The Research On LncRNA NR₁20420 For Early Diagnosis Of Acute Cerebral Infarction And The Mechanism Of Promotion Of Its Apoptosis

A study shows that the incidence of stroke in China has been increasing by 9% annually in recent years and it has become one of the leading causes of death in China.The morbidity and disability rate of stroke are high.It will bring a heavy burden on individuals and society.It also has a huge socioeconomic impact especially in low-and middle-income countries.Ischemic stroke accounts for 80% of all strokes.Surgical treatment of acute cerebral infarction has made a breakthrough in recent years.Its therapeutic effect and prognosis are closely related to the choice of treatment modalities when the patient is sent to a doctor for the first time.In addition to focusing on the time since stroke onset,the doctor also needs to accurately assess the degree of tissue damage caused by cerebral ischemia in order to choose a specific treatment method.Currently,perfusion MRI and perfusion CT are mainly used to obtain this information,but both of them are not perfect,and each has its own shortcomings.This has prompted academics to search for reliable biomarkers of cerebral infarction to assist diagnosis,guide therapy,and predict prognosis.In recent years,some progress has been made in the research of non-coding RNA.LncRNAs has become a hot topic in academia.A lncRNA is a non-coding RNA whose transcripts are longer than 200 nucleotides.It has been found that it regulates biological processes at multiple levels such as transcription,post-transcriptional control,and epigenetic inheritance.It may play a regulatory role by shielding gene transcription initiation region,hybridizing with the binding site of transcription factors,and enhancing the binding between transcription factors and their binding sites.Some studies found that a group of lncRNAs could be used as biomarkers of a variety of diseases,especially of tumor,some of which could also regulated their function.At present,preliminary studies have shown that lncRNAs is closely related to cerebral infarction,but studies of the relationship between them is still at an early stage.There is a theory that the increase of inflammatory cytokines and reactive oxygen free radicals in the brain tissue after acute cerebral infarction will make tight junction of the cerebral microvascular endothelial cells degenerate and break the integrity of the blood brain barrier.The nucleic acid with high expression in cerebral cells will enter the peripheral blood through disintegrated blood brain barrier after cerebral infarction.Therefore,we hypothesized that some of the lncRNAs that had undergone significant changes in peripheral blood after cerebral infarction might have specifically high expression in neurons.They may have the potential of being biomarkers of cerebral infarction.Biomarkers of cerebral infarction may include lncRNAs participating the regulation of cerebral function after infarction.The main purposes of this study were as follow: 1,to explore the diagnostic value of peripheral blood lncRNAs with differential expression as biomarkers of acute cerebral infarction;2,to study the mechanism of lncRNAs' regulation of apoptosis after cerebral ischemic injury which has differential expression in the neural cells.The main techniques and methods involved in this study were: extraction of total RNA from peripheral blood and adherent cells,lncRNAs microarray,bioinformatics analysis technology,quantitative PCR,culture and passage of adherent cell,OGD model establishment,transfection of siRNAs for adherent cell,CCK8 cell viability assay,TUNEL apoptosis assay,flow cytometry,Western blotting,medical statistics.Part ? Microarray differential expression profiles and bioinformatics analysis of circulating blood lncRNAs after acute cerebral infarctionObjectives: To establish differential expression profiles of lncRNAs and mRNAs in peripheral whole blood between patients with acute cerebral infarction and healthy volunteers,and to analyze the function,pathways,transcription factors involved in differentially expressed lncRNAs.Methods: Peripheral whole blood was collected from 6 patients with acute cerebral infarction and 3 healthy volunteers respectively.The total RNA was extracted with PAXgene Blood miRNA Kit.Quality control,labeling,hybridization,scanning,original signal extraction,and data standardization were performed according to the standard protocol of Agilent Human lncRNA Microarray 4 * 180 K V5 chip.The lncRNAs with a p?0.05 and a fold change?2.0 were defined as differentially expressed lncRNAs.Co-expression analysis was performed with the lncRNAs with the fold change ranked in the top 400 and the complete mRNAs database of the microarray,followed by functional prediction,pathway prediction,and analysis of its association with transcription factors.Results: 1,A total of 1,990 differential mRNAs were detected in the patients with cerebral infarction compared with healthy volunteers,of which 1468 were up-regulated and 522 were down-regulated.A total of 5686 lncRNAs were detected,of which 4641 were up-regulated and 1045 were down-regulated.2,GO analysis showed mRNAs related to differentially expressed lncRNAs after acute cerebral infarction mainly involved the negative regulation of NF-?B transcription factor activity and the positive regulation of programmed cell death.3,Pathway analysis based on KEGG database showed that mRNAs related to differentially expressed lncRNAs after acute cerebral infarction mainly involved apoptosis,neurotrophin signaling pathway and so on.4,MRNAs related to differentially expressed lncRNAs after acute cerebral infarction mainly involved PBX3,STAT1,and other transcription factors.Conclusion: Microarray detection could reveal the unique whole blood lncRNAs expression profile of acute cerebral infarction.The threshold of the probe of microarray could be used to detect lncRNAs in whole blood.The majority of the differentially expressed mRNAs and lncRNAs were up-regulated.Subsequent GO analysis showed that abnormally expressed lncRNAs involved the negative regulation of NF-?B transcription factor activity and positive regulation of programmed cell death.Pathway analysis showed that they were closely related to apoptosis.Data from transcription factors correlation analysis could facilitate the study of lncRNAs transcriptional regulation.Part ? Value of circulating lncRNAs in the early diagnosis of acute cerebral infarction and its relationship with subtype and prognosisObjectives: 1,The reliability of lncRNAs microarray differential expression profiles of whole blood was confirmed by quantitative PCR;2,To further investigate whether lncRNAs biomarkers were independent diagnostic factor of acute cerebral infarction;3,The diagnostic accuracy of lncRNAs biomarkers for acute cerebral infarction was studied;4,To further explore the relationship between the expression of lncRNAs biomarkers and cerebral infarction subtype and prognosis.Methods: 1,Some differentially expressed lncRNAs in the microarray of the whole blood were selected for further verification.The expression of these lncRNAs was detected by quantitative PCR in another 39 patients with acute cerebral infarction and 39 healthy volunteers;2,Binary logistic regression was used to investigate correlation of the differentially expressed lncRNAs and the status of cerebral infarction;3,ROC curve was used to determine the diagnostic accuracy of candidate lncRNAs biomarkers for acute cerebral infarction;4,The difference of lncRNAs biomarkers candidate's expression between patients with cerebral infarction in different OCSP subtype and healthy volunteers was compared.The difference of candidate lncRNAs between the groups with high and low MRS scores in the 3-month follow-up was compared.Results: 1,Nine lncRNAs were selected from the microarray differential expression profiles to verify their difference by quantitative PCR.Six lncRNAs were increased and three were decreased in the microarray among them.Quantitative PCR showed that the expression of these nine lncRNAs was all increased in patients with cerebral infarction.NR₁20420 and lnc-GCH1-2:3 were statistically different,while the remaining seven lncRNAs had no statistical difference;2,All clinical features,NR₁20420,and lnc-GCH1-2:3 were analyzed by logistic regression analysis.NR₁20420,age,and hyperlipidemia were included in the regression model,and lnc-GCH1-2:3 did not enter it.NR₁20420 was statistically significant;3,The AUC of NR₁20420 for diagnosis of cerebral infarction of TACI subtype was 0.861.The sensitivity and specificity were 85.7% and 84.6% respectively.The AUC of lnc-GCH1-2:3 for diagnosis of cerebral infarction of TACI subtype was 0.802.The sensitivity and specificity were 85.7% and 82.1% respectively;4,The expression of NR₁20420 and lnc-GCH1-2:3 in cerebral infarction patients of TACI subtype was significantly higher than that in healthy volunteers.The expression of NR₁20420 and lnc-GCH1-2:3 in the poor prognosis group?MRS 3-6?was significantly higher than that in the good prognosis group?MRS 0-2?.Conclusion: The overall changed trend of quantitative PCR and microarray were consistent.The expression of NR₁20420 and lnc-GCH1-2:3 in cerebral infarction patients and healthy volunteers was significantly different.Logistic regression analysis showed that NR₁20420 could be an independent diagnostic factor for acute cerebral infarction.Both of these two lncRNAs had high sensitivity and specificity for the diagnosis of cerebral infarction.The expression of these two lncRNAs in peripheral blood was correlated with the OCSP criteria and the prognosis of cerebral infarction patients.Part ? Mechanism of lncRNA NR₁20420 of its promotion of apoptosis after ischemia injury in nerve cellObjectives: 1,To detect whether two lncRNAs from the second part of this study have corresponding change in neuronal OGD model and select specific way of modeling and siRNAs tools;2,Whether the two lncRNAs candidate regulated apoptosis of the model was studied;3,To investigate the change of target factors in OGD model after knockdown of lncRNAs candidate;4,To confirm whether there was a causal relationship between the change of apoptosis after knockdown of lncRNAs candidate in this model and the changes of target factors.Methods: 1,The change of lncRNAs candidate in SH-SY5 Y cells was detected by quantitative PCR after they were treated with OGD for 0,4,8,12 hours and reperfused for 24 hours after OGD for 4,8,12 hours respectively.The change of corresponding lncRNAs in SH-SY5 Y cells was detected by quantitative PCR after they were transfected with the designed six siRNAs respectively;2,The cell models were established by OGD for 8 hours after they were transfected with the four siRNAs with the qualified knockdown efficiency.The cell viability of them was detected by CCK8 assay,and the apoptosis of them was measured by TUNEL staining and flow cytometry analysis?Annexin V-FITC/PI?;3,The change of protein expression of p-P65,p65,p-ERK,ERK,p-AKT,and AKT was detected by Western blotting after knockdown of NR₁20420;4,The cell models were treated with and without PDTC after they were transfected with siNR₁20420.Then the cell viability and apoptosis of them was detected.Results: 1,The expression of these two lncRNAs candidate in OGD-treated cells increased.Their expression was highest after OGD for 8 hours and then gradually decreased with longer OGD time.The expression of them after OGD for 4 hours and 8 hours has statistical differences.The knockdown efficiency of siNR₁20420 ?,siNR₁20420?,siGCH1-2: 3?,and siGCH1-2: 3? was more than 60% in the six siRNAs,and these four siRNAs were selected as tools for knockdown of lncRNAs candidate in the subsequent experiment;2,Decreased cell viability,increased percentage of TUNEL-positive cells,and increased percentage of flow cytometry-apoptotic cells in SH-SY5 Y cells after OGD model was established suggested that it was successful at the functional level.Knockdown of NR₁20420 increased the cell viability of the model and decreased the percentage of TUNEL-positive cells and flow cytometry-apoptotic cells.Knockdown of lnc-GCH1-2:3 had no obvious effect on the cell viability of the model and the percentage of TUNEL-positive cells and flow cytometry-apoptotic cells;3,The protein expression of P65,p-ERK,ERK,p-AKT,and AKT did not change significantly in this model after knockdown of NR₁20420,and the protein expression of p-P65 was significantly reduced after knockdown of NR₁20420;4,Transfection with siNR₁20420? and siNR₁20420? could increase the cell viability of the model and reduce the percentage of TUNEL-positive cells and flow cytometry-apoptotic cells,and PDTC could abolish the above effects.Conclusion: In this part of the study,relative reasonable and reliable research design and lncRNAs intervention tools were selected.NR₁20420 and lnc-GCH1-2:3 had significant changes in this model,but only knockdown of NR₁20420 could inhibit the apoptosis of this model.NR₁20420 had a damaging effect on cerebral infarction model.The protein expression of NF-?B?p-P65?was significantly reduced in this model after knockdown of NR₁20420.NF-?B inhibitor could abolish the regulation of apoptosis by NR₁20420 in this model.Summary: Microarray detection could reveal unique whole blood lncRNAs expression profiles of acute cerebral infarction.Subsequent GO analysis and Pathway analysis showed that aberrantly expressed lncRNAs were involved in apoptosis and NF-?B activity.The expression of NR₁20420 and lnc-GCH1-2:3 was significantly different between patients with cerebral infarction and healthy volunteers,and NR₁20420 could be used as an independent diagnostic factor for acute cerebral infarction.Both of these two lncRNAs had high sensitivity and specificity for the diagnosis of cerebral infarction.The expression of these two lncRNAs in peripheral blood was correlated with the OCSP criteria and the prognosis of cerebral infarction.NR₁20420 and lnc-GCH1-2:3 had significant changes in OGD model of nerve cells,but only knockdown of NR₁20420 could inhibit the apoptosis of this model.The protein expression of NF-?B?p-P65?was significantly reduced in this model after knockdown of NR₁20420.NF-?B inhibitor could abolish the regulation of apoptosis by NR₁20420 in this model.This study identified two potential lncRNAs biomarkers for cerebral infarction with high diagnostic value and preliminarily clarified the specific mechanism of NR₁20420 of its promotion of apoptosis after ischemia injury in nerve cell.These achievements were expected to improve the diagnosis and treatment of acute cerebral infarction in China.