| Background and ObjectivemiR-184-U(mutant miR-184,r.57 c > u)has been found in a variety of familial hereditary eye diseases,but the biological function and pathogenesis of mir-184-U in these eye diseases are still unknown.Previous studies have shown that circRNAs can be used as a sponge for certain miRNAs.However,no studies have investigated whether miRNAs or their mutant forms affect the expression of circRNAs.Therefore,in this study,whole-transcriptome of the m RNAs/circRNAs of each group was sequenced,and data analysis and experimental verification were performed after transfection of the chemical-synthesized miR-184-U mimics and miR-184-C(wild type miR-184)mimics into human lens epithelial cells,to preliminarily explore the biological function of miR-184-U and the pathogenesis of eye diseases caused by miR-184-U.Previous studies have confirmed that the terminal structure of RNA affects its function,but there are few studies on the flat-terminated double-stranded RNA.With the application of chemically synthesized RNA in more and more fields,there are ever-increasing attempts to adjust RNA structure.Therefore,we designed a new flat-terminated double-stranded RNA(dsRNA-184-U),whose sequence is similar to miR-184-U.Its seed region contains U bases,but its 3' end has no bases overshoot,andits biological function is studied in lens epithelial cells,to explore whether changing the terminal structure of RNA can make it acquire new targets and functions.Retinal neovascular disease seriously endangers vision and eye health.Previous studies have shown that a variety of miRNAs play an important role in the mechanism and regulation of ocular angiogenesis.The potential therapeutic effects of miRNAs are receiving special attention from academia and biotechnology companies.Our previous study found that both miR-184-U and dsRNA-184-U can reduce the level of cellular adenosine-triphosphate(ATP),and the process of retinal neovascularization requires the consumption of a large amount of ATP.Therefore,we designed animal experiments to investigate the effects of miR-184-U and dsRNA-184-U on retinal neovascularization.MethodsIn the first part of the study,whole-transcriptome of m RNAs/circRNAs from lens epithelial cells treated with miR-184-C,miR-184-U and a negative control were sequenced using two different RNA libraries.In order to identify the new genes affected by miR-184-U,we performed an integrated analysis of the data and verified the genes with significant differential expression between the miR-184-U group and the control group by RT-q PCR and western blot.Flow cytometry was used to detect the level of apoptosis in each group.According to the results of the validation experiment,we detected the intracellular ATP levels after transfection of miR-184-U,dsRNA-184-U,and control RNA in the second part of the study.The target genes of miR-184-U and dsRNA-184-U were detected by the luciferase reporting assay.Direct interactions between dsRNA-184-U and intracellular proteins were detected by the RNA pull-down assay and mass spectrometry.The expression and localization of the target protein were detected in situ by immunofluorescence,and the transfer of the target protein was verified by WB detection after the separation of the membrane protein and the cytoplasmic protein.Finally,the direct interaction between dsRNA-184-U and the target protein was simulated by computer software.In the third part of the study,we used oxygen-induced retinopathy model mice todetect the effect of intravitreous injection of miR-184-U and dsRNA-184-U on retinal neovascularization.Fitc-dextran fluorescein was used to display retinal blood vessels in retina-stretched preparation,and the neovascular area was quantified by computer image analysis.ResultsThere were 16 significantly differentially expressed genes(DEG)between miR-184-U treated Human lens epithelium cells and miR-184-C treated HLE cells.There were 47 significant DEGs between the miR-184-C and NC groups.There were 63 significant DEGs between the miR-184-U and NC groups.Compared with wild-type miR-184-C,miR-184-U down-regulated wider range genes.The apoptosis rates of miR-184-C,miR-184-U,and NC groups were not significantly different.miR-184-U can reduce the m RNA and protein expression levels of ALDH5A1 and GABRA3,both of which are related to the alanine,aspartic acid,and glutamate metabolic pathway.The circRNAs expression pattern is globally variable in a single cell type.Under our experimental conditions,the total circRNAs expression and the number of circRNAs chromosome origins also seemed to be random.The interactions between these miRNAs and circRNAs are compatible rather than exclusive.circRNAs are abundant in ALU sequence and have binding sites of miR-184-C and miR-184-U.Both dsRNA-184-U and miR-184-U can down-regulate the levels of ALDH5A1 and ATP in human lens epithelial cells.miR-184-U directly binds to the 3'UTR of ALDH5A1 m RNA,while dsRNA-184-U does not.There is a physical interaction between dsRNA-184-U duplex and DHX9.At the same time,dsRNA-184-U p2(a single strand of dsRNA-184-U)also has physical interactions with ATP5 A and PKM2.The overexpressed dsRNA-184-U can directly act on ATP5 A and release it from the membrane into the cytoplasm.Compared with the control group,the proportion of neovascular area in the total retinal area of the miR-184-U group was significantly reduced.However,the proportion of neovascular area in the total retinal area of the dsRNA-184-U group did not reach a statistically significant level,although there was a decreasing trend.ConclusionsOverexpression of miR-184-U in HLE cells directly inhibited the expression of ALDH5A1 and indirectly down-regulated the expression of GABRA3.The targets may be alanine,aspartic acid,and glutamate metabolic pathways,and may further affect the tricarboxylic acid cycle.This provides a new perspective for understanding the mechanism of progression of eye diseases related to miR-184-U.dsRNA-184-U and miR-184-U have different mechanisms for down-regulating ALDH5A1 expression and decreasing ATP production.miR-184-U binds to the 3'UTR of ALDH5A1 m RNA and silences its expression,also possibly reducing ATP production by affecting the tricarboxylic acid cycle.DHX9 may be used as the duplex identification factor of dsRNA-184-U to unscrew the dsRNA-184-U duplex into a single strand.The released single strand(the seed region containing the U base)interacts with ATP5 A,causing the release of ATP5 A from the mitochondrial membrane into the cytoplasm.This may have resulted in impaired ATP synthase activity,thus resulting in reduced ATP production and indirectly reducing ALDH5A1 expression.dsRNA-184-U provides a new possible mechanism for regulating the activity of ATP synthase and may be helpful for the discovery of therapeutic agents that regulate the abnormal expression of ATP.The intravitreous injection of miR-184-U can significantly inhibit retinal neovascularization.The possible mechanism by which this occurs is through affecting the proliferation and migration of vascular endothelial cells by interfering with the generation of ATP,further inhibiting the neovascularization.Although dsRNA-184-U can also inhibit the production of intracellular ATP,it may not significantly inhibit the neovascularization in this experiment due to the lower inhibition degree than miR-184-U and the complex internal environment of animals.However,dsRNA-184-U may still have the potential of clinical applications for other diseases caused by ATP disorders.Further in vivo and in vitro experiments are needed to confirm our hypothesis. |
PDF Full Text Request