Systems Biology Unraveled the Relationship of lncRNA OIP5-AS1 with CD25 and its Co-Expression Analysis in Cancers

Background: Treg cells function in the immune homeostasis, these cells express high level of CD25. Even though the molecular mechanisms of CD25-mediated signaling pathways has been reported, some questions are still unclear, e.g. the relationship and function of the relative lncRNA. It is known that the CD25 expression levels are various among different cancers. Thus, we intended to dissect systems biology of a lncRNA pertained to CD25 and CD25 protein interactors-targeting miRNAs. Methods: Apart from using the available RNA-seq data, the co-expression analysis of the lncRNA pertained to some cancers was performed. Our analysis was done for protein interactors of CD25 by STRING 11.0, ShinyGO v0.60 and KEGG web servers were used for enrichment and network analysis of CD25. TargetScan 7.2, miRTargetLink Human and mirDIP were applied for determining the CD25 and CD25 interactors-targeting miRNAs. To nd the lncRNA-miRNA and lncRNA-protein interactions, starBase v3.0, LncBase Predicted v.2 and SFPEL-LPI were recruited, respectively. Also, using Co-LncRNA, the co-expressed lncRNA analysis and the relative signaling pathways in some cancers including bladder, breast, head and neck, kidney, liver, lung, prostate and thyroid cancers using RNA-seq data were achieved. Results: OIP5-AS1 was shown to have the interaction with CD25 and CD25 protein interactors-targeting miRNAs. In addition, the co-expression of OIP5-AS1 in cancers and their signaling pathways was identied. Conclusions: Possibly, OIP5-AS1 can effect on CD25 expression in all relative signaling pathways of these cancers.


Background
Treg cells play an important role in immune homeostasis. These CD4+ Foxp3+ Tregs express high levels of CD25 (known as IL-2RA). Tregs are the solitary immune cell type identi ed to express the full heterotrimeric receptor including CD25, CD122 (IL-2RB), and CD132 (IL-2RG), constitutively [1]. The IL-2 functions via high and low a nity in these cell surface receptors. The high a nity receptor complex is begun by binding the IL-2 to CD25 and next, CD122 and CD132 are engaged in the process. The CD122 and CD132 form a receptor with a 10-100 fold lower a nity for IL-2, in the absence of CD25 [2]. Notably, the IL-2 signaling is essential for the generation, survival and function of Treg cells. The heterotrimeric receptor is able to turn on MAPK/ERK, PI(3)K and STAT5 pathways [3]. Even though much is recognized about the molecular mechanisms of CD25 signaling, some questions are remained. Nowadays, some bioinformatic tools have been extended to carry out the functional annotations. The most well-known bioinformatic tool called ORA is recruited to gain the signi cant functional data (enrichment) from sets of related genes/proteins. This tool is used to detect the related and over-represented biological and functional annotations that are signi cantly enriched in a list of genes/proteins [4]. Also, another important bioinformatic tool consists of the network analysis describing and visualizing the proteinprotein interactions of signaling pathways related to the reference genes/proteins list [5]. In these cases, we aimed to describe the enrichment and network analyses of CD25. Also, using the prediction of miRNA targets we intended to describe the most signi cant relative miRNAs for CD25 and its protein interactors.
Through the most signi cant and annotated data, we dissected the relative and regulator lncRNA pertained to CD25 and CD25 protein interactors-targeting miRNAs. It was reported that the CD25 expression level was changed in some cancers [6,7]. With this aim, exploiting the available RNA-seq data, the co-expression analysis of the lncRNA pertained to some cancers was performed and the relative systems biology was dissected. Remarkably, these analyses were done for the rst time to explain the predicted and annotated data of enrichment and network signaling pathways, non-coding RNAs and coexpression analysis in some cancers related to CD25 expression.

Protein-protein interaction (PPI) analysis
The PPI of human CD25 was evaluated using STRING web server version 11.0 (https://string-db.org). In this web server, the medium con dence and the max number of interactors in rst shell were optioned as 0.4 and no more than 20 interactors, respectively.

MiRNA prediction
For predicting the engagement of miRNA and CD25, TargetScan 7.2 (http://www.targetscan.org), miRTargetLink Human (https://ccb-web.cs.uni-saarland.de/mirtargetlink) and mirDIP (http://ophid.utoronto.ca/mirDIP/index.jsp) were utilized. The miRNA targets of CD25 were gained by the options including weaker evidence and predicted interactions in miRTargetLink Human. Also, the targets of CD25 were gainedbased on the score class (very high, high and medium) in mirDIP. The mirDIP web server was used for the CD25 protein interactors-targeting miRNAs.

Results
The PPI outcome of CD25 The PPI result of CD25 was depicted by the number of nodes= 21, number of edges= 182 and PPI enrichment p-value< 1E-16 ( Fig. 1aand 1b).

The enriched signaling pathways and network of CD25
This analysis showed JAK-STAT (Fig. 1c) and In uenza A signaling pathways with the lowest Enrichment FDR= 6E-30 and the highest Enrichment FDR= 0.000025 utilizing KEGG option were placed in the resulted Table 1, respectively. Also, the network signaling pathways pertained to CD25 was depicted (Fig. 1d).

LncRNA-protein interaction outcomes
Based on the results of SFPEL-LPI web server, it was found that OIP5-AS1 possessed the interaction with some proteins. The most score was 0.999 pertained to ELAV-like protein 1 (Table 3).

Discussion
The purpose of systems biology is to combine comprehensive biological data from varied experimental approaches to realize complex interactions at the molecular stage [11]. One of the important protein components of the human immune system is CD25 expressed on cell surface of Treg cells [1]. CD4+CD25+ Tregs cells inside TME known as TI-Tregs possess the essential function in cancer immune escape [12]. Albeit the molecular mechanisms of CD25-mediated signaling pathways has been revealed, some inquiries are still unanswered. Using systems biology approach, the molecular interactors including lncRNAs, miRNAs and proteins for signi cant protein of CD25 will be unraveled. Revealing the interactions between CD25 and other molecules in TME may be promised to overcome the cancer immune escape and gain the cancer successful treatment [13].
This lncRNA is an important non-coding RNA involved in many cellular processes. In fact, the lncRNA with NONCODE ID: NONHSAT041930 or OIP5-AS1 abbreviated from OIP5 antisense RNA 1, is a mammalian lncRNA functioning in the cytoplasm [14]. The OIP5-AS1 has been focused for its role in the development of brain and eye [15]. Kim et al. (2016) reported that the lncRNA could inhibit HuR binding to target mRNAs. Therefore, it repressed the HuR-elicited proliferative phenotypes. In fact, they reported as the study of HeLa cells that OIP5-AS1 sponges ELAVL1 [16]. Also, Kim et al. (2017) illustrated that the lncRNA had the interaction with GAK mRNA, advancing GAK mRNA decay and sodecreasing GAK protein levels and reducing cell proliferation [17]. Also, Zhang et al. (2019) concluded OIP5-AS1 played as a ceRNA to make proliferation, migration and invasion of primary HemECs via regulating miR-195-5p/NOB1 axis. Indeed, ceRNAs perform as the molecular sponges for a particular miRNA via their miRNA binding sites [18]. Because of this function, also known as MREs, they de-repressed all target genes from the respective miRNA family [19].
In another point of view, the present co-expression analysis showed that OIP5-AS1 was co-expressed in normal vs. tumor bladder cancer based on the RNA-seq data. Clearly, this lncRNA was co-expressed in aminoacyl-tRNA biosynthesis pathway with p-value= 3.33E-15 and Bonferroni correction= 6.02E-13. Also, this cancer had the co-expressed OIP5-AS1 in other four top predicted pathways such as DNA replication, RNA degradation, cell cycle and spliceosome (Table 4). This analysis in breast cancer showed that pathways including pathways in cancer, neurotrophin signaling pathway, spliceosome, purine metabolism and aminoacyl-tRNA biosynthesis possessed the co-expressed OIP5-AS1 in the normal vs. tumor tissue (Table S4). This characteristic in head and neck cancer was engaged in these cellular processes including endocytosis, T cell receptor signaling pathway, neurotrophin signaling pathway, MAPK signaling pathway and lysosome (Table S5). In case of kidney cancer, purine metabolism, prostate cancer pathways, insulin signaling, endocytosis and RNA degradation were involved by p-value of 1.32E-12, 5.94E-12, 1.89E-11, 3.66E-10 and 3.87E-10, respectively (Table S6). The liver cancer showed that the regulation of actin cytoskeleton, neurotrophin signaling pathway, focal adhesion, pancreatic cancer pathway and ribosome were engaged in OIP5-AS1 co-expression (Table S7). For lung cancer, MAPK signaling pathway, purine metabolism, lysosome, huntingtons disease and pyrimidine metabolism demonstrated the co-expressed OIP5-AS1 (Table S8). For prostate cancer, focal adhesion, MAPK signaling pathway, chemokine signaling pathway, regulation of actin cytoskeleton apoptosis and apoptosis were shared the co-expression of OIP5-AS1 (Table S9). Notably, the thyrioid cancer illustrated that neurotrophin signaling pathway, RNA degradation, lysine degradation, aminoacyl-tRNA biosynthesis and renal cell carcinoma pathways had the shared feature from the point of view of OIP5-AS1 coexpression by p-value of 5.55E-16, 3.75E-14, 2.82E-11, 1.06E-10 and 1.48E-10, respectively (Table S10).
As a recent report, OIP5-AS1 lncRNA could adjust cell proliferation and apoptosis by miR-410 and its target KLF10/PTEN/AKT [20]. In addition, the researchers recognized a putative ceRNA network for lncRNAs of AC008124.1, OPI5-AS1 and NEAT1 in breast tumors [21]. Also, it was studied in two human osteosarcoma cell lines, MG63 and SaOS2, that OIP5-AS1 led cisplatin resistance via provoking the LPAATβ/PI3K/AKT/mTOR signaling pathway as the sponge for miR-340-5p [22]. In another study in osteosarcoma tissues and cells, the silencing of OIP5-AS1 inhibited the proliferation and also speeded up the apoptosis, and G0/G1 cycle arrest. Indeed, OIP5-AS1/miR-223/CDK14 performed the modulation on the tumorigenesis of osteosarcoma [23]. It was resulted that the over-expression of miR-367-3p, piR-30188 and PIWIL3 or knockdown of OIP5-AS1 effected on the suppression of glioma progression [24]. In undifferentiated oral tumors, the over-expression of OIP5-AS1 could be proposed for the poor clinical result and elevated cancer stemness [25]. Also, OIP5-AS expression was signi cantly reduced in nonsmall cell lung cancer tissues against adjacent non-cancerous tissues in whole samples and in male patients [26].

Conclusion
Taken together, OIP5-AS1 had the important role in CD25-mediated signaling pathways in a whole systems biology. This role was pertained to CD25 and CD25 protein interactors-targeting miRNAs. According to our analysis, it was shown that OIP5-AS1 had no direct interaction with CD25 protein. However, this lncRNA may play a role as molecular sponge for CD25 and CD25 protein interactorstargeting miRNAs. Also, in the current study, the co-expression of OIP5-AS1 in bladder, breast, head and neck, kidney, liver, lung, prostate and thyroid cancers and their signaling pathways was identi ed. Possibly, OIP5-AS1 can effect on CD25 expression in all relative signaling pathways of these cancers. These systems biology data may be useful to nd out the interactions between CD25 and other molecules in TME to overcome the cancer immune escape and gain the cancer successful treatment. However, the in vitro and in vivo investigations should be performed to verify these bioinformatic data.

Limitations
The validation of these bioinformatic analyses should be done both in vitro and in vivo. Also, These analyses should be carried out about other cancers.