Overlaying of Core Network 2

As can be seen in Table 29, Core Network 2 was generated as having 27 molecules from the dataset of All Top Regulated Genes and miRNAs being placed into a network of 70 molecules. The top diseases and functions found to be associated with molecules in Core Network 2 were cell cycle functioning, cancer, and organismal injury and abnormalities.

As can be seen in Figure 35, 54 of the curated genes and miRNAs were mapped onto Core Network 2. The distribution of these genes and miRNAs is widespread, with most of the molecules lying as inter-connectors between defined nodes. Of note, SMAD4, KAT2B, Histone h3, RNA polymerase II, and CCND1 (all outlined by a blue box), are all present in the curated list of genes and miRNAs, and they appear ad nodal genes in Core Network 2. Each of these nodal genes clearly interacted with a number of genes, which are not present in the starting list of genes.

Figure 35: Core Network 2 overlaid with Starting Genes and miRNAs. Of the 866 Starting Genes and miRNAs mapped in IPA, 54 IDs were found in Core Network 2, composed of 207 molecules.

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Interestingly, CCND1 also illustrates direct interactions with genes from the curated genes and miRNAs list, namely HIST2H2BE, RIF1, PTTG1, RUFY3, and STOM (orange boxes).

Four genes, HNRNPK, MYBL2, RBL1, and IGFBBP5 (all outlined by a light green box) are also shown here to be present in the starting list of curated genes and miRNAs, but form integral connections between Histone h4 and MYC (both outlined by a red box), which are two genes not present in the starting list of genes.

Figure 36 was generated by overlaying Core Network 2 with the list of all interacting miRNAs and genes obtained from the analysis of the curated genes and miRNAs, in miRTarBase, and miRecords. Most of the molecules in Core Network 2 were shown to consist of genes and miRNAs from the list of interactions. However, eight genes (outlined by blue boxes), were present in the curated list of genes (see Figure 35), but were not found to interact with the list of miRNAs curated from literature.

Figure 36: Core Network 2 overlaid with Interacting Genes and miRNAs. Of the 2969 Interacting Genes and miRNAs mapped in IPA, 157 IDs were found in Core Network 2, composed of 207 molecules.

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Figure 37 is overlaid with the ‘All Top Regulating miRNAs and associated genes’ list. Of the six top regulating miRNAs selected to build the core networks (see 11.8), none of the miRNAs were overlaid above. A total of 27 genes, which were shown to interact with the top regulating miRNAs, were overlaid in Figure 35. Four genes, HNRNPK, MYBL2, RBL1, and IGFBP5 (all outlined by a light green box), were shown here to be regulated by the Top Regulating miRNAs. Interestingly, these four genes, were also overlaid with the starting list of lupus- associated genes and miRNAs (see Figure 35), as well as by the list of interacting molecules (see Figure 36). Thus, these four genes can be said to be lupus- associated in terms of literature source, as well as associated with lupus, because they have been identified as interacting with known lupus-associated miRNAs. Figure 37: Core Network 2 overlaid with All Top Regulating miRNAs and associated genes. Of the 116 All Top Regulating miRNAs, and associated gene targets mapped in IPA, 27 IDs were found in Core Network 2, composed of 207 molecules.

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Figure 38 is overlaid with the ‘All Top Regulating genes and associated miRNAs’ list. Of the seven top regulating genes selected to build the core networks (see 11.9), three genes can be seen above. Of the 33 miRNAs shown in 11.9 to be regulated by the seven top regulating genes, one miRNA miR-145-5p can be identified Figure 38, outlined by a blue box. Compared to Core Network 1 (Figure 32), Core Network 2’s overlay with the All Top Regulated Genes, shows much fewer of this subset of data being present in Figure 38.

Figure 38: Core Network 2 overlaid with Top Regulated Genes and associated miRNAs. Of the 40 All Top Regulating genes, and associated miRNA targets mapped in IPA, 4 IDs were found in Core Network 2, composed of 207 molecules.

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Figure 39: Core Network 2 overlaid with All Top Regulated Genes and miRNAs and associations. Of the 156 All Top Regulating Genes and miRNAs, and associated targets mapped in IPA, 28 IDs were found in Core Network 2, composed of 207 molecules.

Figure 39 shows Core Network 2 overlaid with all the Top Regulated genes, miRNAs, and their corresponding interactions, as determined by analysis of miRTarBase and miRecords. All of the genes highlighted by the overlay above appear in either Figure 37 or Figure 38.

Figure 40: Core Network 2 overlaid with All Genes and miRNAs, IPA-added molecules in Pink. Of the 3687 All Genes and miRNAs, and associated targets mapped in IPA, 25 IDs were not overlaid in Core Network 2, and thus allowed for the identification of the molecules added by IPA.

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Figure 40 was overlaid with the complete list of all curated genes and miRNAs, and their corresponding interactions. Those genes not overlaid by the dataset, but added in by the IPA database, were selected and highlighted in pink.

A cluster consisting of five genes (outlined by a blue box) namely RBM39, BTG3, INS, ASF1B, AHSP, were all shown to interact with a known lupus-associated gene, Histone h3 (as seen in Figure 35), and also with Histone h4, a new gene added in by IPA to complete this section of Core Network 2.

A number of genes (outlined by a light green box), were also added by IPA, and can be seen here to interact either directly or indirectly, with a starting- and interacting gene named CCND1, and MYC- which was only found to interact with a known lupus-associated miRNA.

Of importance, Figure 40 shows how, along with Histone h4 already described, HDAC9, TFAP2A, YWHAQ, and estrogen receptor (all outlined by a red box), are new genes added by IPA, and appear to be nodal genes, interacting with numerous genes from the complete dataset of starting, and interacting genes and miRNAs.

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