| Background Due to the characteristics of high morbidity,high recurrence,high disability and high fatality rate,acute cerebrovascular disease（ACVD）has been the chief culprit to be serious threat to physical and mental health of human and to cause heavy family and social burdens,therefore it gets a lot of attention.Micro ribonucleic acid（miRNA）is a kind of non-coding ribonucleic acid（RNA）of the length of about2025bp,which lies in eukaryotic cells widely and is highly conserved.RNA polymerase II is responsible for translating the gene of encoding mi RNA into primitive mi RNA（pri-miRNA）.Pri-miRNA possesses thousands of base pairs in length and consists of one or more than one hairpin loops.After being recognized and cleaved by RNA enzymesⅢDroshaand double-stranded RNA binding protein Pasha,pri-miRNA generates a precursor miRNA（pre-miRNA）of 7090 nucleotides with the help of DiGeorgecritical regiongene 8（DGCR8）.Pre-miRNA is transported from the nucleus to the cytoplasm through the function of exportin-5.In the cytoplasm,pre-miRNA undergoes the decorating by RNA enzymesⅢDicer to form the mature double-stranded miRNA.Next,this maturedouble-stranded miRNA will soon be integrated into the miRNA-induced silencing complex（RISC）.Usually one chain is degraded and the mature single-stranded miRNA can be retained in the RISC.Then,the mature single-stranded miRNA can combinate3′-UTR basesequence of target messenger RNA（mRNA）through the principle of complementary base pairing,which regulates the activity of target mRNA expression and inhibits its translation or causes its degradation after transcription.Therefore,miRNA can participate in a series of important pathophysiological processes including cell cycle regulation,apoptosis,differentiation and immune inflammation and so on.Lin-4 was the first miRNA found in nematodes in 1993,which could control temporal development in the life cycle.It is estimated that vertebrate genomes encode at least 1,000 unique mi RNAs,which are expected to regulate the expression of at least 50%genes.Although more than735 human miRNAs species have been found,accounting for about 1 to 4%of human genes,it is reported that the vast majority of the miRNAs can regulate more than 200 mRNAs,which makes mi RNA as the biggest regulatory factor of gene expression.However,there were still many issues to be resolved about their exact function and role in cells.One miRNA can regulate the expressions of multiple target mRNAs,likewise,the combination of several miRNAs can become a miRNA gene cluster to regulate the expression of certain target mRNA accurately.At the same time some mRNAs and or products of their protein coding can also regulate the expressions of some miRNAs through feed-forward,as a result,which lead to the lack or decrease of corresponding protein synthesis and lead to the occurrence of diseases.Studies have confirmed that miRNA exists in all kinds of body fluids besides cells,such as blood,urine,saliva,cerebrospinal fluid,pleural fluid,ascites and so on.At the same time,the same miRNA can be found in several different body fluids,while some mi RNAs exist only in one kind of body fluid,such as miR-224 in plasma,thereinto the specific miRNAs in plasma are the most.That miRNA abounds in the blood and is relatively stable can be connected with the mechanism of against the endogenous Ribonuclease（RNase）activity and RNase degradation resistance,which leads to the long-term stable existing in the blood circulation.And all kinds of processing methods of boiling,acid and alkali environment,repeated freezing and thawing,long-term preservation and so on,do not cause the loss of miRNA.Therefore,serum or plasma miRNA as a potential biomarker,is of great value in the clinical application.MiRNA-related researches cover a wide range of fields,such as cancer,cardiovascular disease,ACVD,diabetes,lung disease,immunity,and so on.ACVD includes acute ischemic stroke（AIS）and acute hemorrhagic stroke（AHS）,thereinto the former includes acute cerebral infarction（ACI）and transient ischemic attack（TIA）.Although the clinical manifestations vary,there are many similarities in the pathogenesis.Oxidative stress,inflammatory response,edema formation,neuronal apoptosis and so on,are important factors involving in secondary brain injury of ACVD.It was found that there were differential expressions of miR-29 b and mi R-424 associated with inflammatory response and oxidative stressin ACVD patients,suggesting that they could be involved in regulating the damage or protection mechanism of brain tissue in ACVD.However,different research results are also controversial,for example some studies suggest that the blood and or brain tissue miR-29 b is up-regulated,while other experimental results support down-regulation of miR-29 b.In addition,the expressions of miR-424 in different time points of cerebral ischemia reperfusion are also different.Therefore,what is the expression levels of miR-29 b and mi R-424 in the blood circulation of ACVD patients?Are they possible to be biological markers for early diagnosis of ACVD?Can they be used for early differential diagnosis of ACI and TIA?Are they related to the short-term prognosis of ACVD?Are there difference expressions in different subgroups analysises of ACVD?Still further research is needed.For this reason,this paper will be used to research and discuss the above issues from two parts,as outlined below.Part One Correlation Between Blood Circulation MiR-29 b,Mi R-424 and Classification of Acute Ischemic Stroke and Its Short-term Prognosis Objective To analyze the expression levels of blood circulation miR-29 b and miR-424 in patients with acute ischemic stroke（AIS）and to explore the correlation between miR-29 b,miR-424 and classification of AIS and its short-term prognosis.Methods 102 cases of hospitalized patients with AIS were selected as the research object from May 2016 to May 2017 within 24 h after the onset of symptom who were admitted to emergency department or neurology department in our hospital（ACI group 70 cases and TIA group 32 cases）.During the same period 30 cases of healthy physical examination persons were selected as the control group.The expression levels of blood circulation mi R-29 b and miR-424 among three groups were detected by real-time fluorescent quantitative PCR（RT-qPCR）,and serum tumor necrosis factor alpha（TNF-α）concentrations were detected by using emzyme linked immunosorbent assay（ELISA）.At the same time blood routine was tested to calculate platelet/lymphocyte ratio（PLR）.At the end of one month follow-up after onset,the short-term prognosis of patients with ACI were evaluated according to Glasgow outcome scale（GOS）（GOS score 4 to 5 points for good outcome,GOS score 1 to 3 points for poor outcome）.ACI group were divided into good outcome group and poor outcome group for subgroup analysis.ACI group were also divided into lacunar cerebral infarction group（LI group）and non-lacunar cerebral infarction group（Non-LI group）for subgroup analysis based on whether the diameter of infarct was greater than 2cm.The relative expression levels of miRNA to the control group（fold change）were calculated by 2-ΔΔCTmethod.Statistical software SPSS19.0 was used for statistical analysis of all data.If the data satisfied the normal distribution and homogeneity of variance,the t-test of independent samples was adopted between mean of the two samples.If the data did not satisfy normal distribution or homogeneity of variance,t/test was adopted.Single factor analysis of variance（ANOVA）was used to compare the mean among multiple samples.If the data meeted homogeneity of variance,pairwise comparisons were performed by using LSD test.On the contrary,Tamhane method was used if the data did not meet the homogeneity of variance.All the results of measurement data were showed by the mean and the standard deviation.Because of the data did not satisfy the normal distribution,the correlation was analyzed by Spearman correlation test.The differences between the classification variables were compared by Chi-squared test.The test levelαwas 0.05,and a p value less than 0.05 indicated statistical significance.The differences of blood circulation miR-29 b,miR-424,serum TNF-αand blood PLR were compared among ACI group,TIA group and the control group,respectively.The differences of blood circulation miR-29 b,miR-424,serum TNF-αand blood PLR were compared between different prognosis groups of ACI patients,respectively.The differences of blood circulation miR-29 b,miR-424,serum TNF-αand blood PLR were compared between different subgroups of ACI patients（LI group and Non-LIgroup）,respectively.At the same time the correlations were analyzed between blood circulation miR-29 b,miR-424 and inflammatory factors（TNF-αand PLR）and the classification of AIS and the short-term prognosis of ACI.Results The relative expression levels of blood circulation miR-29 b and mi R-424 in patients of ACI group were significantly lower than those of TIA group and the control group（all P<0.05）,while the levels of serum TNF-αwere significantly higher than those of TIA group and the control group（all P<0.05）.However,there were no significant statistical differences on miR-29 b,miR-424 and TNF-αbetween TIA group and the control group（all P>0.05）.The levels of blood PLR in patients of ACI group were significantly higher than that of TIA group and the control group（all P<0.05）.Furthermore,the PLR levels in TIA group were significantly higher than that of the control group（P<0.05）.The relative expression levels of blood circulation miR-29 b and miR-424 in poor outcome group of ACI were significantly lower than that of good outcome group,and the PLR levels were obviously higher than that of good outcome group（all P<0.05）,whereas there was no significant statistical difference on TNF-αbetween different prognosis groups of ACI（P>0.05）.There were no significant differences on mi R-29 b,miR-424,TNF-αand PLR between LI group and Non-LI group of ACI subgroups（all P>0.05）.Both miR-29 b and miR-424 in the ACI group were negatively correlated with TNF-αand PLR（rs=-0.265,P=0.027,rs=-0.322,P=0.007,rs=-0.469,P=0.000,rs=-0.683,P=0.000）,and were positively correlated with the GOS score at the end of one month after onset（rs=0.350,P=0.003,rs=0.282,P=0.018）.Conclusion Compared with the healthy control group,the relative expression levels of blood circulation miR-29 b and miR-424 are significantly down-regulated in ACI group,which could be used as a potential biological marker for the diagnosis of ACI.The relative expression levels of blood circulationmi R-29 b and miR-424 are significantly different between ACI group and TIA group,which could be preliminarily used for the early classification of AIS and differential diagnosis.There are no significant statistical differences in the relative expression levels of miR-29 b and miR-424 between LI group and Non-LI group,suggesting that the levels of mi R-29 b and miR-424 could not be used to identify LI or Non-LI.The relative expression levels of blood circulation miR-29 b andmiR-424 are negatively related to the inflammatory factors of ACI,and are positively correlated with the GOS score at the end of one month after onset,furthermore there are significant differences of miR-29 band miR-424 between different prognosis groups of ACI,which can preliminarily be used to evaluate its short-term prognosis.Part Two The Expressions of Blood Circulation MiR-29 b and MiR-424 in Acute Cerebral Hemorrhage and Their Clinical Significance Objective To analyze the expression levels of mi R-29 b and miR-424 in patients with acute cerebral hemorrhage（ACH）and to explore the correlation between miR-29 b,miR-424 and ACH.Methods 58 cases of hospitalized patients with ACH were selected as the research object from August 2016 to October 2017 within 24 h after the onset of symptom who were admitted to emergency department,or neurology department or neurosurgery department in our hospital.During the same period,35 cases of healthy physical examination persons were selected as the control group.The expression levels of blood circulation miR-29 b and miR-424 were detected by real-time fluorescent quantitative PCR（RT-qPCR）on the third day after the onset of symptom of ACH group and at the physical examination day of the control group,respectively.In the same way,serum neuron specific enolase（NSE）and S100 protein concentration were detected by using emzyme linked immunosorbent assay（ELISA）on the third day after the onset of symptom of ACH group and at the physical examination day of the control group,respectively.At the end of one month follow-up after onset,the short-term prognosis of patients with ACH were evaluated according to Glasgow outcome scale（GOS）（GOS score 4 to 5 points for good outcome,GOS score 1 to 3 points for poor outcome）.ACH group were divided into good outcome group and poor outcome group for subgroup analysis.ACH group were divided into secondary cerebral infarction group（SCI group）and non-secondary cerebral infarction group（Non-SCI group）for subgroup analysis based on whether there was secondary cerebral infarction after cerebral hemorrhage or not on the third day after the onset of symptom.The relative expression levels of miRNA to the control group（fold change）were calculated by 2-ΔΔCTmethod.Statistical software SPSS19.0 was used for statistical analysis of all data.If the data satisfied normal distribution and homogeneity of variance,the t-test of independent samples was adopted between mean of the two samples.If the data did not satisfy normal distribution or homogeneity of variance,t/test was adopted.All the results of measurement data were showed by the mean and the standard deviation.Because the data did not satisfy normal distribution,the correlation was analyzed by Spearman correlation test.The differences between the classification variables were compared by Chi-squared test.The test levelαwas 0.05,and a p value less than 0.05 indicated statistical significance.The differences of blood circulation miR-29 b,miR-424,serum NSE and S100 protein were compared between ACH group and the control group,respectively.Likewise,the differences of blood circulation miR-29 b,miR-424,serum NSE and S100 protein were compared between different subgroups of ACH（SCI group and Non-SCI group）,respectively.The differences of blood circulation mi R-29 b,miR-424,serum NSE,S100 protein and brain surgery intervention ratio were compared between different prognosis groups of ACH,respectively.At the same time the correlations were analyzed between blood circulation miR-29 b,miR-424 and NSE,S100 protein,the short-term prognosis（GOS score）and brain surgery intervention of ACH.Results The relative expression level of blood circulation miR-424 in ACH group was significantly lower than that of the control group（P<0.05）.The levels of serum NSE and S100 protein in ACH group were significantly higher than those of the control group（all P<0.05）.While the level of blood circulation miR-29 b in ACH group was slightly lower than that of the control group,but there was no statistically significant difference（P>0.05）.The relative expression level of miR-424 in SCI group of ACH subgroups was obviously lower than that of Non-SCI group（P<0.05）.Though the relative expression level of miR-29 b in SCI group was slightly lower than that of Non-SCI group,there was no significantly statistical difference（P>0.05）.Similarly,there were also no significant statistical differences of NSE and S100 protein between the two groups（all P>0.05）.The relative expression levels of miR-29 b and miR-424 in the poor outcome group were significantly lower than those of the good outcome group（all P<0.05）.However,there were no statistically significant differences of NSE and S100 protein between the different prognosis groups of ACH（all P>0.05）.The ratio of brain surgery intervention in the poor outcome group was significantly higher than that of the good outcome group（81.0%vs24.3%,P=0.000）.MiR-29 b in ACH group was negatively correlated with NSE,and was positively correlated with GOS score at the end of one month after onset,furthermore there were statistically significant difference（rs=-0.290,P=0.027,rs=0.400,P=0.002）.Although it was negatively correlated with S100 protein and brain surgery intervention,there were no statistically significant differences（rs=-0.199,P=0.134,rs=-0.153,P=0.251）.MiR-424 in ACH group was negatively correlated with NSE,S100 protein and brain surgery intervention,and was positively correlated with GOS score.Furthermore there were statistically significant difference（rs=-0.289,P=0.028,rs=-0.267,P=0.043,rs=-0.317,P=0.015,rs=0.311,P=0.017）.Conclusion Compared with the healthy control group,the relative expression level of blood circulation miR-424 is significantly down-regulated in ACH group,which could be used as a potential biomarker for the diagnosis of ACH.The relative expression level of blood circulation mi R-424 is significantly different between SCI group and Non-SCI group,which could be preliminarily used for the prediction of SCI after ACH.The relative expression levels of miR-424 in the different prognosis group of ACH are negatively correlated with brain surgery intervention,and the level of mi R-424 could be preliminarily used for the retrospective prediction of whether brain surgery intervention was needed after ACH.The relative expression levels of blood circulation miR-29 b and miR-424 are negative correlated with NSE and S100 protein in patients with ACH,and are positively correlated with GOS score at the end of one month after onset.Furthermore,there are significant differences of mi R-29 b and miR-424 in different prognosis groups of ACH,which can preliminarily be used to evaluate its short-term prognosis. |
