br During analysis of GO MF no molecular
During analysis of GO MF, no molecular function was found as significant, but endocytic vesicle lumen (GO: 0071682) was the only cellular component found significant in GO CC 2018 analysis (Fig. 5c). Interestingly, progesterone_CTD_00006624 was the major hit among
Stage Microarray common upregulated genes Microarray common downregulated genes
Stage III and Stage
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Fig. 2. Number of selected RNAs using Venn diagram.
The figure depicts numbers of mRNAs selected from GSE29817 (2a), TCGA (2b) and GSE52903 (2c) individually and 2d shows mRNAs common to the above three datasets while 2e shows the numbers of lncRNAs selected from TCGA.
other top ten significant compounds during DSigDB analysis (Fig. 5d) with combined score of 123.59. A few other significant compounds were, 5-Fluorouracil_CTD_00005987, Decitabine_CTD_00000750 and estradiol_CTD_00005920. Adjusted p value, Z score and gene names of each analysis were provided in supplementary spreadsheet. Hs Cyto-plasmic Ribosomal Proteins pathway, Hs Electron Transport Chain pathway, Hs Oxidative Phosphorylation, Hs Proteasome Degradation and Hs EGF/EGFR signaling were found to be the most enriched pathways associated with selected lncRNAs from lncRNA-mRNA co-expression network analysis for pathway enrichment. Hs DNA Re-plication and Hs Focal Adhesion were also of significance in CC pro-gression (Fig. 5e).
Although biomarker selection can be achieved through many methods, its correlation with disease progression is more important for therapeutic applications. Hence, the role of individual markers was also taken into consideration in this study besides global selection of the staging markers. Among the mRNAs selected from TCGA, Hgb-α-globin (HBA1) and Hgb-β-globin (HBB) are overexpressed in SiHa, CaSki cells than normal cells and HBB is thought to act as an antioxidant, reducing oxidative stress-induced damage (Li et al., 2013) and HBA1 was also found to be upregulated in severe cases of CC (Starodubtseva et al.,
2019). SLC2A1 or GLUT1 (solute carrier family 2) acts as a uniporter protein and its high expression is associated with inferior progression-free as well as overall survival in CC (Kanjanapan et al., 2017). SLC2A1 expression was also positively correlated with CC tumor stage and lymph-vascular space involvement (Iwasaki et al., 2015). Although SLC2A1 expression could not be positively correlated with initial grade of histologic differentiation and FIGO staging, increased expression of SLC2A1 was significant in recurrent CC than that in metastatic lymph node (Yen et al., 2004). CXCL10 was also found to reduce E6 and E7 oncoproteins in HeLa and CaSki cell lines and it is known to have an-tiviral and antiangiogenic properties (Wang et al., 2009). CXCL10 was also reported to increase radio sensitivity through RVX-208 redis-tribution in HeLa cells (Yang et al., 2012) and combination of CXCL10 gene therapy and radiotherapy showed improved therapeutic efficacy in xenograft models (Zhao et al., 2015a). S100A7, the selected mRNA for stage IV, has been reported to promote migration, invasion and metastasis through epithelial mesenchymal transition (Tian et al., 2017; Bhatt et al., 2019) and thus confirms the proposed RNA selection pro-cess to be biologically relevant. From mRNAs selected using micro-array, KRT5 showed decreased protein expression (Serafín-Higuera et al., 2016) and significantly so in early stages (Güzel et al., 2018). NKG7, as found in this study as an important staging marker, was also found to be correlated with HPV infection in CC (Weidhaas et al., 2009). Another biomarker found in this study during stage I vs stage II
Fig. 3. Oncoprint, DNA methylation heatmap (of selected mRNAs having genetic alterations of > 5%) and KM plots of selected mRNAs as well as interacting miRNA partners of selected mRNAs and lncRNAs. 3a depicts oncoprint of mRNAs from microarray and 2b indicates DNA methylation heatmap of mRNAs while 2c and 2d show oncoprint and heatmap of DNA methylation of mRNAs from TCGA, respectively. 3e shows KM plot for a combinatorial marker (LAMC2, MMP7, DST,COL7A1, COL8A1) and that of selected prognostic mRNAs is shown in 3f for SLC2A1 and 2g for PMEPA1. KM plots for interacting miRNA partners of selected mRNAs are shown in 3h for hsa-miR-150-5p and those for interacting miRNA partners of selected lncRNAs are shown in 3i (hsa-miR-188-3p) and 3j (hsa-miR-378a-3p).
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Fig. 4. Identification of enriched miRNA families from mRNA-miRNA and lncRNA-miRNA interaction networks and stage specific mRNA-miRNA-lncRNA association network generation. miRNA-mRNA interaction networks are being depicted for miRNA family enrichment of validated interacting miRNA partners of (4a) mRNAs selected from microarray, (4b) mRNAs selected from TCGA and (4c) a selected lncRNA LINC00263. Panel d shows Venn diagram of enriched miRNA candidates of let-7 family. Stage I specific mRNA-miRNA-lncRNA interaction network is shown in 4e and Stage III specific mRNA-miRNA-lncRNA interaction network is shown in 4f. During stage specific mRNA-miRNA-lncRNA triad generation, for experimentally valid interaction in some CC cell lines, miRNAs are shown in orange and mRNAs in pink colour in 4e and for the other valid interactions miRNAs were shown in yellow, while mRNAs were depicted in blue. For all conditions, lncRNAs were depicted in red. In 4a, enriched miRNA families were shown in specific colors like miR-30 were shown in yellow, miR-17 in green, let-7 in pink and miR-130 in cyan. In panels a and b, red color shows mRNA and blue depicts miRNAs. In 4b and 4c, green points depict candidates of let-7 family miRNA, while in 4c, red points depict the lncRNA, LINC000263. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)