| Objective:Age-related cataract(ARC)is one of the major causes of blindness worldwide.With the deepening of the aging of the population in China,the prevalence of ARC is also increasing and has therefore become the main medical public problems.Nowadays,circular RNA(circRNA)has become a current research hotspot because of its ability to participate in multiple regulatory processes in various ocular diseases.However,the expression profile,regulatory roles and underlying mechanisms of circRNAs in ARC remain largely unknown.This study deep-sequenced circRNAs of anterior lens capsules from normal and ARC lenses,and preliminarily explore the possible functions of differentially expressed circRNAs as mi RNA sponge molecules in the occurrence and development of ARC.Methods:(1)Anterior lens capsules were obtained from ARC patients who underwent ARC surgery at our hospital(18 eyes),and we used these samples as the disease group.Transparent anterior lens capsules from healthy donor eyes were used as normal controls(9 eyes).(2)RNA was extracted from all samples.After extracting and quantifing RNA from all samples,the library was sequenced by next generation sequencing(NGS)and circRNA sequencing data were obtained.(3)A standard threshold(fold change≥2.0,p-value ≤0.05)was set to obtain differentially expressed circRNAs between the control group and the disease group.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed on the host genes of differentially expressed circRNA.mi RNA targets for circRNAs and m RNAs were predicted using target prediction software based on mi Randa,Targetscan and star Base.circPrimer1.2 and UCSC Genome Browser were used to annotate the circRNA composition and its location on the parental genes.(4)q RT-PCR was adopted to check the expression of circRNAs and mi RNA in ARC.A dual-luciferase reporter assay was performed to confirm the direct binding of circRNA-mi RNA.Results:(1)Totally,23,787 circRNA candidates were detected across all samples.Among them,63.3% were overlapped with published ones obtained from circBase,while 36.8%circRNA candidates were newly identified in our study.A total of 466 circRNAs were significantly differentially expressed.(2)GO and KEGG enrichment analyses of significantly up-regulated circRNA host genes showed that 434 GO terms and 14 KEGG terms were enriched(P<0.05).Among them,the GO function terms included 293 Biological Process(BP)terms,61 Cell Component(CC)terms and 80 Molecular Function(MF)terms.The significantly down-regulated circRNA host genes were enriched into 180 GO terms(114 BP terms,26 CC terms and 40 MF terms)and 62 KEGG terms.Many of these functional terms were involved in oxidative stress and apoptosis-related signaling pathways.(3)q RT-PCR was used to verify expression levels of randomly selected circRNAs and candidate circRNAs,which showed a strong agreement to the patterns detected by RNA-Seq.The expression trends of circZNF292 and mi R-23b-3p in ARC tissues are opposite.The circRNA-mi RNA targeting region prediction databases and dual luciferase reporter assay showed that circZNF292 interacts with mi R-23b-3p.Conclusion:(1)Compared with the normal group,the expression of circRNAs in ARC group were significantly different.Significantly differentially expressed circRNAs can participate in various important cell signaling pathways related to the pathogenesis of ARC.(2)circZNF292 could regulate the expression of antioxidant genes in LECs by sponging the mi R-23b-3p,providing a potential target for ARC prevention and treatment. |
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