FALL 2015
Special topics in Bioengineering
microRNA, Cancer Epigenetics and Bi
oinformatics
Jasmin Šutković
27th October 2015
Outline
• Introduction
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Micro RNA s
Epigenetic
DNA Methylation
In silico study – Bioinformatics
Examples
Epigenetic refers to heritable changes in gene expression
(active versus inactive genes), that does not involve changes to
the underlying DNA sequence; a change in phenotype without a
change in genotype.
Or
“Epigenetics” refers to covalent modification of DNA, protein, or
RNA, resulting in changes to the function and/or regulation of
these molecules, without altering their primary DNA sequences.
Micro RNA molecules
MicroRNAs (miRNAs) comprise species of short noncoding RNA (18-25 bp)t
hat regulate gene expression post-transcriptionally.
Recent studies have demonstrated that epigenetic mechanisms, including
DNA methylation and histone modification, not only regulate the expression
of protein-encoding genes, but also miRNAs, such as let-7a, miR-9, miR-34a,
miR-124, miR- 137, miR-148 and miR-203.
microRNA processing
Picture taken from: http://discovermagazine.com/~/media/i
mport/images/0/2/6/micrornadiag.jpg
DNA methylation
Epigenetic factor that occurs by covalent addition of a methyl group (CH3)
on the 5’ position of cytosine.
• Alters the expression of genes in cells as cells divide and differentiate
from embryonic stem cells into specific tissues.
• This event typically occurs in CpG dinucleotide content where 60% to 90
% of all CpGs are methylated in mammals (Varley et al., 2013).
• Regulation of microRNA expression is influenced by DNA methylation a
nd abnormal methylation is playing an important role in the
tumorgenesis (Suzuki et al., 2012).
DNA methylation
Picture taken from: https://drjanephilpott.wordpress.c
om/tag/dna-methylation/
What is bioinformatics ?
• It is a discipline that represents a marriage between
biotechnology and computer technologies and has evolved the
convergence of advances in each of these fields.
• Today bioinformatics is a field that encompasses all aspects of
the application of computer technologies to biological data.
Computers are used to organize, link, analyze and visualize co
mplex sets of biological data.
In silico study – Bioinformatics
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Using compitutional based methods to analyse biological data
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Protein structure and function prediction
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Genome wide screening for epigenetic factors
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Currently several gene banks are publicly available for micrRNA screening and
methylation analysis
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Correlations can be found between genes, noncoding RNA and methylation
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Predictictions often match with in vitro studies
Reference article
• In silico study conducted by Haung et al., 2015.
• RhoGDI2 interacts with the mRNA expression of EGFR (epidermal
growth factor) gene and is associated with the expression of
mir-10a microRNA molecule.
• RhoGDI2 is involved in the EGFR-associated key network in Squamous
cell carcinoma (SCC) directly or indirectly through interaction with
Proto-oncogene tyrosine-protein kinase Src or simply c-Src.
RhoGDI2
Also known as: RhoGDI2 or ARHGDIB2
Rho GDP-dissociation inhibitor 2 that regulates the GDP/GTP exchange
reaction of the Rho proteins by inhibiting the dissociation of GDP from them,
by subsequent binding of GTP
Biological processes:
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Immune response
Regulation of small GTPase mediated signal transduction
Negative regulation of cell adhesion
small GTPase mediated signal transduction
Others
Reference : http://www.uniprot.org/uniprot/P52566
miR-10a microRNA
Also known as: MIRN10A; mir-10a; miRNA10A; hsa-mir-10a
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Location: Chromosome 17
Several studies showed upregulation and its role as tumor supressor
Possible Biomarkers
Downregulated by DNA methylation
Reference : http://www.ncbi.nlm.nih.gov/gene/406902
Specific aims
1. To investigate expression of miR-10a in NSCLC tissues or/and NSCLC cell lines
Literature:
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Several report have shown that miR-10a is disregulated in several cancer types
(Chang et al.,2014, Jia et al.,2014, Markou et al.,2013).
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Overexpression of miR-10a may be used as a prognosis factor for breast cancer
patients (Chang, et al.,2014).
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miR-10a is shown to contribute to abnormal activation of HOXA1 (Homeobox 1)
gene in human bronchial epithelial cell line (Shen, et al., 2009).
2. To investigate the effects of DNA methylation on miR-10a expression
Literature:
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A recent study conducted by Jia et al., 2014, found that the expression of miR-10a in
gastritic cancer (GC) cells was lower than that in normal cells, which was due to the
hypermethylation of the CpG islands upstream of miR-10a.
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The demethylation experiment found an induction of miR-10a, which indicating DNA
methylation is likely to regulate miR-10a expression in the gastritic cancer.
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Furthermore, reintroduction of miR-10a into GC cells was able to inhibit cell prolifera
tion, migration and invasion.
3. To analyze the target protein expression and correlation to miR-10a microRNA
Literature:
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Huang et al., 2015, confirmed the connection between miR-10a and RhoGDI2
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To verify that RhoGDI2 is the direct target of miR-10a, the wild type 3 untranslated
region (UTR) of RhoGDI2 will be fused directly downstream of the luciferase gene
(pLuc). Previous studies have successfully proven the cloning of the successor
miRNA10-a into the pcDNA3.1 expression vector (Xiang-Hua Liu et al., 2012).
Possibile actions :
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In order to discover the molecular mechanism by which miR-10a acts and the
confirmation on the connectivity to RhoDGI2 we will firstly reconfirm the
onnection by commonly cited programs such as TargetScan, PicTar and miRanda.
link for programs : http://mirnablog.com/microrna-target-prediction-tools/)
c
(
4. Investigate cell proliferation effects by miR-10a and RhoGDI2
Literature:
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It is known that deregulation of miRNAs may lead to alterations in cellular
differentiation, proliferation and apoptotic processes that are important in the
development of cancer (Chitwood et al., 2010, Kosik KS, 2010).
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Downregulation of miRNAs is closely associated with tumor initiation, promotion and
progression via the regulation of key oncogenes or tumor suppressors
(Calin and Croce, 2006).
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Inn a recent study, it was shown that the proliferation of gastritic carcinoma cells is
inhibited by the miR-10a suggesting that miR-10a may function as a tumor
suppressor in vitro (Jia et al., 2014)
But…first comes Bioinformatics research and than
verification and confirmation in the lab…
Experimental Design and Methods
Background results
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miR-10a is deregulated in head and neck squamous cell carcinomas and also in
hepatocellular carcinoma (Hui et al., 2010, Varnholt H et al., 2008) .
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miR-10a serves as an oncogene by regulating CHL1 in human cervical cancer ( Long
MJ et al., 2012), down-regulation of miR-10a in chronic myeloid leukemia promotes
CD34+ cells proliferation (Agirre X., 2008).
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RhoGDI2 expression is down regulated in lung cancer, and the lower expression is
strongly correlated with higher malignancy grade, lower cell differentiation, and
greater lymph node metastasis of lung cancer. The expression of RhoGDI2 is inversely
correlated to the activation level of the PI3K/Akt/mTOR pathway, which plays key
roles in lung cancer (Niu H. et al., 2010).
Methods
The canger genome atlas – (TCGA)
– aimed to analyze the genomic features through sequencing of about
30 different types of cancers
– TCGA is a joint project (27 instititutes)
– https://tcga-data.nci.nih.gov/tcga/ - platform for researchers to
search, download, and analyze data sets (methylation data,
expression of genes, SNPs)
Functional
genes
Functionalannotation
annotation ofof
genes
The
Database for Annotation, Visualization and Integrated Discovery
(DAVID)- v6.7- assigns functions to particular genes !
Famous software- more then 10000 citied papers, some papers were
published in many journals of Nature !!!
Link :http://david.abcc.ncifcrf.gov/
KEGG
KEGG
Koyoto Encyclopedia of Genes and Genomes (KEGG) – to annotate
functions of large scale molecular databases generated by
genome sequencing and other high-throughput experimental
technologies
Used published databases of genes or proteins to generate pathway
diagrams…
Link : http://www.genome.jp/kegg/
Experimental Design and Methods
1. Purchase several non-small- cell lung cancer (NSCLC) cell lines
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Growing them under standard conditions in CO2 incubator
2. Do polymerase chain reaction (qRT-PCR).
To investigate Expression of miR-10a and PhoGDI2 in cell lines
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Isolate RNA
qRT-PCR assays for detection of miR-10a and PhoGDI2 expression using ready
kits
To check the PhoGDI2 mRNA expression, total RNA will be reverse transcribed
by RT-PCR
To verify integrity of PhoGDI2 expression, GAPDH gene can be used as an
internal endogenous control - GAPDH is a housekeeping gene commonly used as a reference for
quantification of gene expression.
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The relative levels of PhoGDI2 mRNA can be calculated using the 2-Δct
method.
3. Effect of DNA methylation
To confirm the effect of DNA methylation on miR-10a expression
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Inhibit methyltion in cells by 5-Aza-CdR - DNA methyltransferase inhibitor, can be detected by
qRTPCR ( to check if methylation has reduced the gene expression or not)
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CpG island methylation can be assessed by bisulfite sequencing- to determine the patterns of
methylation .
4. Verification of miR-10a connection to RhoGDI2 expression
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luciferase reporter vector – clononing of the the wild-type 3’ untranslated region (UTR) of RhoGDI
2 into the firefly luciferase gene (pLuc) of pCDNA vector. – to measure or track expression of the
cloned gene.
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New plasmid will be cotransfected into the cell lines and treated with Lipofectamine 2000 reagent
– used to increase the transfection efficiency of the plasmid
5. To investigate cell proliferation affects by miR-10a and RhOGDI2
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Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science) for cell viability.
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MiR-10a inhibitors cells and pCDNA/miR-10a will be transfected to our cells and
allowed to grow in well plates. Appropriate negative controls will used.
Cell proliferation will be documented every 24 h following the manufacturer’s
protocol
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Todetermine whether RhoGDI2 could also affect the cell proliferation we will do
taget knock down of RhoDGI2 expression using RNAi in our cell lines.
RNA interference (RNAi) is a means of silencing genes by way of mRNA
degradation
Expected Result and Potential Problems
• Expexted results are in vitro correlation of RhoDGI2 and miR-10a
• This study will help us to understand the biological roles of the
molecules involved in lung cancers suppression and lead us to find
potential therapeutic target for the lung cancer.
Potential Problems:
Besides miR-10a, we may have other microRNA interaction with RhoGDI2,
References
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Calin GA, Croce CM,“MicroRNA signatures in human cancers,” Nat Rev Cancer, 6(11):pp 857-866, 2010
Chitwood DH, Timmermans MC,“ Small RNAs are on the move,” Nature, 467(7314):415-419, 2010
Eucan, Accessed at 10th April 2015: Link: http://eco.iarc.fr/EUCAN/Country.aspx?ISOCountryCd=70
E. V. Stevens, N. Banet, C. Onesto et al., “RhoGDI2 antagonizes ovarian carcinoma growth, invasion and metastasis,” Small GTPases, vol. 2, no. 4, pp. 20
2–210, 2011
F. R. Hirsch, M. Varella-Garcia, P. A. Bunn Jr. et al., “Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy
number and protein expression and impact on prognosis,” Journal of Clinical Oncology, vol. 21, no. 20, pp. 3798
H. Niu, H. Li, C. Xu, and P. He, “Expression profile of RhoGDI2 in lung cancers and role of RhoGDI2 in lung cancer metastasis,” Oncology Reports, vol. 2
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Hui AB, Lenarduzzi M, Krushel T, Waldron L, Pintilie M, et al, “Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas,” Clin C
ancer Res 16: 1129–1139, 2010
Kosik KS,“MicroRNAs and cellular phenotypy,” Cell, 143(1):pp 21-26,2010
Long MJ, Wu FX, Li P, Liu M, Li X, et al. (2012) MicroRNA-10a targets CHL1 and promotes cell growth, migration and invasion in human cervical cancer
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Tao Huang, Jing Yang, and Yu-dong Cai ,“Novel Candidate Key Drivers in the Integrative Network of Genes, MicroRNAs, Methylations, and Copy Numb
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Varley KE, Gertz J, Bowling KM, Parker SL, Reddy TE, Pauli-Behn F, et al. “Dynamic DNA methylation across diverse human cell lines and tissues,” Geno
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Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, et al,“MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular c
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Xiang-hua Liu, Kai-hua Lu, Ke-ming Wang, Ming Sun, Er-bao Zhang,et al,“MicroRNA-196a promotes non-small cell lung cancer cell proliferation and in
vasion through targeting HOXA5,” BMC Cancer,12:348, 2012.