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CERVICAL CANCER AND GENE EXPRESSION ANALYSIS WITH KEY GENES IDENTIFICATION BY COMPUTATIONAL METHOD

The second most unpropitious female illness, Cervical cancer, has been an alarming disease amongst Malaysian women population. This sight has been proven since twenty years ago in the scene with half a million cases worldwide and more than 50% mortality rate in Asia up till present time. Functional annotation analysis was conducted with a total of 247 gene list at Gene Expression Omnibus (GEO) database, then performed in DAVID (Database for Annotation, Visualization and Integrated Discovery) continued in PANTHER (Protein Analysis Through Evolutionary Relationships database) for cervical cancer gene understanding through biological, molecular function to cellular level. Protein-protein interaction network of 61 upregulated and 187 downregulated gene lists were analyzed in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) server. The network between the gene proteins were represented in nodes and edges with information such as number of networks and PPI enrichment values. As a conclusion, the discovered centre for cervical cancer hub genes were AURKA, UBE2C, TPX2, MCM5 and GINS2 in upregulated gene list and SPRR1B, DSC3, SPRR1A, DSG1 and SPRR2D in downregulated gene list.


INTRODUCTION
Cervical cancer has been the ultimate agony of the female population in Malaysia since the early days of HPV infection broke out. The disastrous malignancy infection has become this nation's second most horrified female cancer, following after breast cancer [1]. Despite being a developed country with heavily subsidized healthcare benefits [2]. Malaysian females are still heavily blinded by the fact of the discriminated and tabooed topic should never be publicly discussed and combat in public. Precancerous and cancerous lesions are generally detected to be worrisome only at the later stages (Stage 3 and Stage 4). Human papillomavirus (HPV) is a devastating sexually transmitted disease affecting both genders, male and female. The infection is fueled by more than 100 diversed strains, but some being a high-risk strain -HPV 16 and 18. The powerful HPV strain 16 is the main culprit behind almost 70% cervical causing cancers [3], primarily being transmitted through unprotected sexual contact. High risk human papilomavirus or alphapapilomavirus is ignited in the human body when the dangerous strain enters a wounded lesion of basal epithelial cells region. 50 to 100 genomes per cell will be generated there and malignant cells spread like wildfire as they duplicates [4]. Cervical cancer do not show any symptoms that can be experienced by affected individual, but signs arises when the disease is at a daunting stage. Pain during sexual intercourse, abnormal watery and foulsmelling vaginal discharge, unexpected bleeding before and after menstruation cycle are among of the signs one should be aware of. Predominantly, HPV virus are transmitted through sexual contact, but there are studies and evidences of mother-newborn cases that happens during delivery.
Gene expression method have been applied to study comprehensively HPV infection in the human body. The method digs into how it affects human biological system and their metabolic pathway is further elucidated. Malaysian government have made continuous effort in taking preventive measures towards the cancerous HPV infection by introducing vaccination (primary prevention) as early as in 1960's till present era [1]. Screenings and treatments of precancerous lesions falls into the secondary prevention method. In addition to that, therapeutic chemotherapy and radiation treatments are aggressively in action as tertiary treatments for diagnosed, invasive cancer. The Ministry of Health are taking their stand on preventing the spread of this deadly but preventable disease by holding nationwide healthcare campaigns, attitude, prevention and knowledge awareness. In 2010, three years after the first HPV vaccine was approved in Malaysia, nationwide, thrice dosage immunization package was introduced for females in Malaysian high schools and they are free of charge. The effort was indeed a maximized collaboration in aggressive approach for cervical cancer prevention [5].
The snowballing public repository demand for microarray experiments that are high-throughput began in 1999 and gene expression omnibus (GEO) was released to the public. This open design server serves as a storage, submission and retrieval of ceaseless amount of data sets that were obtained from antibody array experiments, genomic hybridization and gene expression [6]. At the moment, GEO has a billion of expressed individual genes, that were derived from 1500 laboratories that addresses extensive biological conditions [7]. Differentially expressed genes is an important J.Bio.Innov 9(5), pp: 868-878, 2020 |ISSN 2277-8330 (Electronic) Venkataramanan et al., and crucial step in microarray data analysis as scientific information disseminates across huge datasets and the results are summarized graphically of their functional information for researchers and public [8] certainly the database for annotation, visualization, and integrated discovery (DAVID) gave a wholesome picture of four distinctive categories which are annotation tool, gocharts, kegg charts and domain charts. These categoriess swiftly adjoins descriptive data from multiple open source databases to the gene lists [9]. Proteins interaction in biological pathways, cell to cell communication and the signal regulations displayed between each other or among distinct cells are perfectly potrayed in protein annotation through evolutionary relationship server (PANTHER) is a part of an integral approach in understanding the importance of biological pathway ontologies [10]. The statistical based analysis tool feature in the server gives biologists the opportunity to analyze sequencing, proteomics or gene expression experiments data. The wide range of gene fammilies and subfamilies, including evolutionary relationships are expressed in phylogenetic trees, statistical models (hidden Markov models or HMMs) and multiple sequence alignments [11]. Protein to protein interaction network (PPI) in search tool for the retrieval of interacting genes/proteins server (STRING) provides a system-wide comprehension of the most in depth cellular function details that requires broad functional interactions between the expressed proteins [12]. The connections and associations includes direct (physical) and indirect (indirect) interactions with imported known pathways and complexes of proteins derived from curated databases. The feature of subsets network visualization, enrichment analysis such as gene ontology and metabolic pathways with additional of clustered network hierachy, STRING server maps the protein structures accordingly [13]. Mutually exclusive interactions are also exposed with structural details, nodes (protein) and edges (interactions), after the PPI were constructed [14].

RESEARCH BACKGROUND
This research was conducted in identifying the dominant cervical cancer causing genes and thoroughly expressing them biologically and pathway mechanism wise.

PROBLEM STATEMENT
The HPV gene is not thoroughly comprehended and expressed in cervical cancer disease (Ramakrishnan et al., 2015). The cervical cancer data set with Geo ID : GSE9750 was put into accession box and the results are then seperated into normal and cervical cancer samples, with a total of 66 samples. There were 33 primary tumors, 9 cell lines, and 24 normal cervical epithelium.

GEO2R
GEO2R is a collective web server that distinctively categorizes the cervical cancer samples. The server works on datasets of samples expressed by array expression profiling method. In this study, there were 24 cervical cancer samples and 12 normal sample conditions identified, after the analysis.

DAVID Functional Annotation Clustering
Following after that, DAVID ( Database for Annotation, Visualization and Integrated Discovery) server, as a functional annotation clustering tool was used. The main purpose of this tool was to understand the three major aspects of a HPV gene protein which are biological component, cellular component and molecular function. DAVID enhanced the interpretation of a massive gene list in a gene network context. This data mining tool extends module-centric approach where cervical cancer are studied from gene-to-gene or similar relationships applied to it, with information embedded on the server. DAVID has three major stages where firstly, gene pairs were measured in terms of functional relationship, then proceeded to agglomeration procedure to assemble the likegenes into their respective categories. Finally, the results were visualized in two approaches, graphical and text modes.

PANTHER
(Protein Annotation Through Evolutionary Relationship) is a classification system server that was used with the aim to maximized HPV gene biologically from molecular function, biological component to cellular level. Protein families and subfamilies were annotated with ontology terms and later curated with PANTHER pathways. The understanding of the processess delivered both graphical and statistical results.

STRING
The gene lists of up regulated and down regulated were analysed on their proteinprotein interaction network respectively. The network between the gene proteins were represented in nodes and edges with information such as number of networks and PPI enrichment values were calculated and displayed.

CYTOSCAPE
In addition to that, the upregulated and downregulated gene lists information were thoroughly analysed in this server for the degree of betweenness was set at > 0.4 and centrality at >20.0. The top 5 highest genes from both categories were counted as the hub genes of cervical cancer. The interactions were later visualized as a merged network.

Omnibus GeoTool
The gene list results were obtained from GEO dataset after the Top250 analyzing process were performed. The positive and negative logFC values were differentiated into two separate tables which are upregulated and downregulated gene lists, respectively (Table 1). Innov 9(5), pp: 868-878, 2020 |ISSN 2277-8330

FIGURE 2 :
The gene list report was obtained from DAVID server analysis with aurora kinase (AURKA) as the dominant gene.

FIGURE 3 : Molecular function analysis of upregulated gene.
Additionally, the gene list was carried forward for further analysis in molecular function, biological function and pathway analysis in PANTHER server. The results were displayed in graphical pie chart for all three analysis. The molecular function results showed 44.4% binding, 42.6% catalytic activity, 3.7% molecular function regulator, 5.6% molecular transducer activity and 3.7% structural molecule activity (Figure 3).

STRING server results
The KEGG pathways described fat digestion and absorption pathway, pancreatic secretion and cell cycle pathway. In the reactome pathway, five pathways were exposed, the S phase, G1/S transition, activation of the pre-replicative complex, activation of ATR in response to replication stress and E2F mediated regulation of DNA replication.  The upregulated genes protein-protein interaction network depicted perfectly in the image screen captured below. The discovered centre for hub genes were AURKA, UBE2C, TPX2, MCM5 and GINS2. The downregulated genes protein-protein interaction network depicted perfectly in the image screen captured below. The discovered centre for hub genes were SPRR1B, DSC3, SPRR1A, DSG1 and SPRR2D ( Figure.14). Then, the combination of the hub genes were created with a more dominated and transparent view as shown in the image below ( Figure 15) of the hub genes involving upregulated and downregulated genes.

FIGURE 15 :
The merged network of strongest 10 cervical cancer hub genes from upregulated and downregulated gene lists, performed in STRING server.

DISCUSSIONS
The analysis performed on the gene dataset GSE9750 in DAVID server gave the result of AURKA, a protein coding gene, being highly expressed in it. Aurora kinase A is a cancer related enzyme. This enzyme can be found in the process of microtubule stabilization or formation during chromosome segregation. This gene has the possibility of playing a huge role in tumour progression and development [15]. The gene list potrayed a whole complete results of this specific gene only and this is supported by the previous studies where it is identified as an oncogene in carcinogenic cell proliferation [16] The PANTHER analysis results depicted heavily on molecular function, biological processes and cellular component of gene list. The binding receptors were reported to be 44.4% in upregulated and 37.5% in downregulated gene lists. The human papillomavirus gene mechanism supported this result as the pathway of HPV invasion in the keratinocytes were binding receptorstargeted [17]. HPV gene mechanism occured through clathrin-dependent endocytic mechanism and this confirms the cellular processes results where metabolic process and biological adhesion emerged as the top three highest percentages in biological process analysis [17].
The STRING analysis results showed the interactions of the protein to protein interaction network, the KEGG pathways and biological processes that happened.
In addition to that, in downregulated gene list analysis, five strongest hub genes were small proline-rich protein 1B (SPRR1B), desmosomal glycoprotein III (DSC3), small proline-rich protein 1A (SPRR1A), pemphigus foliaceus antigen (DSG1) and small prolinerich protein 2D (SPRR2D) were dominating. The protein network genes showed interaction of cancer biomarkers identification in the visualization form.

CONCLUSION
In a nutshell, the hub genes was successfully identified from GSE9750 Geodataset via gene expression analysis for its biological, molecular and cellular component. The pathway mechanism was fully elaborated and depicted in hopes that in the future, an indepth pathway study on cervical cancer gene biomarker analysis are broaden.