Detection Of Circulating MiRNAs From Hyperoxia Induced ROP Model SD Rats And The Related Validation Studies

Backgrouds and ObjectivesRetinopathy of prematurity (ROP) is one of the retinal disease with vascular proliferation mainly by premature delivery and low birth weight, and it is main factor for blind children. The main pathological changes of ROP is the formation of retinal neovascularization, reports showed that vascular endothelial growth factor (VEGF) is the strongest angiogenesis factors, and it expression level will change significantly. So it is very important to study the related regulation factors for VEGF more in depth.Recently, microRNA (miRNA) as one of the important non-coding small RNA became biomedical research hotspot since it was discovered. miRNA has been verified key factors in many human disease. The non-invasive circulating miRNA in plasma and serum were also showed to be important in clinical researches. It is necessary to screen the circulating small RNAs and its related target genes for retinal angiogenesis. High-throughput DNA sequencing technology as one of the most promising techniques could be used for miRNA detection in this present study.Although ROP premature with high incidence, it is very difficult to collect full samples for the related detection and study. Model animals such as mouse and rats were often used in these studies and the ROP model mouse and model rats had been successfully constructed by many previous studies. We selected rats as ROP model animal in this study for miRNA detections. Hyperoxia has been verified to be the main important pathogenic factor for ROP, so we will construct hyperoxia-induced model rats for miRNA sequencing and analysis in this present study.In this present study, we will detect the circulating miRNA expression level in plasma of hyperoxia-induced model rats by the next generation sequencing technology, then following the microRNA target prediction, GO function enrichment and KEGG pathway analysis. Molecular biological verification was also carried out and the value of miRNA as biomarker for ROP research and diagnosis was also evaluated. The main research results of the thesis are as follows:Methods and Results1) The construction of ROP model rats and the circulating miRNA deep sequencingIn this study, the newborn SD rats were raising in high oxygen environment for ROP model construction and assessment. Then plasma samples were collected and RNA extracted, the miRNAs were sequenced after library preparation. The main results are as follows:1) the model rats induced by hyperoxia were proved to be successful through typical characteristics of retinopathy. Observed result showed that the diameter of vascular vessels was thin and branches decreased, large part of non-perfusion area shown both in the center and periphery, and more vascular endothelial cells breaking through the retinal internal limiting membrane were found.2) Deep sequencing results showed that some differential expressed miRNAs were sequenced with strict filtering standards. Among them 22 miRNAs were up-regulated and 44 miRNAs were down-regulated, and most of miRNAs involved in the following verification studies. An additional interesting finding showed the ratio of miRNA 5p/3p expression level was also different between model rats and controls.2) High-throughput screening of the retina tissue miRNA from ROP model ratFor more comprehensive analysis of the differential miRNAs, the retina tissue were also collected and total RNA was extracted at the same time for high-throughput screening with Agilent chips and bioinformatics analysis. The results could be used for comparison with the differential expressed circulating RNAs.13 up-regulated miRNAs and 14 down-regulated miRNAs were found by Agilent chip detection with strict filtering standards. The differential miRNA target gene function enrichment of shows responses to low oxygen and other related functions and regulation pathways on the GO bioprocess, it proved to be great value for further analysis of the differential expressed circulating miRNA.3) Analysis of the differential circulating miRNAs and function related validation rsearchesmain aim of this study is investigation of the functional mechanism of miRNAs in ROP and its relation with the genesis and development of ROP. Some of the differential circulating miRNAs were verified by qRT-PCR. VEGF and GABA level in plasma were also detection by ELISA among the model rats and control samples. ROP specific miRNA was obtained by comparing the circulating miRNAs and retina tissue miRNAs, plasma samples from 5 premature were used for verification. The results are as follows:1) The expression level of ten differential miRNAs including high expressed, low expressed, high differences, low differences, up-regulated and low regulated were confirmed by qRT-PCR in additional model rats samples, as well as the miRNA with 5p/3p ratio; 2).594 coexpressed target genes were predicted by TargetScan and PicTar. GO and KEGG analysis results showed that most of the genes related with Embryonic development ending in birth or egg hatching, Cell migration, Cell motion, Chordate embryonic development, Cell morphogenesis, Cell motility, Cell projection organization, Positive regulation of cellular biosynthetic process and the MAPK signaling pathway, GnRH signaling pathway, mTOR signaling pathway, TGF-beta signaling pathway, Vascular smooth muscle contraction.3). The interaction analysis of miRNAs, VEGF and GABA showed that several miRNAs involved in the regulation of VEGF and GABA through coordination and mutual restrictive. Some of the miRNAs such as miR-351, miR-200b and miR-199a etc. which targeted to VEGF and GABA would be potential biomolecules for ROP diagnosis and treatment.4) The co-expressed differential miRNAs were detected by qRT-PCR in 5 premature infants, and was confirmed in 3 of them. The one ROP infants which obtained inconsistent results was showed to be accompanied with congenital heart disease, and it could be inferred that the miRNAs detected might also differential expressed in congenital heart disease.ConclusionsIn summary, some differential miRNAs were obtained from hyperoxia induced model rats by deep sequencing, parts of the differential miRNAs were confirmed by qRT-PCR. These miRNAs were showed to be involved in ROP by bioinformatics analysis of the target genes and functional verification. Several miRNAs which also differential expressed in retina tissues were detected in plasma samples from five infants with typical retinopathy of premature.