Ethical consideration: The study has been carried out after approval from Ethics Review Committee of Ziauddin University (2861120SHPAT).The study was conducted in Multidisciplinary Laboratory Ziauddin University(Figure 1). In our study the microarray dataset was obtained from Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/).

Identification of DEGs: We selected 121 samples, with 62 controls and 58 CRC case which were obtained from NCBI generated microarray dataset GSE164191. The identification of DEGs was done based upon p value to test the differential expression of the genes between the CRC and the control groups. The p value was calculated using the Student’s t-test. The cut‐off criteria were kept at the fold change>2.0 and a corrected p < 0.05.

PPI analysis: To perform the PPI analysis, online database Search Tool for the Retrieval of Interacting Genes was used. The parameter of interactions was set as confidence>0.4. To visualize and analyze the PPI network, Cytoscape software was used. The scattered proteins in cytoscape were removed from the final PPIs. The proteins, which worked like a hub in the network, were selected by CytoNCA on the basis of their interaction with other proteins. The selection was based upon degree centrality, between-ness centrality and sub graph centrality. The degree was set at ≥10 for further selection of hub proteins.

Literature search: Manual literature search was performed to explore the role of the selected hub proteins in CRC.

KEGG Pathway analysis: The selected hub proteins were searched for associated pathways in human cancers using KEGG pathway analysis. 

Figures & Tables

In our study we aimed to find out vital protein interaction networks in colorectal cancer. The comparison of dataset between normal controls and colorectal cancer patients revealed 180DEGs in which 95 were up regulated and 85 were down-regulated. The DEGS were selected for PPI construction which led to identification of two key proteins, heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) and Signal transducer and activator of transcription 3 (STAT3) based on sub graphcentrality, degree and betweenness.On exploring literature, we found association of these two proteins withcolorectal carcinogenesis. The primary role of HNRNPA2B1 has been documented in pathogenesis of amyotrophic lateral sclerosis [12,13]. Studies have reported thatHNRNPA2B1can promotingcolorectal tumour cell invasiveness [14]. It has been suggested that HNRNPA2B1facilitates tumor metastasis through extracellular regulated protein kinases (ERK) pathway [15]. Moreover, the expression and splicing of RAF kinase has been documented to be regulated by HNRNPA2B1 [16]. Moreover, the heterogeneous nuclear ribonucleoproteins (HNRNPs) have been reported to be associated with JAK STAT signaling pathway with up regulation of HNRNPs via STAT3 activation [17,18]. The STAT3 gene is a member of the STAT protein family which   regulates cellgrowth and proliferation [19]. Among the family of STAT proteins, STAT3 has been reported to be overexpressed in about 70% cancer[20]. KEGG pathway analysis highlights the association of STAT3 with human cancers.Activation of STAT3 may be due to phosphorylation signals by cytokines like IL6, activated Janus kinases (JAK), activated epidermal growth factor receptor (EGFR) or by mitogen activated pathway (MAP) kinases. Activated STAT3 can lead to increased transcription of target genes including cell-cycle regulator genes, proto-oncogenes, and anti-apoptotic genes. In this era of precision medicine,anti-EGFR drugs have gained popularity with EGFR being a possible therapeutic target in CRC patients especially in patients where surgery does not offer complete cure [21]. However, the development of resistance to EGFR-tyrosine kinase inhibitor drugs has narrowed their scope in CRC therapeutics [21]. Several kinases associated signalling pathways have beenimplicated as possible mediators of resistance to anti-EGFR targeted therapy [22,23] .Zulkifli et al documented upon the role of STAT3 signaling in providing tumour cells with an escape mechanism to inhibitory effects of anti-EGFR drugs [23]. Our search of KEGG pathways highlightsthe association of STAT3 activation with EGFR which indicates that STAT3 is an important protein contributing to stepwise accumulation of genetic events incolorectal Carcinogenesis. Hence anti EGR therapy resistance may be due to downstream activation of STAT3. Moreover, the role of STAT3 in CRC chemotherapy resistance has been studied by STAT3 inhibition which can sensitize colorectal cancer cells to 5-Florouracil therapy through down-regulating cyclinD1 [24]. This resistance may be attributed to STAT3 activation through cytokine receptor activation. The phosphorylation of cytokine receptors is triggered  by Janus kinase which leads to activation of cytokine receptor associated kinases namelyEGFR, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and receptor-associated kinases that activate STAT3 [25]. JAK2 and STAT3 activation may play a significant role in promoting CRC metastasis [26]. Tsai et al. demonstrated that progression of colon cancer can be attributed to JAK2 and STAT3 activation by IL-6 [27]. Furthermore, the negative regulators of STAT3 like suppressors of cytokine signaling are believed to be perturbed in malignancy [28]. Studies have reported that STAT3 levels are higher in dedifferntiated colon cells and that there is a negative correlation between high levels of STAT3 and prognosis of CRC [4].

Clinical trials are ongoing to explore efficacious target therapy based on inhibiting STAT3. However STAT3 inhibitors have not been marketed as yet. Rational bioinformatics toolsalong with reliable assays are critical for attaining this aim. Because of the crosstalk of STAT3 and other oncogenic signaling pathways drug repurposing approach for chemotherapy may lead to better cancer management. Furthermore, the development of STAT3 inhibitors in combination with other therapies may help in formulating effective cancertreatment [29]. Our analysis highlightsSTAT3 as the hub gene which is associated with CRC. Theassociation of STAT3 with CRC related pathways highlights the need of further research on STAT3 as a possible therapeutic target. This may help in overcoming the treatment failure associated with drug therapy such as anti-EGFR therapy resistance.

Author Contributions

Sobia Hassan: conceptualization ,data acquisition,  manuscript writing and editing, correspondence
Talat Mirza: designing the study, editing, revising and final manuscript approval 
Ambrina Khatoon: data acquisition ,analysis and interpretation, manuscript writing
Uzma Bukhari: data analysis and interpretation, manuscript writing and editing
Fouzia Shaikh : manuscript writing and editing.

Conflict of Interest

The authors declare that there is no conflict of interest.

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