus(control AAV)were injected in the M1 and CPU regions of the mouse brain bilaterally,followed by modeling.To investigate the cerebral protective effect of upregulated Snhg1 in IS animals,adeno-associated virus(Snhg1 AAV)with upregulated Snhgl and control virus(Con AAV)were injected in the M1 and CPU regions of the mouse brain bilaterally,followed by modeling.5.Behavioral assays.The rotarod test and the pole-climbing test were used to test the motor function of mice,and the Barnes Maze was used to test the memory function of mice.6.Bioinformatics analysis and validation.Prediction of Snhgl binding proteins using the starBase v2.0 website.The PROMO website was used to predict the transcription factors of Bcl2 and Snhg1.Bcl2 transcription factor and Snhg1 transcription factor were scored using JASPAR.WB results and RT-qPCR were used to detect Bcl2 expression levels after downregulation of YY1(YY1 transcription factor,YY1)and Snhg1 expression levels after downregulation of YY1 by RT-qPCR.Dual luciferase reporter genes were used to detect the regulatory effects of YY1 on the Bcl2 and Snhg1 promoters.Chromatin isolation by RNA Purification(CHIRP)was used to detect the binding of Snhgl to the Bcl2 promoter region.Results1.Neuronal expression of Snhgl in the injured brain region was elevated after IS,with a peak at 12 hours.Sequencing revealed 12 down-regulated differential genes and 35 upregulated differential genes in the injured brain region within 24 hours after IS,with Snhgl being one of the most up-regulated genes.RT-qPCR results confirmed that Snhgl expression was elevated after IS,with a peak at 12 hours after IS.Snhg1 was observed mainly expressed in neurons.2.The decreased Snhgl level promoted the neuronal apoptosis during IS.In mice,downregulating Snhgl resulted in the increased volume of damaged brain tissue,the increased number of apoptotic cells,the increased motor function impairment and memory impairment after IS.In cultured N2a cells,downregulating Snhgl resulted in the decreased cell viability and increased apoptosis following OGD.3.Reduced Snhgl levels inhibited Bcl2 expression.The elevated Bcl2 level was shown at 2 hours and 12 hours after IS.Downregulation of Snhg1,which was mainly localized in the nucleus,was observed reduce the expression of Bcl2.Snhgl specifically binds to the P1 region of the Bcl2 promoter.PROMO-and JASPAR-based analyses suggested YY1 be a transcription factor of Bcl2,Furtherly,we proved that YY1 positively regulates the expressions of Bcl2.Starbase-based analysis indicated YY1 a Snhgl-binding protein.PROMO-and JASPAR-based analyses suggested YY1 be a transcription factor of Snhg1.Furtherly,we proved that YY1 positively regulates the expressions of Snhgl.4.Upregulating Snhgl level significantly reduced the IS induced brain injury and apoptosis.In cultured cells,upregulation of Snhgl increased the cell viability and reduced the apoptosis after OGD.In IS mice,the increased Snhgl expressing significantly reduced ischemic volume,apoptosis and memory impairment,but did not significantly improve the motor function impairment.Conclusion1.Neuronal Snhgl specifically binds to the P1 region of the promoter of Bcl2,an important suppressor of apoptosis,and positively regulates its expression.The transcription factor YY1 positively regulates the expressions of Snhgl and Bcl2.2.Reduced Snhg1 level aggravates IS induced brain injury.Upregulating Snhgl level might be one potential therapeutic strategy to reduce the brain injury during IS.