Transcriptome analysis identified a novel 3-LncRNA regulatory network of transthyretin attenuating glucose induced hRECs dysfunction in diabetic retinopathy

As the leading cause of vision impairment and loss in the working age population, diabetic retinopathy (DR) is a common and specific microvascular complication characterized by early retinal microvascular dysfunction [1]. In China, the number of DR patients is growing each year [2]. The early stage of DR is exemplified by retina endothelial cells and pericytes death [3]. As DR progresses, vascular leakage becomes evident and may eventually lead to diabetic macular edema, the most common cause of vision loss in DR [4]. Till now, panretinal photocoagulation, vitrectomy and anti-neovascularization drug intraocular injection remain the few therapeutic options [5]. Accumulating evidence implicated that inflammation, cell apoptosis, oxidative stress and autophagy were major causative factors involved in the pathogenesis of DR [6]. Therefore, uncovering potential mechanisms underlying DR pathogenesis, especially revealing hub genes and pathways in inflammation, cell apoptosis, oxidative stress and autophagy is of paramount importance for the development of effective therapeutics for patients with DR.

Transthyretin (TTR) is a 55-kDa homotetramer protein reported to transport thyroxine and retinol through retinol-binding protein (RBP) [7], and it is mainly secreted by human retinal pigment epithelial cells (hRPECs) and choroid in ocular tissue. In vitreous and serum samples of high myopia patients, TTR showed significantly higher expression compared with healthy donors [8]. Interestingly, the expression of TTR in DR patients was significantly lower, compared with healthy donors [8, 9]. In our previous research, an exogenous 4 μM TTR treatment or endogenous TTR overexpression both repressed blood vessel tube forming capability of hyperglycemic cultured human retinal endothelial cells (hRECs), by attenuating pro-angiogenic genes expression, like Angiopoietin 2 (Angpt2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), and Vascular Endothelial Growth Factor (VEGF2) [10, 11]. Additionally, TTR was reported to repress neovascularization by promoting hRECs apoptosis through direct binding to glucose-regulated protein 78 (GRP78) [12]. Surprisingly, the exogenous added TTR efficiently crossed cell membrane and nuclear membrane, and located in both in cytoplasm and nucleus, implicating complex transcriptional and post-transcriptional regulatory role, while the exact mechanism still remains obscure.

Long noncoding RNA (lncRNA) is characterized to be transcripts with a length of more than 200 nt and of no protein-coding capability. Accumulating evidence pointed that lncRNA exerted function in cis by regulating nearby genes, or in trans by modulating distantly located genes [13]. In addition, it’s a putative mechanism that lncRNAs may regulate target gene expression via miRNA response element (MRE), known as competing endogenous RNA (ceRNA) [14]. The aberrant expression of lncRNAs in early diabetic retinopathy was first characterized in 2004 [15]. Subsequent studies of individual lncRNAs playing a transcriptional or post-transcriptional regulatory role in DR pathogenesis and progression had been reported, including Maternally Expressed 3 (MEG3) [16], Myocardial Infarction Associated Transcript (MIAT) [17, 18], HOXA Distal Transcript Antisense RNA (HOTTIP) [19], Metastasis Associated Lung Adenocarcinoma Transcript 1(MALAT1) [20, 21], SOX2 Overlapping Transcript (Sox2-OT) [22], antisense non-coding RNA in the INK4 locus (ANRIL) [23] and BDNF Antisense RNA (BDNF-AS) [24]. However, there have been limited studies of lncRNAs in TTR repressing DR neovascularization.

In this study, we proposed that there may exist certain lncRNAs, through which TTR exerts its function of repressing neovascularization in DR. RNA transcriptome of hRECs treated with low glucose concentration of 5.5 mM (LG), high glucose concentration of 25 mM (HG), and a 25 mM high glucose plus a concentration of 4 μM TTR (HG + TTR) was determined for the first time. The impact of high glucose and TTR on hRECs neovascularization was evaluated with tube formation assay. Then differentially expressed lncRNAs and mRNAs that induced by high glucose and TTR were identified, respectively. Functional annotation and network analysis of TTR regulated mRNAs significantly enriched pathways and biological processes in DR. Furthermore, the TTR regulated lncRNAs and mRNAs were predicted, and the lncRNA-mRNA regulatory network in cis, trans and ceRNA was constructed. Finally, after qRT-PCR validation in hRECs, vitreous humor and serum samples, a 3-lncRNA centered TTR response coding-non-coding network with potential diagnosis performance was proposed to explain the protective role of TTR, which would provide basis for the future potential application of TTR and lncRNAs in DR diagnosis and treatment.

Recently, it was reported that dysfunction of TTR was bound up with system diseases and the intervention of TTR improved multiple clinical manifestations of hereditary transthyretin amyloidosis [49, 50]. TTR was also involved in various biological processes such as proteolysis, nerve regeneration, autophagy, glucose homeostasis and angiogenesis regulation [51]. In ophthalmic diseases, abnormal TTR expression was reported in high myopia and diabetic retinopathy patients. Exogenous TTR treatment inhibited hRECs proliferation, migration and tube forming capability and induced apoptosis in vitro [12, 13]. However, the underlying mechanism still remains unknown. In this study, through RNA-sequencing, the basic transcriptome characteristics of hRECs underlying hyperglycemia and TTR with high glucose conditions were first determined. Then, through functional annotation, protein-protein interaction and co-expression analysis, the protective role of TTR in hRECs dysfunction including signaling pathways, biological processes and trait modules were further explored. Furthermore, potential targets of TTR related lncRNAs were predicted and 3 coding-non-coding networks were constructed based on putative lncRNA acting mode. Finally, a 3-lncRNA centered regulatory hub network with clinical significance was proposed to explain the function of TTR attenuating neovascularization.

The functional annotation of TTR related mRNAs corroborated previous reports of relative decreased expression of TTR in DR patients and neovascularization function. In our study of TTR function in hRECs, biological processes involved in cell proliferation, migration and neovascularization were significantly enriched, in lining with studies that TTR modulated human umbilical vein endothelial cells (HUVECs) apoptosis and inhibit migration, regulation of hRECs apoptosis, proliferation and migration. In addition, biological processes and pathways like response to oxidative stress, inflammatory signaling like IL2-STAT5, and regulation of autophagy were also enriched and reported for the first time. The intricate cross-talking between oxidative stress, inflammatory signaling pathway, autophagy and apoptosis were reported in melatonin ameliorating DR progression [52]. Thus, we speculated that there may exist a similar regulatory network, composed of oxidative stress, inflammation signaling, autophagy and apoptosis, linking the protective function of TTR and DR. The 5 protein-protein interaction sub-networks in Fig. 4 implied that TTR might orchestrate pathways in transcriptional and post-transcriptional levels. Genes in sub-network 1 implied that TTR might regulate cell cycle via ubiquitin mediated proteolysis, and the function of sub-network 3 suggested that TTR modulates MAPK and NF-κB pathway through RNA transportation, splicing and translation, post-transcriptionally. In transcriptional level, the function of genes in the sub-network 5 was focused on DNA repair, an important link of oxidative stress, inflammatory signaling, autophagy and apoptosis [53, 54, 55]. Hence, the functional annotation provides a comprehensive direction for further research of how TTR rescuing high glucose induced neovascularization abnormality.

Another important part of our work was the investigation of TTR related lncRNAs and their potential regulatory role in DR. As non-coding RNAs, the functional mode of lncRNAs via regulating mRNA expression in cis, trans or as miRNA sponge has been well recognized in angiogenesis and vascular diseases [56]. LncRNA MEG3 knockdown aggravated retinal vessel dysfunction by the activation of PI3k/Akt signaling [16]. Through regulating p38-MAPK pathway, HOTTIP improved diabetic retinal microangiopathy [19]. As an important inflammation regulator, lncRNA MALAT1 was reported to affect retinal endothelial cell proliferation, migration, and tube formation through cross-talking with p38 MAPK signaling pathway [20, 21]. As miR-29b and miR-150-5p sponge or VEGF ceRNA, lncRNA MIAT (Myocardial infarction associated transcript) was reported to regulate endothelial cell apoptosis [18]. Another role that well studied in cardiovascular diseases is lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), which regulated VEGF expression and function in diabetic retinopathy via miR-200b, and EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit) of the PRC2 (Polycomb Repressive Complex 2) complex [23]. However, the role of lncRNAs in TTR ameliorating hRECs dysfunction has not yet been reported. In this paper, a 3 lncRNA-mRNAs regulatory network and 5 lncRNAs-mRNAs sub-networks in cis, trans and ceRNA were constructed for the first time. The networks would contribute to understanding the underlying mechanism of TTR functions in DR from the perspective of different lncRNAs acting mode.

In addition, expression of 10 lncRNAs in lncRNA-mRNAs networks were validated by qRT-PCR and 3 lncRNAs including 2 up-regulated novel lncRNAs, i.e., MSTRG.15047.3 and AC008403.3, 1 down-regulated lncRNA, i.e., FRMD6-AS2 (FRMD6 Antisense RNA 2). The 3 lncRNAs formed a complicated potential interacting network, including 11 mRNAs and 2 miRNAs. Based on the network, the novel predicted lncRNA MSTRG.15047.3 might interact with nearby up-located CBFTA2T2 in a cis-regulatory way, trans-regulates cell cycle and neovascularization via UBE2N (Ubiquitin Conjugating Enzyme E2 N), PPP2R5C (Protein Phosphatase 2 Regulatory Subunit B′ Gamma), MCM7(Minichromosome Maintenance Complex Component 7), TAF6 (The TATA-Box Binding Protein Associated Factor 6) and PBRM1 (Polybromo 1), or just sponge miR-4651 and promote FGF13 (Fibroblast Growth Factor 13) expression. Gene UBE2N encoded a member of the E2 ubiquitin-conjugating enzyme family and was reported to be involved in cell cycle and error-free DNA repair pathway [57]. The UBE2V1 (Ubiquitin Conjugating Enzyme E2 V1)-UBE2N heterodimer acts in concert with TRIM5 (Tripartite Motif Containing 5) to activate the MAP 3 K7/TAK1 complex and induce expression of NFκB and MAPK-responsive inflammatory genes [58, 59]. PPP2R5C gene belongs to the phosphatase 2A regulatory subunit B family. The PP2A (Protein Phosphatase 2 Phosphatase Activator) -PPP2R5C holoenzyme may specifically dephosphorylate and activate TP53 (Tumor Protein P53) and play a role in DNA damage-induced inhibition of cell proliferation, as well as regulate the ERK signaling pathway through ERK dephosphorization [60]. MCM7 acts as component of the MCM2–7 complex (MCM complex) and may be involved in the formation of replication forks and in the recruitment of other DNA replication related proteins [61]. The TATA-Box Binding Protein Associated Factor 6 (TAF6) are components of the transcription factor IID (TFIID) complex and modulates cell apoptosis in a TP53 dependent [62] and independent [63] way. PBRM1 encodes a subunit of ATP-dependent chromatin-remodeling complexes. In clear cell renal cancer, concomitant loss of PBRM1 rescues VHL-induced replication stress, maintaining cellular fitness and allowing proliferation [64]. Fibroblast Growth Factor 13 possesses broad mitogenic and cell survival activities, including embryonic development, cell growth, morphogenesis and invasion [65]. For AC008403.3, in addition to sharing 4 trans targets of TAF6, PPP2R5C, PBRM1 and FGF13 with MSTRG.15047.3 may act as a ceRNA of NAA60 (N (Alpha)-Acetyltransferase 60, NatF Catalytic Subunit) and SLC5A5 (Solute Carrier Family 5 Member 5) via sponging miR-6884-5p. NAA60 encodes an enzyme that localizes to the Golgi apparatus and transfers an acetyl group to the N-terminus of free proteins [66]. SLC5A5 encodes a member of the sodium glucose cotransporter involved in thyroxine metabolism, a shared molecular function of TTR [67]. As the antisense RNA and potential cis-regulator of FRMD6 (FERM Domain Containing 6), a known hippo pathway kinase regulator [68], lncRNA FRMD6-AS2 might interact with PBRM1, PPP2R5C and ASB (Arylsulfatase B) to regulate cell proliferation and neovascularization. Further studies will be conducted to explore the exact role of 3-lncRNA regulatory network linking TTR and DR neovascularization.

Finally, lncRNAs MSTRG.15047.3, AC008403.3 and FRMD6-AS2 showed significant differential expression levels in aqueous humor and serum samples of DR patients and normal controls (patients without diabetes), made possible 3 lncRNAs as diagnostic biomarkers in DR. Several studies investigated the clinical significance of lncRNAs in DR, like MIAT [17] and MALAT1 [21]. In this study, we proposed 3 of 10 lncRNAs potentially involved in DR progression, and we would explore their function and molecular mechanism in the future. Additionally, the correlation of lncRNAs expression with clinical parameters, and especially performance as DR biomarkers would be studied, with more samples, including healthy donors and other ocular diseases.

In summary, it was concluded that TTR repressed hRECs neovascularization, via a 3-lncRNA regulatory network. This investigation was mainly based on glucose and exogenous TTR treatment of hRECs, RNA sequencing, bioinformatics analysis and qRT-PCR validation in clinical samples. There are several limitations in this study. First, hRECs cell line was used as the DR cell model, and the tube forming assay demonstrated that TTR repressed neovascularization. Whether the neovascularization repressing effect of TTR and 3-lncRNA regulatory network were restricted to hRECs, or represents a general mechanism in microvascular endothelial cells is an important question. In fact, Kun Shan .et al. employed other microvascular endothelial cell lines, like human umbilical vein endothelial cells (HUVECs), EA.hy.926 and human coronary artery endothelial cells (HCAECs) to investigate retinal vascular dysfunction [69]. In addition, using primary human retinal microvascular endothelial cell (HRMECs) would help to illustrate the point. Secondly, this study was conducted in vitro, and investigation of TTR effect in DR animal models, like streptozotocin (STZ) administration, pancreatectomy and genetic models [70] would provide more solid evidence to support the conclusion. Thirdly, an exogenous TTR was employed to examine the function and a vector or virus mediated TTR overexpression approach would provide more details about the endogenous function of TTR. Lastly, we speculated that the 3-lncRNA regulatory network play a pivotal role in TTR function, and more experiments, like rescue assays of 3 lncRNAs knockdown or overexpression in TTR treatment cells, would help to support the point.

In summary, we investigated (1) the basic characteristics and landscape of RNA transcriptome in TTR and glucose treated hRECs; (2) the function of TTR-mRNAs from signaling pathway, biological process, subnetworks and hub modules; (3) lncRNA-targets networks and functional sub-networks in cis, trans and ceRNA regulatory way; (4) a 3-lncRNA centered hub network that might mediate the comprehensive function of TTR and serve as potential diagnostic biomarkers in DR. The research would pave the way for understanding the complex biology of TTR in DR progression.