J. Appl. Environ. Biol. Sci.,8(7)36-45,2018 | ISSN: 2090-4274 |
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BiotechnologyProgramme, DepartmentofMathematics and NaturalSciences, BRACUniversity, 66 Mohakhali, Dhaka-1212, Bangladesh
Received: January 19, 2018 Accepted: April 19, 2018
Breast cancer is an important public health issue since it has become the reason of 69% cancer caused death in women throughout the whole world and 15% of cancer death in Bangladesh. According to recent findings, bi-omarker studies can help in the betterment of diagnosis, treatment and recurrence problem by constructing a bi-omarker panel. The project was designed to find the basic properties -structure, sequence motif and expression level ofpotential biomarker molecules of breast cancer. Onlyprotein and miRNA were selected as biomarkers since they were easily collectable from body fluid. 11 proteins and 7 miRNAs were selected, as they were the one showing high specificity and sensitivity as biomarkers. The protein molecules were -ER, ER Beta, PR, TTR, Ki67, HSP60, Her2,CyclinD1,Cyclin E, P53 andCEA. The miRNAswere-miR10b, miR21,miR145,miR155, miR191, miR 382 and miR425. Bioinformatics approach was the fundamental base of this research to detect properties of these bi-omarkers. For structure SWISS MODELWorkspace (protein), mfold (miRNA), for sequence motif MEME, and to check expression level GEO Profiles were used. In the end of this study it was seen that CEA, TTR, ER, PR, ER Beta, Cyclin E and Ki67 were the proteins that could be a potential biomarker for breast cancer screening panel. CyclinD1, Her2, P53 along with miR155 are potentialbiomarkers for breast cancer staging and miR10b, miR21 can be potentialbiomarker for ER silencing treatment. KEYWORDS:miRNA,cancer biomarker,P53, CEA,Cyclin E, Ki67
Being a significant contributor to overall morbidity and mortality, breast cancer is by far the most frequent cancer among women in both developed and developing countries with an estimated 1.38 million new cancer cases (Kulasingam, 2008). And with 15% cancer death in women in Bangladesh (Sacha, 2014) and incidence rate 22.5 per 100000 in females.(Rai, 2012). It has become a hidden burden in Banglqdesh. Studies have proved that, this condition is the result of the absence of suitable diagnostic or screening test for an early detection of clinically relevant breast cancer. The current screening methods used to detect breast tumors either benign or malignant, include clinical breast examination (CBE), mammography and ultrasound. (Kulasingam, 2008). All processes have their own limitations and also averyhigh false negetiverates(Kulasingam, 2008). One of the most promising ways to achieve methods with improved sensitivity and specificity is through the use of cancer biomarkers. (Kulasingam, 2008). A joint venture on chemical safety, led by WHO with the United Nations and the International Labor Organization has defined abiomarker as “anysubstance, structure or process that can be measured in the bodyor its products and influence or predict the incidence of outcome or disease.”(Mandal, 2013.) Among all the different molecules in a human body-Protein and miRNA are known to perform the best as bi-omarker for specific diseases. Protein molecules are known as the best ofbiomarker molecules since they can easily be traced, studied and evaluated (Gam,2010). In the present study11 protein molecules and seven miRNAs were selected (Table 1 & 2) to predict their structure, sequence motifs and expression level in cancer state. Protein molecules were :Estrogen Receptor (ER), Estrogen Receptor beta (ERbeta), Progesterone (PR), Transthyretin (TTR), Cyclin E, Cyclin D1, Her2( Human Epidermal Growth Receptor),Carcinoembryonic Antigen (CEA), P53, Heat Shock Protein60 (HSP60). When it comes to miR-NA, Patterns of miRNA expression plays a very important role in oncogenesis. Because of their distinct patterns of expression associated with cancer type, remarkable stabilityin blood and other bodyfluids, miRNAs are considered to be highly promising cancer biomarkers (Zhao, 2010). Among the miRNAs, seven were selected for this study – miRNA10B,miRNA21, miRNA145,miRNA155,miRNA191, miRNA382, miRNA425.
Corresponding author: Romana Siddique, Biotechnology Programme, Department of Mathematics and Natural Sciences, BRAC University, 66 Mohakhali, Dhaka-1212,Bangladesh.Email: romanasiddique@gmail.com Telephone:+880-2-8824051-4Ext.4060 Fax:+880-2-58810383
Theaim of this studywas to explore the structure,motifs and expression levelof someselected cancer biomarkers so that these can beused as apossibletherapeutics in cancer treatmentsand for earlybrest cancer screening.
Table1:Protein markersassociated withBreastcancer
Protein | Accessionnumber | Taxonomicname |
---|---|---|
ER | AAI28574.1 | Estrogen receptor 1 |
PR | BAC06585.1 | Progesteronereceptor |
HER2 | AAA75493.1 | human epidermal growth factor receptor 2 |
CEA | CAE75559.1 | carcinoembryonic antigen |
KI67 | NP_002408.3 | Ki-67 protein |
CYCLIN D1 | AAH23620.1 | CyclinD1 |
CYCLIN E | NP_001229.1 | G1/S-specific cyclin-E1 |
ERBETA | AAV31779.1 | estrogen receptor 2 (ER beta) |
TTR | CAG33189.1 | Transthyretin |
P53 | BAC16799.1 | Tumor protein p53 |
HSP60 | AAF66640.1 | heat shock proteinHSP60 |
Table2:miRNA markersassociated withBreastcancer
miRNA | GInumber | Taxonomicname |
---|---|---|
MIR10B | 262206216 | Homo sapiens microRNA 10b |
MIR21 | 262205659 | Homo sapiens microRNA 21 |
MIR145 | 262205329 | Homo sapiens microRNA 145 |
MIR155 | 269846817 | Homo sapiens microRNA 155 |
MIR191 | 262205347 | Homo sapiens microRNA 191 |
MIR382 | 262206264 | Homo sapiens microRNA 382 |
MIR425 | 262205357 | Homo sapiens microRNA 425 |
In the current study,all thesequences information for the miRNAwere retrievedfrom miRBase (www.mirbase.org) and sequence information for proteins wereretrievedfromNCBIdatabse( https://www.ncbi.nlm.nih.gov/) andUniprot(www.uniprot.org/) .In silico analysis for structure, expression level and sequence motifs were carried out for selected proteins and miRNAs.
Mfold web server version 3.5 (unafold.rna.albany.edu/) was used to predict secondary folded structure of miRNA (Zucker M., 2003). To predict protein structures, first the FASTA sequences were retrieved from a database, Uniprot (www.uniprot.org/). The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. UniProtis a collaboration between the European Bioinformatics Institute (EMBL-EBI), theSIB Swiss Institute of Bioinformatics and the Protein Information Resource (PIR)(www.uniprot.org/). Then blast was done using the Basic Local Alignment Search Tool (BLAST) (https://www.ncbi.nlm.nih.gov/BLAST/) with these sequences to find their best suited templates. After that alignment was checked with this sequence and their template with Clustal omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). And finally this alignment result was given as an input in the homology modelling website, Swiss model workspace (https://swissmodel.expasy.org/workspace/) which is aweb-basedintegrated service dedicated to protein structurehomology modelling. It assists andguides the user in buildingprotein homology models atdifferent levels of complexity(Arnold K, 2006).
J. Appl. Environ. Biol. Sci., 8(7)36-45, 2018
MEME Suite (meme-suite.org/) was used to discover sequence motif in both the cases of protein and miRNA molecules. The MEME Suite is a software toolkit with a unified web server interface that enables users to perform four types of motif analysis: motif discovery, motif–motif database searching, motif-sequence database searching and assignment of function. It offers a significantly expanded set of programs for these tasks compared with the earlier web server (Bailey,T. L.,2006).
To find out the expression level of the selected proteins and miRNA molecules in different conditions of breast cancer, GEO (https://www.ncbi.nlm.nih.gov/geo/) of NCBI was used. GEO represents to Gene Expression Omnibus. TheGene Expression Omnibus (GEO) is an international public repository that archives and freely distributes mi-croarray, next-generation sequencing, and other forms of high-throughput functional genomic data sets(Barrett T,2013 TheGEO Profiles (www.ncbi.nlm.nih.gov/geoprofiles/)database stores gene expression profiles derived fromcuratedGEO Datasets(https://www.ncbi.nlm.nih.gov/gds). EachProfileispresented asachart that displays the expression level of onegene across allSampleswithin theDataset.
3.1StructurePrediction formiRNAs
Basically in this study, finding the secondary hairpin like structure of the miRNAs was the main focus. Knowing this kind of structure specifically is important because the folding in a certain way leads to the solution of different unanswered question in binding and functioning of that miRNA molecule. Secondary structure for seven miRNA moleculaes werepredicted usingMfold(Fig.1)
Fig1:Hairpin structureofa)miR10Bb) miR21 c)miR145d) miR155 e) miR191 f) miR382g)miR425
For seven selected miRNAs, motif wasobserved(Fig.2) usingMEMESuite(meme-suite.org/).Motif means asequence that can have specialimportancebiologicallyor functionally. Knowing motifs are important because they arerecurrent and theyindicatebinding sitesor functional sequence of that molecule.
Fig2:Important sequence motif of a)miR10Bb) miR21 c)miR145d) miR155 e) miR191 f) miR382g)miR425
GEO tool was used to observe expression of seven miRNA molecules to observe expression pattern in different conditions of breast cancer. This is a trickyone to observe because this involves a multiple step to be expressed and also because this is an actual indicating property that makes the miRNA molecules a biomarker. Out of seven three miRNA molecule (miR10B, miR21, miR155) showed significant changes in their expression level. The expression level was measured in normal breast cancer patient and in patient whom were treated with ER mutation. It is seen that miR10B expression level is higher in the ER mutated cells and miR21 is low expressed in the ER mutated cells (Fig. 3).
This expression profile ofmiR155 showed expression in two differentbreast cancer celllinesalong with acontrol. It was seen that in both cancerous cell lines (MDA-MB-436 and HCC 1954) miR155 is expressed differently than the normal cell line. It is expressed more in theMDA-MB-436 cell line and less in the HCC1954 cellline (Fig. 3).
J. Appl. Environ. Biol. Sci., 8(7)36-45, 2018
Fig3:Expression levelofa)miR10B b) miR21 c)miR155
Homology modelling was done using For the 11 protein biomarkers using Swiss model workspace (https://swissmodel.expasy.org/workspace/) (Fig.4). Predicting the homology model of these molecules can help in determining the structural motifs as well as site directed mutagenesis that might make them a candidate for bi-omarker panel ofbreast cancer.
Fig4:Homology modelofa)CEAb)Cyclin D1 c)CyclinEd)ER e)ERBeta f)HSP60 g)HER2h)Ki67i)P53j)PRk)TTR
Motifs for protein were also observed using MEME software (meme-suite.org/) was used and for each protein at least five motifs were observed. From these most significant motifs were selected (Fig.5). Knowing protein motifs are important becausetheygive aclear information aboutthe effects ofsequence variation,protein interaction etc.
J. Appl. Environ. Biol. Sci., 8(7)36-45, 2018
Fig5: ImportantSequenceMotif ofa)CEAb)Cyclin D1 c)Cyclin E d)ER e)ERBeta f)HSP60g)HER2h)Ki67i)P53 j)PR k)TTR
The expression of CEA, CyclinE, ER, ER Beta, HSP60, Ki67 and TTR were almost similar. All of them were over-expressed in both the breast cancer cell lines (MDB-MB-436 and HCC 1954) (Fig.6). On the other side expression level ofCyclin D1, Her2 and P53 were almost similar (Fig.6). They were more expressed in the HCC 1954 cell line and less expressed in the MDB-MB-436 cell line. Lastly PR was differently expressed than all as it was under ex-pressedin both of the celllines than thenormal one.
Fig6: Expression level ofa)CEAb)Cyclin D1 c)Cyclin E d)ER e)ERBeta f)HSP60 g)HER2 h)Ki67i)P53 j)PR k)TTR
In the structure section secondary structure of miRNA andtertiary structure ofproteins are shown. All the homology models are validated with QMEAN and the QMEAN score says all of them are more than 0.77% in a scale of 0 to 1.Sequence motif is the sequence that might have biological or functional importance. (Timothy, 1994).For each molecule maximum five motifs were commanded to be found and the standard length is from 6 to 50. Most of the motifs are present in two sites, but only a few are present in more than two sites. In the expression level observation results were taken from GEO Profile. From the huge store of different comparisons suitable results were taken. All these different cell lines are presented in the bottom light pink bars. And the diseased state are shown in the second bottom green bars. Above these two line of bars, ash colored bars show the name of the samples. In the long red lines that represents the transformed count of the expression level. This transformed count is from the actual experiment results as they were performed in affymetrix systems. And the blue squares presents their percentile rank among all the samples. If these results are combined together a better biomarker panel could be decided with CEA, Cyclin E, ER, ER Beta, HSP60, KI67, TTR andPR. On the other hand Cyclin D1, Her2, P53 along with miR155can make abiomarker panel for breast cancer staging. mi10B and miR21 can playbiomarker role in the ER silencing treatment systems.
J. Appl. Environ. Biol. Sci., 8(7)36-45, 2018
Breast cancer is aglobal curse. This is the most commonly encountered cancer in our countryas well as in thewhole world (Y. Baskin, 2010). With an objective to add a little help in the findings of better treatment and diagnosis system this study was designed to know more about breast cancer biomarker molecules like protein and miRNA. It is hoped that individuallythe information of these biomarker molecule can help in finding anew therapeutic agent, site directed mutagenesis, virtual screening. Also together they can make a panel of biomarkers with better specificity and sensitivitythat isneeded themost at this moment. (Li, 2002).
Theauthors declare that there is no conflict ofinterests regarding the publication of this paper.
Theauthors wouldlike to thank Department ofMathematics andNaturalSciences,BRACUniversity.
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