Investigation Of The Differential Expression And Correlation Between MicroRNA-21-5p And MicroRNA-125b-5p In Patients With Mild Cognitive Impairment In Type 2 Diabetes Mellitus

Objective:To investigate the role of microRNA-21-5p（miR-21-5p）and microRNA-125b-5p（miR-125b-5p）in Type 2 Diabetes Mellitus,Differential expression and correlation of Mild Cognitive Impairment（MCI）in patients with mild cognitive impairment（T2DM）were studied to further evaluate its clinical diagnostic value.Methods:Part Ⅰ:Clinical data of patients with T2DM（n=8）hospitalized in Endocrinology Department of Taizhou People’s Hospital from March 2022 to August 2022 were collected,Such as age,sex,duration of disease,years of education,Body Mass Index（BMI）,waist circumference,hip circumference,Fasting Plasma Glucose（FPG）,Glycosylated hemoglobin（Hb A1c）,Total Cholesterol（TC）,Triglycerides（TG）,High Density Lipoprotein Cholesterol（HDL-C）,Alanine Transaminase（ALT）,Aspartate Transaminase（AST）,Blood Urea Nitrogen（BUN）,Creatinine（CR）,Uric Acid（UA）and other biochemical indicators,The patients were divided into T2DM with MCI（T2DM+MCI）group（n=4）and T2DM without MCI（T2DM+n MCI）group（n=4）according to the results of Mo CA scale.Whole blood was collected for second-generation high-throughput sequencing,and differentially expressed miRNAs were screened out.Bioinformatics analysis was conducted to analyze the main biological functions of the differential genes.miR-21-5p and miR-125b-5p were finally selected as target miRNAs for sample size expansion verification according to the results of the second generation of high-throughput sequencing and combined with the literature.Part Two:Clinical data of 60 patients with T2DM hospitalized in Endocrinology Department of Taizhou People’s Hospital from March 2022 to August 2022 were collected,including age,sex,disease course,BMI,waist circumference,hip circumference,FPG,Hb A1c,TC,TG,HDL-C,ALT,AST,BUN,CR and UA.They were divided into T2DM+MCI group（n=30）and T2DM+n MCI group（n=30）according to Mo CA scale score.Real-time fluorescence quantitative Polymerase Chain Reaction（q RT-PCR）was verified in whole blood of the two groups.Comparative CT value method(2^{-△△CT})was used to analyze the differential expression of miR-21-5p and miR-125b-5p in the two groups,and Spearman statistical method was used to analyze the correlation between their expression levels and the Mo CA scale and clinical indicators.Results:1.The second generation of high-throughput sequencing results in|log2Fold Change|>1,P<0.05 for the standard,T2DM+MCI patients compared to patients with T2DM group+n MCI outside comprehensive blood samples,there are 39differentially expressed miRNAs,expression level of miRNAs has 28,There were 11miRNAs with down-regulated expression.2.GO enrichment analysis showed that,from the level of Biological Processes（BP）,differentially expressed miRNAs were mainly related to the regulation of nervous system functional development,cell metabolism and cell surface receptor signaling pathways.At the Cellular Components（CC）level,differentially expressed miRNAs are mainly related to neuronal projection and synapses.From the Molecular Functions（MF）level,differentially expressed miRNAs are mainly related to sequence specificity of transcriptional regulatory region and nucleic acid binding of regulatory region.3.KEGG pathway analysis showed that some signaling pathways related to differentially expressed miRNAs mainly included type II diabetes mellitus,phosphoinositol metabolism,tumor necrosis factor signaling pathway,AGE-RAGE signaling pathway and Fox O signaling pathway in diabetic complications.4.The expressions of miR-21-5p and miR-125b-5p in the two groups were verified by q RT-PCR,and 2^{-△△CT} was calculated according to CT values.Statistical analysis showed that the expressions of the two verified miRNAs,miR-21-5p and miR-125b-5p,were up-regulated in T2DM+MCI group.It was consistent with the trend of the second-generation high-throughput sequencing results,and the difference was statistically significant,which verified the reliability of the sequencing results.5.Clinically,there were statistically significant differences in age,course of disease,years of education and Hb A1c between T2DM+MCI group and T2DM+n MCI group（P<0.05）;Gender,BMI,waist circumference,hip circumference,FPG,TC,TG,HDL-C,ALT,AST,BUN,CR and UA were not statistically significant（P>0.05）.6.Spearman correlation analysis showed that the relative expressions of miR-21-5p and miR-125b-5p were negatively correlated with Mo CA in T2DM+MCI group（P<0.05）.It was positively correlated with age,course of disease and Hb A1c（P<0.05）.7.Receiver operating characteristic curve（ROC）analysis indicated that miR-21-5p had an area under the curve of 0.724,sensitivity of 0.633 and specificity of 0.833.The95%confidence interval was 0.5955-0.854,and the optimal truncation value was 7.457.The area under the curve of miR-125b-5p was 0.686,the sensitivity was 0.6,and the specificity was 0.767.The 95%confidence interval is 0.549-0.822,and the optimal cut-off value is 4.523.Conclusion:1.Based on the analysis of second-generation high-throughput sequencing and biological information technology,the differential expression of miRNAs was found in T2DM+MCI patients compared with T2DM+n MCI patients,providing theoretical basis for further discussion on the pathogenesis,clinical diagnosis and treatment of T2DM+MCI patients.2.The up-regulated expression of miR-21-5p and miR-125b-5p in T2DM+MCI was verified by q RT-PCR,and the results were consistent with the second-generation high-throughput sequencing results.Correlation analysis showed that the lower the Mo CA score in T2DM+MCI group,the higher the expression of miR-21-5p and miR-125b-5p.3.miR-21-5p and miR-125b-5p have certain diagnostic value for T2DM+MCI.