Differentially expressed miRNAs in premature infants with retinopathy-a bioinformatics analysis

1Department of Neonatology, Children’s Hospital Affiliated to Nanjing Medical University, Nanjing 210008, Jiangsu Province, China

2Department of Neonatology, Jiangsu Provincial People’s Hospital Affiliated to Nanjing Medical University, Nanjing 210029, Jiangsu Province, China

Abstract· AlM: To reveal the role of miRNAs in retinopathy of prematurity (ROP) by bioinformatics analysis.· METHODS: The raw data of this study came from the researches of Wang et al and Zhao et al who analyzed the microRNA (miRNA) expression profile between ROP and controls. Based on the identified differentially expressed miRNAs, the related target genes, lncRNA and circRNA were predicted. Then we performed functional enrichment analysis to further analyze the functions of target genes.· RESULTS: Hsa-miRNA-128-3p and hsa-miRNA-9-5p showed significantly different expression in both studies.LncRNA of POLDlP2, GAS5, NEFL and UHRF1, circRNA of ZNF280C_hsa_circ_001211 and SlAE_hsa_circ_002083,tar get gene of QKl showed meaningful differential expression in ROP. Enrichment analysis showed that TGF-β signaling pathway, Pl3K-Akt signaling pathway and MAPK signaling pathway might play important roles in the prog ress of ROP.· CONCLUSlON: This research may provide a comprehensive bioinformatics analysis of differentially expressed miRNAs which are possibly involved in ROP.

INTRODUCTION

Retinopathy of prematurity (ROP) is a potentially preventable neurovascular disorder, which is estimated to make 20 000 infants blind or severely visually impaired each year worldwide[1]. No matter the gestational age at birth,and no matter the ultimate severity of the retinopathy, the onset of ROP seems to start at approximately 32wk gestation[2]. With the maternal age growing, very premature infants gradually increased. Therefore, patients with ROP become common in clinical practice despite developed neonatal care technology and good management of oxygen inhalation. Although the assistance of cryotherapy, laser photocoagulation and antivascular endothelial growth factors (VEGF) drugs has resulted in a remarkable reduction in mobility and improved prognosis,treatment of this dis ease has remained difficult. The key problem for pediatricians is the physiopathologic mechanism of ROP remains unclear yet.

microRNAs (miRNAs) are small non-coding RNAs of 18-26 nucleotides in length that regulate gene expression through sequence-specific base pairing with target miRNAs. miRNA has been reported to be important in various biological processes including vascular angiogenesis, cell growth, embryonic development, cell proliferation, tissue differentiation and apoptosis[3-6]. It is well documented that miRNAs could regulate vascular angiogenesis[7]. And the regulation of VEGF, IGF-2 and HIF-1α by miRNA-126 is associated with retinal vascular angiogenesis under conditions of oxygen-induced retinopathy(OIR)[8], which suggested miRNA’s possible role in ROP.

By carefully reviewing the literature in databases (PubMed and Embase), there’re only two miRNA profile papers reporting identification of differential miRNAs in ROP newborns.One of them is our previous report[9], with the assistance of microarrays and subsequent reverse transcription-polymerase chain reaction (RT-PCR), we first revealed the involvement of miRNA in ROP by screening miRNAs expression in retinal tissue of mice. In another miRNA article, Zhao et al[10] described an initial use of deep sequencing to comprehensively profile miRNAs in the plasma of ROP rats induced by hyperoxia.

Because miRNA sequence is well conservative among species,it gives us a good opportunity to look for common significant miRNAs by using these raw data. In the present study, we took advantage of previous data to analyze the miRNA expression pro file between ROP group and control group. Besides, basedon the miRNAs with differential expression, we predicted the relevant target genes, lncRNA and circRNA. In addition, we performed further functional enrichment analysis to analyze the functions of target genes. We aim to explore the involvement of miR NAs in the progression of ROP.

SUBJECTS AND METHODS

Data Source The data in our study came from the study of Wang et al[9] and Zhao et al[10]. Wang et al[9] analyzed the miRNA profile data of retinal tissue between 40 C57BL/6 neonatal mice with ROP and 40 without ROP (controls)on postnatal day 17. Finally, 67 differentially expressed miRNAs were identified. Zhao et al[10] compared miRNAs in neonatal Sprague-Dawley rats. They found 66 differentially expressed plasma miRNAs by deep sequencing technology on postnatal day 14. Based on the above data, we perform a stricter screening standard which is the significant miRNAs are selected from the intersection of the above two articles (Wang et al[9] and Zhao et al[10]). And fold change must fulfill greater than two in both studies. In addition, miRNA sequence must be well conservative between human and rodent. After screening,only 1 up-regulated and 1 down-regulated miRNAs passed the test, and were further selected into this study (Table 1). Based on these findings, we try to identify the roles of miRNA in ROP.Target Gene Prediction The target genes of miRNAs were predicted by following seven published databases, including miRanda (http://microrna.sanger.ac.uk)[11], Mir-Target2(http://nar.oxfordjournals.org/cgi/content/abstract/34/5/1646)[12],PicTar (http://pictar.bio.nyu.edu)[13], Probability of Interaction by Target Accessibility (PITA; http://genie.weizmann.ac.il/pubs/mir07)[14], Target Scan (http://targetscan.org)[15],miRecords (http://miRecords.umn.edu/miRecords)[16], Mirwalk(http://mirwalk.uni-hd.de/)[17]. The miRNA-gene pair would be reserved when it was predicted by no less than four of the databases above.

Functional Enrichment Analysis The Database for Annotation, Visualization and Integrated Discovery (DAVID;http://david.niaid.nih.gov)[18] has been developed for mapping genes to associated pathways and Gene Ontology (GO) terms.We performed GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to analyze the target genes using the DAVID online database. P＜0.05 was considered as significance.

miRNA-lncRNA Regulatory Relationship Prediction StarBase v2.0 (http://starbase.sysu.edu.cn/)[19] database is used to identify the RNA-RNA and RNA-protein interaction networks systematically (miRNA-pseudogene, miRNA-lncRNA, miRNA-mRNA, miRNA-circRNA and protein-RNA) from 108 CLIP-Seq (CLASH, PAR-CLIP, iCLIP,HITS-CLIP) generated datasets. ChIPBase (http://deepbase.sysu.edu.cn/chipbase/)[20] is a database for annotating and discovering trans criptional regulatory relationships of miRNAs and lncRNAs binding maps from ChIP-Seq data. All the interaction networks of miRNA-lncRNA in our study were downloaded from starBase v2.0 and ChIPBase. The subnetworks related to the differentially expressed miRNAs were further selected.

miRNA-circRNA Regulatory Relationship Prediction In our study, all the interaction networks of miRNA-circRNA were analyzed and downloaded from starBase v2.0, and then, the sub-networks related to the differentially expressed miRNAs were selected at last.

RESULTS

Differentially Expressed miRNAs and Target Genes Three differentially expressed miRNAs passed the preliminary screening (fold change ＞2 in both articles), including 1 upregulated and 2 down-regulated ones (Table 1). However,the sequence of miRNA-377-3p is not conservative among species. So, only miRNA-9-5p (miRNA-9a-5p) and miRNA-128-3p were included into our further study. The target genes were reserved when it was predicted by not less than 4 of the databases above.

Function Enrichment Analysis of hsa-miRNA-9-5p and hsa-miRNA-128-3p Target Genes The target genes of hsamiRNA-9-5p and hsa-miRNA-128-3p were performed GO enrichment analysis and the result was shown in Table 2. The target genes of hsa-miRNA-9-5p were mainly enriched in negative regulation of transcription from RNA polymerase II promoter. And the target genes of hsa-miRNA-128-3p were mainly enriched in positive regulation of transcription, DNA-templated.

Pathway Enrichment Analysis of hsa-miRNA-9-5p and hsa-miRNA-128-3p Target Genes The signaling pathways enriched by the target genes of hsa-miRNA-9-5p and hsamiRNA-128-3p were shown in Table 3. The target genes of hsa-miRNA-9-5p were enriched in Ras signaling pathway,cancer pathway, MAPK signaling pathway. And the tar get genes of hsa-miRNA-128-3p were mainly enriched in TGF-β signaling pathway and PI3K-Akt signaling pathway.

LncRNAs Targeted by hsa-miRNA-9-5p and hsa-miRNA-128-3p From the predicted lncRNAs of hsa-miRNA-9-5p and hsa-miRNA-128-3p, we found that some lncRNAs were regulated by both miRNAs, such as RP6-24A23.7. In addition,some common target genes regulated by both lncRNA and miRNA were found, such as IGF2BP1, QKI and TNRC6 in hsa-miRNA-9-5p; such as QKI, UPF1 and SFRS1 in hsamiRNA-128-3p (Table 4).

circRNAs Interaction with hsa-miRNA-9-5p and hsamiRNA-128-3p As for the predicted circRNAs of hsamiRNA-9-5p and hsa-miRNA-128-3p, we finally found 35 and 27 candidates from StarBase including ZNF280C_hsa_circ_001211, SIAE_hsa_circ_002083 and etc (Table 5).

DISCUSSION

ROP, which afflicts premature infants often, is characterized by abnormal retinal vasculature. Onset of ROP occurs when the neurosensory retina is quite immature. Its pathogenesis has not been very clear yet. But the effect of oxygen toxicityon the retina on the immature vasculature has been reported in two important stages: 1) vasoconstrictive stage: this occurs during exposure to hyperoxia and there is also suppression of the normal anterior ward vascularization of the retina.This mechanism of vasoconstrictive and obliterative effect of oxygen is often seen in the developing retinal vessels; 2)vasoproliferative stage: it involves dilatation and tortuosity of the existing larger vessels with neovascularization and proliferation of new vessels into the vitreous[21]. Consequently,the irreversible vascularization is the core mechanism of ROP.In the past several years, few reports have shown that miRNA expression is associated with ocular physiologic and pathological processes and displays tissue- and spatiotemporal specific expression[22-23]. By our further analysis, miRNA-128-3p and miRNA-9-5p are the only up and down regulated miRNAs in ROP among reported profile studies[9-10]. As for miRNA-128-3p, which received less investigation, was generally reported in hepatocellular carcinoma[24] and also involved in is chemia-induced brain injury[25]. About its related lncRNA, POLDIP2 is a molecule that has been shown to be involved in cell migration of vascular cells and angiogenesis[26].It’s also involved in a variety of processes of organ fibrosis lesions[27]. Another lncRNA-GAS5 seems play an important role in angiogenesis and migration, in colorectal cancer it acts as tumor suppressor by inhibiting IL-10 and VEGF expression[28].Mechanistically, GAS5 regulated endothelial cells and vascular

smooth muscle cells function through β-catenin signaling[29].So, though obvious evidences are missing, it also suggested the possible roles of miRNA-128-3p and its lncRNA in the aspect of angiogenesis in ROP.

It’s worthy noting that PI3K-Akt signaling pathway and TGF-β signaling pathway passed enrichment analysis of hsamiRNA-128-3p. Di et al[30] found CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygeninduced retinopathy. TGF-β signaling is also essential for the differentiation of retinal pericytes during vascular development of the retina. Kimsa et al[31] once found treatment of proliferative vitreoretinopathy is associated with processes regulated by TGF-β, such as inflammation, proliferation,epithelial-mesenchymal transition, and fi brosis.

The sequence of miRNA-9-5p is conserved among species and less reported in the previous references. Inspite of the abundant expression of the brain-specific miRNA (miRNA-9-5p/3p) in both proliferating and postmitotic neurons, most functional studies have focused on their role in neuronal development. Sim et al[32] found miRNA-9-5p has an important role in hippocampal synaptic plasticity and memory.In addition, miRNA-9-5p dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. It has been described differentially expressed in the RHO-P347S mouse retina, a model for a common form of inherited blindness[33].

As for the lncRNA of miRNA-9-5p, NEFL and UHRF1 have been studied the most widely. Kim et al[34] found that mRNA levels of previously described ganglion cell markers, e.g.NEFL was highly enriched in the isolated retinal ganglion cell layer. Liu et al[35] found NEFL is correlated with human corneal fi broblasts in ocular in fl ammatory diseases. These data provide the first evidence that NEFL and UHRF1 function are required for retina development and perfection. Enrichment analysis of miRNA-9-5p was mainly located in MAPK signaling pathway.For MAPK signaling pathway, the reactive oxygen speciesdependent phosphorylation of EGFR/MAPK is an important signaling pathway for Aurano fi n-induced inhibition of retinal pigment epithelium cell survival, and may have the potential for treatment of abnormal survival of retinal pigment epithelia cells in proliferative vitreoretinopathy[36].

In conclusion, our data provided a bioinformatics analysis of differentially expressed miRNAs which are involved in ROP. hsa-miRNA-128-3p and hsa-miRNA-9-5p showed significantly differential expression between ROP group and control group. LncRNA of POLDIP2, GAS5, NEFL and UHRF1, circRNA of ZNF280C_hsa_circ_001211 and SIAE_hsa_circ_002083, tar get gene of QKI and enrichment analysis of TGF-β signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway may play important roles in the prog ress of ROP. Here, we want to remind readers to pay attention to the physiological and pathological differences between animal model and human. Therefore, further genetic and experimental studies with larger sample size are still needed to con fi rm these results.

ACKNOWLEDGEMENTS

Conflicts of Interest: Yang Y, None; Pan JJ, None; Zhou XG, None; Zhou XY, None; Cheng R, None.

1 Hellström A, Ley D, Hansen-Pupp I, Hallberg B, Ramenghi LA,Löfqvist C, Smith LE, Hård AL. IGF-I in the clinics: use in retinopathy of prematurity. Growth Horm IGF Res 2016;30-31:75-80.

2 Hansen RM, Moskowitz A, Akula JD, Fulton AB. The neural retina in retinopathy of prematurity. Prog Retin Eye Res 2017;56:32-57.

3 Kuhnert F, Mancuso MR, Hampton J, Stankunas K, Asano T, Chen CZ,Kuo CJ. Attribution of vascular phenotypes of the murine Eg fl 7 locus to the microRNA miR-126. Development 2008;135(24):3989-3993.

4 Chen Y, Gorski DH. Regulation of angiogenesis through a microRNA(miR-130a) that down-regulatesantiangiogenic homeobox genes GAX and HOXA5. Blood 2008;111(3):1217-1226.

5 Kuehbacher A, Urbich C, Zeiher AM, Dimmeler S. Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis. Circ Res 2007;101(1):59-68.

6 Johnson CD, Esquela-Kerscher A, Stefani G, Byrom M, Kelnar K,Ovcharenko D, Wilson M, Wang X, Shelton J, Shingara J, Chin L, Brown D, Slack FJ. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res 2007;67(16):7713-7722.

7 Shen J, Yang X, Xie B, Chen Y, Swaim M, Hackett SF, Campochiaro PA. MicroRNAs regulate ocular neovascularization. Mol Ther 2008;16(7):1208-1216.

8 Bai Y, Bai X, Wang Z, Zhang X, Ruan C, Miao J. MicroRNA-126 inhibits ischemia-induced retinal neovascularization via regulating angiogenic growth factors. Exp Mol Pathol 2011;91(1):471-477.

9 Wang Y, Wu S, Yang Y, Peng F, Li Q, Tian P, Xiang E, Liang H, Wang B, Zhou X, Huang H, Zhou X. Differentially expressed miRNAs in oxygen-induced retinopathy newborn mouse models. Mol Med Rep 2017;15(1):146-152.

10 Zhao R, Qian L, Jiang L. Identi fi cation of retinopathy of prematurity related miRNAs in hyperoxia-inducedneonatal rats by deep sequencing.Int J Mol Sci 2014;16(1):840-856.

11 Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS.MicroRNA targets in Drosophila. Genome Biol 2003;5(1):R1.

12 Wang X, EI Naqa IM. Prediction of both conserved and nonconserved microRNA targets in animals. Bioinformatics 2007;24(3):325-332.

13 Krek A, Grün D, Poy MN, Wolf R, Rosenberg L, Epstein EJ,MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M, Rajewsky N.Combinatorial microRNA target predictions. Nat Genet 2005;37(5):495-500.14 Kertesz M, Iovino N, Unnerstall U, Gaul U, Segal E. The role of site accessibility in microRNA target recognition. Nature Genetics 2007;39(10):1278-1284.

15 Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB.Prediction of mammalian microRNA targets. Cell 2003;115(7):787-798.16 Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T. miRecords: an integrated resource for microRNA-target interactions. Nucleic Acids Res 2009;37(Database issue):D105-D110.

17 Dweep H, Sticht C, Pandey P, Gretz N. miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes. J Biomed Inform 2011;44(5):839-847.

18 Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4(1):44-57.

19 Li JH, Liu S, Zhou H, Qu LH, Yang JH. starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data. Nucleic Acids Res 2014;42(Database issue):D92-D97.

20 Yang JH, Li JH, Jiang S, Zhou H, Qu LH. ChIPBase: a database for decoding the transcriptional regulation of long non-coding RNA and microRNA genes from ChIP-Seq data. Nucleic Acids Res 2013;41(Database issue):D177-D187.

21 Shah PK, Prabhu V, Karandikar SS, Ranjan R, Narendran V, Kalpana N. Retinopathy of prematurity: past, present and future. World J Clin Pediatr 2016;5(1):35-46.

22 Karali M, Peluso I, Marigo V, Ban fi S. Identi fi cation and characterization of microRNAs expressed in the mouse eye. Invest Ophthalmol Vis Sci 2007;48(2):509-515.

23 Ryan DG, Oliveira-Fernandes M, Lavker RM. MicroRNAs of the mammalian eye display distinct and overlapping tissue specificity. Mol Vis 2006;12:1175-1184.

24 Huang CY, Huang XP, Zhu JY, Chen ZG, Li XJ, Zhang XH, Huang S,He JB, Lian F, Zhao YN, Wu GB. miR-128-3p suppresses hepatocellular carcinoma proliferation by regulating PIK3R1 and is correlated with the prognosis of HCC patients. Oncol Rep 2015;33(6):2889-2898.

25 Mao G, Ren P, Wang G, Yan F, Zhang Y. MicroRNA-128-3p protects mouse against cerebral ischemia through reducing p38α mitogen-activated protein kinase activity. J Mol Neurosci 2017;61(2):152-158.

26 Katsumura S, Izu Y, Yamada T, Griendling K, Harada K, Noda M,Ezura Y. FGF suppresses Poldip2 expression in osteoblasts. J Cell Biochem 2017;118(7):1670-1677.

27 Brown DI, Lassègue B, Lee M, Zafari R, Long JS, Saavedra HI,Griendling KK. Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fi broblasts.PLoS One 2014;9(5):e96657.

28 Li Y, Li Y, Huang S, He K, Zhao M, Lin H, Li D, Qian J, Zhou C, Chen Y, Huang C. Long non-coding RNA growth arrest specific transcript 5 acts as a tumour suppressor in colorectal cancer by inhibiting interleukin-10 and vascular endothelial growth factor expression.Oncotarget 2017;8(8):13690-13702.

29 Wang YN, Shan K, Yao MD, Yao J, Wang JJ, Li X, Liu B, Zhang YY,Ji Y, Jiang Q, Yan B. Long noncoding RNA-GAS5: a novel regulator of hypertension-induced vascular remodeling. Hypertension 2016;68(3):736-748.

30 Di Y, Zhang Y, Nie Q, Chen X. CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy. Int J Mol Med 2015;36(6):1507-1518.

31 Kimsa M, Strzalka-Mrozik B, Kimsa-Dudek M, Kruszniewska-Rajs C, Gola J, Adamska J, Mazurek U. Transforming growth factor β-related genes in human retinal pigment epithelial cells after tacrolimus treatment.Pharmacol Rep 2016;68(5):969-974.

32 Sim SE, Lim CS, Kim JI, Seo D, Chun H, Yu NK, Lee J, Kang SJ, Ko HG, Choi JH, Kim T, Jang EH, Han J, Bak MS, Park JE, Jang DJ, Baek D,Lee YS, Kaang BK. The brain-enriched microRNA miR-9-3p regulates synaptic plasticity and memory. J Neurosci 2016;36(33):8641-8652.

33 Palfi A, Hokamp K, Hauck SM, Vencken S, Millington-Ward S,Chadderton N, Carrigan M, Kortvely E, Greene CM, Kenna PF, Farrar GJ. microRNA regulatory circuits in a mouse model of inherited retinal degeneration. Sci Rep 2016;6:31431.

34 Kim CY, Kuehn MH, Clark AF, Kwon YH. Gene expression pro fi le of the adult human retinal ganglion cell layer. Mol Vis 2006;12:1640-1648.

35 Liu L, Walker EA, Kissane S, Khan I, Murray PI, Rauz S, Wallace GR. Gene expression and miR profiles of human corneal fibroblasts in response to dexamethasone. Invest Ophthalmol Vis Sci 2011;52(10):7282-7288.

36 Chen X, Tzekov R, Su M, Hong H, Min W, Han A, Li W. Auranofin inhibits retinal pigment epithelium cell survival through reactive oxygen species-dependent epidermal growth factor receptor/ mitogen-activated protein kinase signaling pathway. PLoS One 2016;11(11): e0166386.

Correspondence to: Rui Cheng. Department of Neonatology,Children’s Hospital Affiliated to Nanjing Medical University,Nanjing 210008, Jiangsu Province, China. chengrui@163.com; yy860507@126.com

DOl:10.18240/ijo.2018.05.09

Citation: Yang Y, Pan JJ, Zhou XG, Zhou XY, Cheng R. Differentially expressed miRNAs in premature infants with retinopathy-a bioinformatics analysis. Int J Ophthalmol 2018;11(5):773-779