Study On The Serum Content Of MiR-let-7f In Patients With Alzheimer's Disease And Its Mechanism Of Oxidative Damage To SHSY-5Y Cells

Alzheimer's disease(AD)is a common clinical chronic neurodege-nerative disease,including mental behavior,social function and cognitive function and other obstacles or abnormalities.At the same time,the patient's disease course is characterized by progressive development and irreversibility.The 2015 report on AD on a global scale showed that there may be 1 new clinical patient every 3 seconds worldwide,and the number of AD patients in the world may rise to 131.5 million by 2050.At the same time,among them Approximately 70%of patients are from developing countries.The different increase in the number of clinical AD patients and the increasing incidence have brought a huge burden and heavy pressure to the economy and society of all countries in the world.In 2015,the cost of care and care for AD patients in the world was 820 billion.About US dollars,and the cost can increase to about 2 trillion US dollars by 2030.At the same time,the above report also pointed out that the region with the most AD patients in the world is East Asia.my country is the country with the largest population and largest geographic area in East Asia.Especially in recent years,with the continuous increase of the domestic aging population,AD is important to my country's social economy,The community and the patient's family have brought huge challenges and burdens.Recent studies have shown that the pathogenesis of AD is closely related to micro RNAs(miRNAs).In the normal nervous system in the body,miRNAs participate in regulating the differentiation of neural stem cells,the development of the nervous system,neuroprotection,neuron proliferation and apoptosis,and the production of dendrites.Different signal transduction pathways and different physiological processes such as spines and synaptic plasticity.Therefore,this study analyzed the expression of miR-let-7f in Alzheimer's disease patients and the mechanism of oxidative damage to SHSY-5Y cells,in order to provide a lot of laboratory evidence and theoretical support for the diagnosis and treatment of clinical patients.This study will conduct research from three aspects.The first part explores the serum content of oxidative stress indicators in patients with Alzheimer's disease and its correlation with cognitive dysfunction,and clarifies the abnormal serum content of oxidative stress indicators in patients with Alzheimer's disease.The second part is to analyze the expression and clinical significance of miR-let-7f in Alzheimer's disease patients to clarify the possible role of miR-let-7f in AD patients.The third part is to clarify the potential role of miR-let-7f in the oxidative damage of SHSY-5Y cells,which is expected to further clarify the molecular mechanism of miR-let-7f in the pathogenesis of AD.Part One Serum content of oxidative stress indicators in patients with Alzheimer's disease and its correlation with cognitive impairmentObjective:To analyze the serum content of oxidative stress indicators in patients with Alzheimer's disease and their correlation with cognitive dysfunction.Methods:A retrospective analysis of 30 cases of AD patients treated in this hospital was used as the AD group,and 30 healthy patients who received physical examination in this hospital during the same period were selected as the control group.Record the patient's clinical data,including name,marital history,gender,family history,age,contact number,weight,height,and vital signs.Record the patient's serum oxidation stress(Mn SOD,GSH-Px,CAT,SOD,MDA,Hcy)levels and CDR and MMSE scores.Correlation analysis was performed on the serum oxidation stress and CDR and MMSE scores of AD patients.Results:1.Patients in the AD group were between 58 and 69 years old,with an average of(63.18±10.17)years old.There were 16 males and 14 females.The BMI index ranged from 21.9 to 26.8 kg/m²,with an average of(25.58±3.20)kg/m².The education period was 4 to 16 years,with an average of(11.01±6.14).12 cases had a history of smoking,12 cases had a history of drinking,and complications:8 cases had heart disease,15 cases had hyperlipidemia,and 10 cases had diabetes.Patients in the control group were between 55 and 70 years old,with an average age of(66.94±10.84)years,15males and 15 females,with a BMI index ranging from 21.4 to 26.9 kg/m²,with an average(24.64±4.10)kg/m²,and were educated Time 4 to 15 years,average(9.27±5.10),13 cases have a history of smoking,14 cases have a history of drinking,comorbidities:9 cases have heart disease,14 cases have hyperlipidemia,9 cases have diabetes,two groups There were no statistically significant differences in patient age,gender,comorbidities,smoking and drinking history,education time,and BMI index(P>0.05).2.The serum Mn SOD,GSH-Px,CAT and SOD levels in the AD group were(7.65±1.36)ng/ml,(152.66±40.13)?mol/L,(18.32±2.17)U/ml,(62.01±4.99)U/ml,lower than the control group(12.49±2.18)ng/ml,(199.74±41.82)?mol/L,(18.32±2.17)U/ml,(69.17±4.31)U/ml,serum MDA and Hcy in AD group The contents were(6.41±1.60)?mol/L and(29.10±6.47)mol/L,which were higher than those in the control group(4.69±1.74)?mol/L and(23.70±7.52)mol/L;the CDR score of the AD group was(2.61±0.35)points,higher than 0 points of the control group,MMSE scores(22.57±3.18)points,lower than the control group(27.79±3.15)points,the differences were statistically significant(P<0.05).3.The serum Hcy,MDA content and CDR score of AD patients were positively correlated(P<0.05),and the serum Mn SOD,GSH-Px,CAT and SOD content and CDR score of AD patients were negatively correlated(P<0.05).The serum Hcy,MDA content and MMSE score of AD patients were negatively correlated(P<0.05),and the serum Mn SOD,GSH-Px,CAT and SOD content of AD patients were positively correlated with MMSE score(P<0.05).Conclusions:1.Compared with healthy people,serum oxidative stress indicators of Alzheimer's disease patients are significantly abnormal;2.Cognitive impairment and serum oxidative stress indicators of Alzheimer's disease patients are obviously mutually related;3.The damage of oxidative balance in Alzheimer's disease patients may be a factor leading to cognitive decline.Part Two The serum content and clinical significance of miR-let-7f in patients with Alzheimer's diseaseObjective:To analyze the expression and clinical significance of miR-let-7f in Alzheimer's disease patients to clarify the possible role of miR-let-7f in AD patients.Methods:A retrospective analysis of 30 cases of AD patients treated in this hospital was used as the AD group,and 30 healthy patients who received physical examination in this hospital during the same period were selected as the control group.Collect 5ml of fasting venous blood from the patient and place it in a dry test tube without anticoagulation.Centrifuge at 4000rpm for10min.The supernatant is aliquoted into EP tubes and stored in a refrigerator at-80?.Detection of biochemical indicators:serum adiponectin(ANP),total cholesterol(TG),homocysteine(Hcy),triglycerides(TC),high-density lipo-protein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)It is detected by a fully automatic biochemical analyzer.The detection of various indicators is carried out according to the instructions of the instrument and kit.In order to control the quality of the detection,all the samples in this study are carried out by the staff of the laboratory in the same machine.The test is performed on the instrument,and each sample is tested 3 times.miRNA microarray analysis of various miRNA expression levels in AD patients;record the serum miR-let-7f expression in the AD group and the control group;record the serum ANP,Hcy,TC,TG,HDL-C,LDL-in the AD group and the control group C content and MMSE score;record the correlation between serum miR-let-7f content and serum ANP,Hcy,TC,TG,HDL-C,LDL-C content and MMSE score in AD patients;two classifications of factors affecting AD Logistic regression analysis.Results:1.Compared with the control group,there were 11 types of differentially expressed miRNAs in AD patients,including 6 miRNAs,namely miR-242,miR-301,miR-134,miR-let-7i,miR-let-The expression of 7f and miR-34a increased significantly,while the expression of 5 miRNAs,namely miR-18a,miR-188,miR-214,miR-328,and miR-3135a,decreased significantly.2.The expression of serum miR-let-7f in the AD group was(1.84±0.32),which was higher than that of the control group(1.02±0.14),and the difference was statistically significant(P<0.05).3.The contents of TC,TG and HDL-C in the AD group were(1.71±0.84)mmol/L,(21.63±1.77)mmol/L,(1.31±0.40)mmol/L,and the control group(1.79±0.95)mmol/L/L,(22.04±1.81)mmol/L,(1.34±0.39)mmol/L,the difference was not statistically significant(P>0.05).The serum ANP content and MMSE score of AD group were(6.80±2.81)?g/ml and(22.57±3.18)points,which were lower than those of the control group(11.91±3.06)?g/ml and(27.79±3.15)points.In AD group,the serum levels of Hcy and LDL-C in the patients were(29.10±6.47)?mol/L and(2.85±0.68)mmol/L,respectively,which were higher than those in the control group(23.70±7.52)?mol/L and(2.37±0.54)mmol/L,The difference was statistically significant(P<0.05).4.There was no correlation between the serum miR-let-7f content and HDL-C,TG and TC content in AD patients(P>0.05).The expression of serum miR-let-7f in AD patients was negatively correlated with MMSE score and ANP content(P<0.05),and positively correlated with Hcy content and LDL-C content(P<0.05).Conclusions:miR-let-7f is highly expressed in the serum of patients with Alzheimer's disease,and may be involved in the onset and progression of patients.Part Three Study on the mechanism of miR-let-7f on oxidative damage of SHSY-5Y cellsObjective:To clarify the potential role of miR-let-7f in the oxidative damage of SHSY-5Y cells,and hope to further clarify the molecular mechanism of miR-let-7f in the pathogenesis of AD.Methods:Culture SHSY-5Y cells to prepare H₂O₂ injury-induced oxidative stress injury model,MTT to detect cell viability,flow cytometry to detect cell apoptosis,miR-let-7f mimic,miR-let-7f-1 inhibition Transfection of SHSY-5Y cells with q RT-PCR and Western blot to measure miR-let-7f,AKT-2 and apoptosis-related proteins,luciferase reporter gene to detect miR-let-7f and AKT-2 targets To relationship.Results:0,50,100,200,500mol/L H₂O₂ caused SHSY-5Y oxidative damage.The results showed that compared with untreated cells,the cell viability of each dose group was significantly reduced(P<0.05),and another200mol was selected/L of H₂O₂ is used in subsequent experiments.Flow cytometry detection of apoptosis showed that compared with the control group,the apoptosis rate of SHSY-5Y cells treated with 200mol/L H₂O₂ was significantly increased(P<0.05).Compared with the control group,the expression of Bcl-2 protein in the H₂O₂ treatment group decreased,and the protein expression of Bax,Caspase-3,and Caspase-9 increased(P<0.05).SHSY-5Y cells were transfected with miR-let-7f mimic,miR-let-7f inhibitor,mimic NC,and inhibitor NC respectively.q RT-PCR showed that compared with mimic NC,miR-let-7f mimic group miR-let-The expression of 7f was significantly increased;compared with inhibitor NC,the expression of miR-let-7f in the miR-let-7f inhibitor group was significantly decreased(P<0.05).Compared with the control group,the cell viability of the H₂O₂treatment group was significantly reduced,while the apoptosis rate was significantly increased(P<0.05);compared with mimic NC,the cell viability of the miR-let-7f mimic group was significantly increased,while the apoptosis rate Significantly decreased(P<0.05);compared with inhibitor NC,the cell viability of miR-let-7f inhibitor group was significantly decreased,while the apoptosis rate was significantly increased(P<0.05).After clarifying that miRNA-let-7f is a potential molecule to improve the oxidative damage caused by H₂O₂,extensive biological information analysis was carried out to determine the target gene of miRNA-let-7f.Searching various databases(micro RNA.org and Target Scan)showed that ALT-2 3'-UTR has a corresponding pairing and miR-let-7f binding application,as shown in Figure9.Luciferase reporter gene assay was used to detect the relationship between miR-let-7f and AKT-2.The relative luciferase activity of co-transfected SHSY-5Y cells was significantly reduced.Use miR-let-7f and AKT-2 3'-UTR(P<0.05).Conclusions:Up-regulation of miR-let-7f alleviates the oxidative damage induced by H2O2 to SHSY-5Y cells by targeting AKT-2.These findings provide a new perspective on the role of miR-let-7f in the pathogenesis of oxidative damage during AD.