| Objective:1.To analyze the abnormal expression of LncRNA NEAT1 in serum of acute cerebral infarction and its diagnostic value in acute cerebral infarction.2.To analyze the relationship between abnormal expression of NEAT1 and the inflammatory response to acute cerebral infarction.3.To analyze whether NEAT1 can be used as an index to evaluate the severity of acute cerebral infarction and predict the prognosis.Methods:This experiment is divided into two parts: clinical study and cellular experiment.Clinical study:In a previous study,we have high-throughput sequencing obtained that LncRNA NEAT1 is abnormally elevated in patients with cerebral infarction.1.A casecontrol group was used to include 50 patients between 18 and 75 years of age diagnosed with acute cerebral infarction(acute cerebral infarction group)and 30 healthy controls on physical examination during the same period(control group)who visited the emergency department of Taizhou People’s Hospital between June 2021 and December 2021).General clinical data such as age,gender,BMI(Body Mass Index),smoking history,hypertension and diabetes mellitus were collected and compared between the two groups.2.The expression level of NEAT1 in the blood test at the time of admission was measured in all subjects,and the ROC curve was plotted to assess the diagnostic value of LncRNA NEAT1 in acute cerebral infarction;3.The expression of inflammation-related factors Creactive protein,Calcitoninogen,Interleukin-6 and Nuclear Factor κΒ p65 in the peripheral blood of patients with acute cerebral infarction and healthy controls was detected by enzyme-linked immunosorbent assay,and the correlation between the expression levels of LncRNA NEAT1 and CRP,PCT,IL-6 and NF-κΒ p65 in the peripheral blood of patients with acute cerebral infarction was analyzed by applying Pearson correlation;4.To assess and record all patients’ The National Institute of Health stroke scale(NHISS)scores of all patients at admission were evaluated and the prognosis of patients was assessed using the Glasgow Outcome Score(GOS)to count survival and recurrence within 6 months.NEAT1 expression correlated with NHISS score and GOS score.Cellular experiment: 1.Human umbilical vein vascular endothelial cells were used,and they were divided into LncRNA NEAT1 sh RNA group(sh RNA group),LncRNA NEAT1 sh RNA negative control group(NC group)low and blank control group(Control group)according to the transfectants;2.The proliferation ability of cells in each group was detected using CCK-8 method;3.Apoptosis was detected in each group using Flow Cytometry;4.RT-q PCR was used to detect the cells;4.RT-q PCR to detect the difference in expression of apoptosis-related genes Bcl-2,Bax and Caspase-3 in each group of cells,and WB(Western blot,protein blotting)to detect the difference in expression of Bcl-2,Bax,Caspase-3 and NF-κΒ p65 at the protein level in each group of cells;5.ELISA to determine the expression level of IL-6 in the cell supernatant.Results:Clinical trial results: 1)General data of patients with acute cerebral infarction were not statistically different from healthy controls;2)Serum NEAT1 expression level was higher in patients with acute cerebral infarction group(3.03±1.46)than in healthy controls(1.01±0.57),p<0.001.area under the ROC curve AUC=0.8753,specificity was 96.67%,sensitivity was 76%,95% confidence interval 0.7999-0.9507,best diagnostic value 1.680.NEAT1 has diagnostic value in acute cerebral infarction.3)Peripheral blood inflammatory factors CRP(11.47 ± 11.15 vs.1.39 ± 1.35,P< 0.01),PCT(0.667 ± 0.434 vs.0.165 ± 0.184,P < 0.01),IL-6(68.61 ± 33.20 vs.19.29 ± 9.52,P < 0.01),NF-κΒ p65(3.87 ± 0.71 VS 3.27 ± 0.05,P< 0.05)were higher than those in healthy controls;NEAT1 expression levels were positively correlated with IL-6(P< 0.01);with PCT(P < 0.01);with p65(P< 0.01);and with CRP(P > 0.05);4)NEAT1 expression was positively correlated with NHISS score was positively correlated(r=0.7334,P<0.01);and negatively correlated with GOS score(r=-0.741,P<0.01).Cellular assay results:(1)CCK-8 assay showed that: the cell proliferation capacity and cell activity was enhanced compared with NC group and blank control;2)Flow cytometry showed that the number of apoptotic cells in sh RNA group was reduced compared with NC group and Control(7.96 VS 13.16 VS 13.44,P< 0.01).The expression of Bcl-2,which inhibits apoptosis,was increased in the LncRNA NEAT1 sh RNA group;the expression of Bax and Caspase-3,which promote apoptosis,was decreased in the sh RNA group.4)The expression of IL-6 in the supernatant of cells in the sh RNA group228.792±23.016 VS 145.892±16.276 VS 142.579±20.516,P<0.01) was decreased compared with the NC and Control groups.Conclusions:1.increased expression of LncRNA NEAT1 in the peripheral blood of patients with acute cerebral infarction has the potential to become a diagnostic marker for acute cerebral infarction.2.the expression level of LncRNA NEAT1 was positively correlated with inflammatory factors PCT,IL-6,NF-κΒ p65 and not with CRP,and may play a role in acute cerebral infarction by regulating the inflammatory response.3.the expression of LncRNA NEAT1 correlates with the severity of acute cerebral infarction and may predict the prognosis of ACI patients.4.LncRNA NEAT1 may be involved in the pathological process of acute cerebral infarction by promoting apoptosis and inhibiting vascular endothelial cell proliferation... |
PDF Full Text Request