| Object:Diabetic retinopathy(DR)is a common complication of diabetes and is a major cause of vision loss in middle-aged and elderly people,with the increasing numbers of people with diabetes living longer,the number of people with diabetic retinopathy is rising worldwide.So far,the treatment of DR is limited and costly,therefore,early screening and prevention are crucial.Recently,the potential of exosomal miRNAs serve as biomarkers are being discussed.Derived from almost all cells,exosomes are a class of extracellular vesicles with a diameter of 40 to 160 nm and can be found in all body fluids such as blood and urine.They also contain nucleic acids and other substances,which have biological functions and can be absorbed by the recipient cells to realize the material transportation and information transmission.For all these,making exosomal miRNAs to be the candidate biomarkers for disease pathogenesis such as DR.Here,we aim to investigate the potential circulating exosomal miRNAs associated with DR.Our studies will provid a better understanding of the pathological process in DR and provided a theoretical basis for further research.Methods:1.Collection of blood samples: patients with diabetes who meet the criteria was selected from Wuhan Union Hospital from September 2020 to January 2021,we use medical vacuum blood collection tubes for blood drawing,after centrifuge,the upper aqueous phase will be transferred to a new collection tube and stored at-80℃ for later experiment.2.We use the international "gold standard" ultracentrifugation method for extracting exosomes to extract serum exosomes,and then identify serum exosomes as well as miRNA sequencing.3.Using R4.0.3 software to perform gene enrichment analysis,GO analysis,KEGG pathway analysis and target gene prediction for differential miRNAs based on the sequencing results.4.After reverse transcription and amplification,real-time fluorescent quantitative PCR(q PCR)is used to detect and verify differential miRNAs.5.Use R4.0.3 software to statistically analyze the correlation between serum exosomal differential miRNA and clinical characteristics.Results:1.After the serum is subjected to ultracentrifugation to extract exosomes,the products are identified by NTA,TEM,and WB.The appearance of cup-or sphere-shaped morphology can be seen under the transmission electron microscope,and the NTA result indicates the size of the nanoparticles is basically in the distribution range of 30-200 nm,and the average particle size is 140 nm.The WB results showed that three specific markers of CD9,CD63 and TSG101 were detected in the exosomes samples.The results confirmed that the isolated circulating nanoparticles were exosomes..2.With the DM group as the control and the DR group as the experimental group,the extracted exosomes were subjected to miRNA sequencing,and a total of 1059 miRNA results were screened,18 of which were statistically significant,and their expressions were all up-regulated.3.Perform gene enrichment analysis,KEGG,GO on the selected exosomal miRNAs,and predict the biological functions involved and the enriched signal pathways.The results show that the exosomal miRNAs in the DR group target to many genes that are mainly concentrated in signal transduction and transcriptional regulation;as for cell components,they mainly concentrated in membranes,cytoplasm and so on;and in molecular functions,these genes mainly concentrated in protein binding,metal ion binding.At the same time,KEGG obtained a total of268 related pathway information,48 of which were statistically significant.Subsequently,the miRNA targeted gene prediction of the differential miRNA miR-3976-p3 was performed,and the most significant miRNA:m RNA pair in the miR-3976-p3 target gene enrichment analysis results was composed of a network diagram.4.After qPCR verification,there was no difference in whole blood miRNA,and serum exosomal miRNA in the DR group was significantly higher than that in the DM group5.We used Pearson’s correlation coefficient test to explore the correlation between the expression of different miRNAs in the population and clinical characteristics.The results showed that the expression of miR-3976-p3 in DR patients was significantly correlated with HDL levels.In the DR group,the expression of pc-5p-39533 is significantly correlated with height.Conclusions:1.Compared with the DM group,it is the serum exosomal miRNA,not the whole blood miRNA,was significantly increased in the DR group.2.miR-3976-p3,PC-5p-39533,PC-3p-37421 may play an important role in the occurrence and development of diabetic retinopathy,as well as their potential to become the biomarkers of DR.3.Low level of HDL or poor islet function may be the risk factors of diabetic retinopathy. |
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