MicroRNA204-5p Is Involved In Early Diabetic Retinopathy Via Regulating Microtubule-associated Protein 1 Light Chain 3 ?

Background:Diabetes mellitus(DM)is one of the most important non-communicable diseases that threaten the healthy of global human currently.Diabetic retinopathy(DR)also become a serious issue as diabetes patients continue to increase,accounting for 5% of all blind patients.DR is one of the most common microvascular complications of diabetes.The main features are occlusion and exudation of retinal microvasculature,causing macular edema,ischemia and hypoxia to promote retinal neovascularization,and proliferative diabetic retinopathy(PDR).what can make the retinal vitreous hemorrhage easily,and resulting in tractional retinal detachment(TRD),than,the vision of patient is damaged severely,and even lead to blindness.At present,most studies believe that the main reason for the development of DR is a series of metabolic abnormalities caused by persistent hyperglycemia,which damage to the retinal microvascular system,change permeability,and destruction of the blood-retinal barrier.However,strict control of blood glucose levels does not completely prevent the occurrence of DR,and DR has a family clustering tendency.The incidence of DR in different ethnically diabetic patients is also different.This shows that the pathogenesis of diabetic retinopathy is very complicated and needs further study.Autophagy is a conservative evolutionary process for the degradation and reuse of intracellular components,and it is also an important process for maintaining cell stability.Under certain conditions,autophagy stimulates the physiological turnover of aging and damaged organelles,thereby controlling the cells through various signaling pathways.Diabetes increases inflammation in the retina and its capillaries,increases advanced glycation end products(AGEs)and oxidative stress;a large body of evidence suggests that there is a gap between oxidative stress and other metabolic abnormalities involved in development of DR.In addition,more and more data highlight the key role of reactive oxygen species(ROS)in autophagy activation.This provides important clues for the study of autophagy in DR.MicroRNAs(miRNAs)are non-encoded single-stranded RNAs that are widely present in organisms and are 20-25 bases in length.miR-204 is expressed by TRPM3 intron 6 located on chromosome 9q21.12.Studies have shown that microRNAs is involved in diabetes,miR-375 and miR-9 play an important role in insulin synthesis and pancreatic cell release.MiR-103,miR-107,miR-143,miR-144 and miR-145 can also regulate peripheral insulin sensitivity in liver tissues and skeletal muscles of the insulin receptor pathway.In ocular structure,miR-204 is involved in the occurrence and development of age-related cataract,diabetic corneal and other diseases.This makes miR-204 and autophagy become the pointcut in this study,providing a new perspective for our research on the molecular mechanisms of diabetic retinopathy.Objective:To determine the expression of miRNA-204 and LC3 in the retina of DR rats,to explore the relationship between miRNA-204 and LC3 that what mechanisms DR is.Methods:The male Sprague-Dawley(SD)rats weighing near 200 g were randomly divided into control group(control group)and diabetic retinopathy group(DR group).The rats of DR group were intraperitoneal injection of 60 mg/kg body weight of STZ,and the rats of control group intraperitoneal injection of equal amount of citrate buffer.The general physiological indicators such as body weight and blood glucose were measured on 72 hour,1 week,4week and 8week.HE staining was performed on the retinal tissue at random on 10 week.The rats were divided into control group,DR0 group,DR group,anti-mir group,and mimic group,with a total of 5 groups,and n=10 for each group.The real-time quantitative PCR(qRT-PCR)was used to detect the expression of miRNA-204.Western blot was used to detect whether LC3 II was altered in the DR.Using the adeno-associated virus(AAV),miR204-5p and anti-miR204-5p were each packaged into viral vector(AAV-U6-Anti-rno-miR-204-5p-CAG-EGFP;AAV-U6-mimic-rno-miR-204-5p-CAG-EGFP);The above-mentioned AAV vectors were transfected to rats and expressed in retina,than,detecting the expression of LC3 II in that retina.Results:The results show that,the expression of miR-204-5p in rat retina tissue was significantly higher in DR group than control group;and the expression of LC3 II was significantly inhibited in DR group than control group.After the AAV vector were transfected in rats,the expression of LC3 II in anti-miR group was significantly inhibited compared to the DR group.Conclusions:In early diabetic retinopathy,miR-204 negatively regulates the expression of autophagy-related protein LC3 II.