Identification And Characterization Of Proliferative Retinopathy-related Long Noncoding RNAs

Objective:Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and vitreoretinal surgery, which can lead to severe vision reduction.Long non-coding RNAs (lncRNAs,>200 nucleotides) constitute a novel class of non-coding RNAs that play critical roles in many biological processes and in the development of diseases. Although recent data reveal that lncRNAs may be associated with several ocular diseases, little is known about the role of LncRNAs in the setting of proliferative vitreoretinopathy (PVR).Methods:Total RNAs were extracted from the epithelial membranes (ERMs) surgically removed from the eyes of PVR patients and the eyes with the secondary ERMs that developed after cataract. LncRNA microarray was conductedto identify differentially expressed lncRNAs between PVR-ERMs and the secondary ERMs.Quatitative reverse transcription polymerase chain reactions (qRT-PCRs) were employed to verify theexpression pattern of some of these differentially expressed lncRNAs. We thus selected a specific lncRNA as the target for thefollowing study.To further determine the expression pattern oftarget lncRNA duringPVR progression, we further collected the clinical samples and extracted the total RNAs from the retinas oftrauma patients, PVR-ERMs, and secondary ERMsdeveloped after cataract surgery. qRT-PCRs were conducted to compare the expression of target lncRNA during the three group samples.Meanwhile, we detected theexpression of PDGFA, PDGFC, Collagen I and KNGlas well as lncRNA-MIAT, RNCR3,TUG1 in the traumatic retinas, secondary ERMs, and PVR-ERMs.The blood samples from different grade PVR cases were collected and divided intothe cellular fraction and plasma fraction. qRT-PCRs were used to detect the target lncRNA expression. The same experiments were also administrated to identify the target lncRNA expression of PVR patients after operation treatment.In intro studies, we investigated the roleof target lncRNA in RPE viability, proliferation and migration upon thestimulus of TNF-α/PDGF-A/IGF-1 through MTT, Tyran blue assay, JC-1 staining and wound healing assay, respectively.Results:Microarray analysis revealed that a total of 78 lncRNA transcripts were differentially expressed in theERMs of PVR patients, including 48 up-regulated and 30 down-regulated lncRNAtranscripts. qRT-PCRs verified 11 differentially expressed lncRNAs in the ERMs of PVR patients.Notably, MALAT1 expression targeted by three different probes was up-regulated. We subsequently focused on lncRNA-MALATl, and investigated its expression pattern in the biofluiod of PVR patients. MALAT1 was significantly up-regulated in the cellular and plasma fraction of the peripheral blood in PVR patients. MALAT1 expression was obviously reduced after PVR operation. Moreover, oligonucleotide-mediated knockdown implicated the role of MALAT1 in regulating RPE cell proliferation and migrationinvolved in the formation of ERMs.Conclusions:LncRNAs are implicated as the modulators of the pathological response of PVR progression. Among them, MALAT1 might be considered as a potential prognostic indicator and a target for the diagnosis and gene therapy for PVR diseases.