br mRNA selection from microarray LAMP was also
mRNA selection from microarray, LAMP3, was also found to be a sur-vival marker at early stages and its upregulation also promoted CC metastasis (Kanao et al., 2005). GAGE5, GAGE2B and GAGE4 are found important in our study during mRNA selection from stage II vs stage III, but a previous study showed that GAGE family genes like GAGE-3/6 were upregulated in CC in late stages (Sarcevic et al., 2003). Emergence of downregulated AKR1C2 as an important mRNA found during stage
III vs stage IV based mRNA selection from microarray in this study may be biologically relevant as HPV16 E7 transactivation on aldo-keto
The results showing the significance of electron transport chain and oxidative phosphorylation pathway in lncRNA associated pathway
Fig. 5. Pathway, gene ontology and DSigDB Analysis of selected RNAs.
Bar plots of (5a) Reactome 2016 (5b) GO Biological Process 2018 (5c) GO Cellular Component 2018 and (5d) DSigDB analysis of selected mRNAs and plot for (5e) Wiki-pathway enrichment of selected lncRNAs and their co-expressed mRNAs.
S. Banerjee and D. Karunagaran
analysis correlate well with E6-mediated activation of genes associated with those pathways (Evans et al., 2016). Emergence of Hs Proteasome Degradation pathway can also be biologically validated as E6 in-activates p53 through proteasomal degradation in CC too (Tomaić, 2016). Oxidative phosphorylation was also found to be the preferential ATP production mechanism from glutamine in HeLa 22978-25-2 (Jose et al., 2011). Receptor tyrosine kinase signaling pathway is another lncRNA associated pathway obtained in the study that was found to be pro-longed due to E6 (Spangle and Munger, 2013). HPV oncogenes also dysregulated the associated collagen and ECM dynamics via transcrip-tional regulation independently and synergistically with estrogen (Spurgeon et al., 2017). Enhanced EGFR internalization by E6 of HPV16 and mTORC1 activation supports the role of HPV in lncRNA mediated
CC progression during pathway identification (Spangle and Munger, 2013). It may be noted from our results that the pathways enriched by selected lncRNAs are mainly modulated by HPV oncogenes, recognized to be the major cause for CC. As many selected mRNAs in this study were found to be hypermethylated like HBB, HBA2, SLC2A1, S100A7, CXCL10 etc., emergence of Decitabine, a DNA hypomethylating agent, beside another convention chemotherapeutic drug 5-Fluorouracil during DSigDB analysis supports the biological significance of this analysis, since E6 and E7 modulate the expression of many proteins by epigenetic regulation (Durzynska et al., 2017). In metastatic CC, Cis-platin- decitabine combination is in Phase II clinical trial (Pohlmann et al., 2002).
miRnet used in this study only considered validated mRNA-miRNA interactions and in all cases miR-let 7 family was found to be the most significant. Let-7 miRNA acts through targeting RAS indirectly affecting downstream MAP kinase signaling cascade and altering the cell pro-liferation (Gomez-Gomez et al., 2013). miR-30 regulated Rb-induced senescence in CC (Martinez et al., 2011) and tumor suppressor activity of miR-30e was exerted by targeting GALNT7 in CC (Wu et al., 2017). Cell proliferation and metastasis were promoted by miR-17-5p mod-ulating Transforming Growth Factor-β Receptor 2 (TGFBR2) (Cai et al., 2018), TP53INP1 (Jose et al., 2011) and by miR −20 via TIMP2 and ATG7 (Zhao et al., 2015a, 2015b). Among the survival markers se-lected, low expression of miR-188 was reported to show reduced sur-vival (Gao et al., 2018).
Only 21% and 4% patients are presented in clinics at stage III and stage IV CC, respectively (Spurgeon et al., 2017), accounting for the low number of samples present in this study at those stages. Functional role of individual lncRNAs (Fang and Fullwood, 2016) and their association with predicted miRNAs as well as with reference to identified stage specific mRNA-miRNA-lncRNA triads in this study also can be explored further for better understanding of CC progression. Experimental vali-dation of the results would be more interesting and relevant. Hence it can be concluded that, the mRNAs and lncRNAs selected in the study by the proposed integrated pipeline are robust from both computational and biological perspectives. DE mRNAs identified from more than one microarray can be used as biomarkers to distinguish FIGO specific CC stages, while mRNAs and lncRNAs identified from TCGA can be used as biomarkers for differentiating FIGO specific CC stages being biologically relevant. miR-let7 family was found to be enriched in both mRNA-miRNA co-expression network using mRNAs selected from microarrays and lncRNA targets. Since Cytoplasmic Ribosomal Proteins pathway, Proteasome Degradation, EGF/EGFR signaling pathway, Oxidative Phosphorylation and Electron Transport chain pathway were found to be the most enriched pathways associated with selected lncRNAs from lncRNA-mRNA co-expression network analysis for pathway enrichment, it can be concluded that selection of lncRNAs are biologically relevant too in CC progression, since cancer or HPV mediated pathways were enriched in the pathway analysis. Proposed integrated approach utilizing of publicly available datasets