Analysis Of LncRNA Expression And Functional In Lens Epithelial Cells Of Age-related Cataract Patients

Objective:Age-related cataract is the most common cause of vision impairment in the elderly worldwide,and it is the result of the combined influence of genetic and environmental factors.Oxidative stress and apoptosis play an important role in its pathogenesis.Recently,it has been found that long non-coding RNA(lncRNA)plays a role in cell function and disease progression,but the expression and functional mechanism of lncRNA in lens epithelial cell of ARC patients are still uncertain.The purpose of this study is to explore the expression profile of lncRNA in lens epithelial cell of patients with ARC,and to further explore the role of significantly differentially expressed lncRNA TCONS＿00018537 in the pathogenesis of ARC.Methods:(1)According to the screening and exclusion criteria,the anterior lens capsule tissues(18 eyes,18 cases)of ARC patients who underwent cataract surgery in Qingdao Eye Hospital affiliated to Shandong First Medical University were taken as the experimental group,and all patients signed the informed consent form.At the same time,9 cases(9 eyes)of transparent lens anterior capsule tissue donated from the eye bank of Shandong Institute of Ophthalmology were used as the control group.Specimens were collected according to the principles of Helsinki Declaration and approved by the ethics committee.(2)The RNA contained in one anterior capsule tissue is not enough for lncRNA sequencing,so we take three anterior capsule tissues as a test sample,namely,6 samples in the experimental group and 3 samples in the control group.Total RNA of each sample was extracted,sequencing library was constructed,and quality control and quantification were carried out.According to Illumina sequencing instructions,150 cycle of sequencing were performed to obtain lncRNA data.(3)The multiple change and p-value between the two groups were calculated,and the differentially expressed lncRNAs were obtained using fold change ≥ 2.0 and p-value ≤ 0.05 as screening criteria.Mi Randa and Target Scan(Cloud-seq,Biotech,Shanghai,China)were used to determine the miRNA targets of lncRNA.(4)Combined with previous studies and sequencing results,qRT-PCR was used to detect the expression levels of three significantly changed lncRNA with miR-30a-3p response elements and miR-30a-3p.To determine the m RNA target gene function of miR-30a-3p,we used Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and genomes(KEGG)pathway analysis.The first 10 enrichment GO entries and the first 16 KEGG pathways were annotated.The relationship between miRNA,lncRNA and m RNA was verified by dual-luciferase reporter assay.Results:(1)lncRNA expression profile: a total of 363 significantly different lncRNAs(FC ≥2.0,p-value ≤ 0.05)were detected between the two groups,among which 267 lncRNAs were significantly up-regulated and 96 were significantly down-regulated.(2)qRT-PCR verification: the qRT-PCR verification results of three lncRNA with miR-30a-3p response elements and significantly up-regulated sequencing results showed that their change trends were consistent with the sequencing results,and had statistical significance.So we initially selected TCONS＿00018537 as the research target.(3)GO and KEGG analysis: the results showed that the m RNA target genes of miR-30a-3p were enriched to 451 GO entries and 47 KEGG entries with statistical significance.Growth regulation(GO: 0048638),trans-Golgi network(GO: 0005802)and histone binding(GO: 0042393)account for the largest proportion in Biological Process,Cell Component,and Molecular Function.There are many items in KEGG pathway analysis related to oxidative stress and apoptosis of cells.(4)Dual-luciferase reporter assay: the results showed that miR-30a-3p may directly bind to TCONS＿00018537 and CASP3 through miRNA recognition sites.Conclusion:(1)The expression profile of lncRNA in LEC of ARC patients was significantly changed compared with that of the control group,providing a new research direction for the pathogenesis of ARC.(2)TCONS＿00018537 is an up-regulated lncRNA in the anterior capsule of lens in patients with ARC.It may induce LEC apoptosis by sponge adsorption of miR-30a-3p,and it plays an important role in the pathogenesis of ARC,may become a new target in the diagnosis and treatment of ARC.