| Objective: In this current thesis,we screened serum specific micro RNA(mi RNA)in patients with diabetic retinopathy(DR)in clinical study section,and detected the expression of candidate mi RNA and its target gene in retina of diabetic rats in vivo experimental research section,meanwhile validated the targeting regulation of candidate mi RNA on its target gene in hyperglycemic human umbilical vein endothelial cells(HUVEC)in vitro experimental research section.The purpose of the current thesis is to investigate the theoretical principle of serum mi RNA as a biomarker for occurrence warning,diagnosis stage and prognostic assessment in patients with DR.Methods: In clinical study section,mi RNA microarray analysis was used to screen a serum specific mi RNA expression profile in patients with DR;Venn diagram analysis was used to determine the candidate mi RNAs of serum specific mi RNA expression profile in patients with DR;real-time quantitative PCR(q PCR)was used to measure the expression level of serum candidate mi RNAs in patients with DR;receiver operating characteristic(ROC)curve was used to evaluate the diagnostic accuracy of serum candidate mi RNAs for patients with DR;bioinformatic analysis was used to predict the biological function and signaling pathways of target genes of serum candidate mi RNAs in patients with DR.In vivo experimental research section,adenosine diphosphatase(ADPase)staining was used to observe retinal morphological characteristics of diabetic rat;hematoxylin-eosin(HE)staining was used to observe retinal histologic characteristics of diabetic rat;q PCR was used to measure the expression level of candidate mi RNA and its target gene m RNA in retinal tissue of diabetic rat;western blot was used to measure the expression level of target gene protein of candidate mi RNA in retinal tissue of diabetic rat.In vitro experimental research section,dual-luciferase reporter assay was used to validate targeting binding effect between candidate mi RNA and its target gene;Cell Counting Kit-8(CCK-8)was used to detect the effect of candidate mi RNA on cell viability of HUVEC in hyperglycemic environment;flow cytometry was used to detect the effect of candidate mi RNA on cell apoptosis of HUVEC in hyperglycemic environment;q PCR was used to detect the effect of candidate mi RNA on the expression level of its target gene m RNA of HUVEC in hyperglycemic environment;western blot was used to detect the effect of candidate mi RNA on the expression level of its target gene protein of HUVEC in hyperglycemic environment.Results: In clinical study section,mi RNA microarray analysis indicated that there was a serum specific mi RNA expression profile in patients with DR;Venn diagram analysis indicated that 4 candidate mi RNAs of serum specific mi RNA expression profile in patients with DR,in which the expression level of serum mi R-18 b,mi R-19 b and mi R-211 were up-regulated,while the expression level of serum mi R-23 a was down-regulated;q PCR indicated that the expression tendency of serum mi R-18 b,mi R-19 b and mi R-211 were consistent with those in serum specific mi RNA expression profile,while the expression tendency of serum mi R-23 a was not consistent with it in serum specific mi RNA expression profile;ROC curve indicated that serum mi R-18 b,mi R-19 b and mi R-211 revealed significant diagnostic accuracy for patients with DR,in which mi R-211 is the most powerful,while serum mi R-23 a revealed no diagnostic accuracy for patients with DR;bioinformatic analysis indicated that 2081 target genes were predicted by mi R-18 b,mi R-19 b and mi R-211 in all,and Sirtuin 1(SIRT1)may be a target gene of mi R-211;biological process of target genes predicted by candidate mi RNA involved transcription,DNA-templated,positive regulation of transcription from RNA polymerase II promoter and regulation of transcription,DNA-templated,etc.,cellular component of target genes predicted by candidate mi RNA involved cytoplasm,nucleus and plasma membrane,etc.,molecular function of target genes predicted by candidate mi RNA involved protein binding,metal ion binding and DNA binding,etc.;signal pathway of target genes predicted by candidate mi RNA involved axon guidance signal pathway,adrenergic signaling in cardiomyocytes signal pathway and endocytosis signal pathway,etc.In vivo experimental research section,ADPase staining indicated that the density of neovascularization was significantly increased in the retina of diabetic rat,and aggravated gradually associated with the diabetic duration;the number of preretinal neovascular cell nuclei was significantly increased in the retina of diabetic rat,and aggravated gradually associated with the diabetic duration;q PCR indicated that the expression level of mi R-211 was significantly up-regulated in retinal tissue of diabetic rat,and increased gradually associated with the diabetic duration;q PCR indicated that the expression level of SIRT1 m RNA was significantly down-regulated in retinal tissue of diabetic rat,and decreased gradually associated with the diabetic duration;western blot indicated that the expression level of SIRT1 protein was significantly down-regulated in retinal tissue of diabetic rat,and decreased gradually associated with the diabetic duration.In vitro experimental research section,dual-luciferase reporter assay indicated that mi R-211 and its target gene SIRT1 represent as target binding effect;CCK-8 indicated that the cell viability of HUVEC transfected with antagomi R-211 was significantly increased in hyperglycemic environment;flow cytometry indicated that the cell apoptosis of HUVEC transfected with antagomi R-211 was significantly decreased in hyperglycemic environment;q PCR indicated that the expression level of SIRT1 m RNA was significantly up-regulated in HUVEC transfected with antagomi R-211 in hyperglycemic environment;western blot indicated that the expression level of SIRT1 protein was significantly up-regulated in HUVEC transfected with antagomi R-211 in hyperglycemic environment.Conclusion: The current clinical study and experimental research imply that there was a serum specific mi RNA expression profile in patients with DR;serum mi R-211 as a novel biomarker with high sensitivity and specificity could be associated with occurrence and progression of DR via targeting SIRT1. |
PDF Full Text Request