my preliminary pagesx - Yimg

skirlorangeΒιοτεχνολογία

1 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

111 εμφανίσεις


i


Computer aided MiRNA Target Prediction
in four frequently Amplified and Mutated
Genes in Lung Cancer




Researcher

Maimoona Ali

12
-
FBAS/MSBI/F08



Submitted as partial requirement for the fulfillment of MS in Bioinformatics at
Department of
Bioinformatics and
Biotechnology, Faculty

of Basic & Applied
Sciences, International

Islamic
University Islamabad




Supervisor: Dr. Sumbul Khalid
31
st

August 2010





ii











In the name of Allah Most Gracious and Most Beneficial
















iii



DECLARATION


I hereby declare that the work presented in the following thesis is my own effort, except where
acknowledged otherwise, and that the
thesis is my own composition. Not

part of the thesis


has
been previously

presented for any other degree or copied from any

other source.



M
a
imoona Ali

















iv


Dedicated to those who believe in Al
-
Quran and the endless
healing powers of Al
-
Quran for the spiritual and physical
sufferings of mankind
as Allah (
SWT
)

says in Al
-
Quran:
“We
sent down (reveled) of the Qur’an
that which is a healing
and mercy to those who believes”



.









v

Certificate

Title of Thesis:


Computer aided MiRNA Target Prediction in four frequently Amplified
and Mutated Genes in Lung Cancer

Name of Student:

M
a
imoona

A
li

Registration No:

12
/FBAS/MSBI/F08


Accepted by the department of Bioinformatics and Biotechnology, Faculty of Basic and Applied
Sciences, International Islamic University Islamabad as a requirement for the fulfillment for
Master of Science in Bioinformatics


Viva Voce
Committee
























Chairman/Director/Head






Dr. Naveeda Riaz







Assistance Professor/
In
-
charge

DBI














External Examiner
















Internal Examiner










Supervisor








Dr. Sumbul Khalid



Assis
tance Professor DBI


Dated: 31
st

August, 2010


vi

TABLE OF CONTENTS


Chapter 1










(1
-
18)


1.1
Introduction










1


C
hapter 2


1.2 Literature
Review








7


Chapter 3









(25
-
32
)


M
ethodology










25


3
.1
Genetic Analysis of Lung
Cancer






26


3.2
Target

Gene Selection








26


3.3 Incorporating Mutations in Genes






26


3.4 Downloading Human MiRNA sequences






27


3.5 Target Prediction via miRanda



__________________
28


3.6 Shuffled vs. Non shuffled Sequences






29


3.6.1 Calculation of Cut off Value






29


3.6.2 Data Parsing____





_
____
_
30



3.6.3 Data Plotting ______________________________________ 30




3.6.4 Data Analysis _____________________________________
_
30


3.7 Results
Verification via RNAhybrid







31


3
.8 Gene Mapping and MSA









31


3.9 Designing miRNA Strand_________________





32


Chapter 4










(33
-
55
)


Results and Discussion



4.1 Genes Involved in Lung Cancer








34


4.2 Target
Genes________________






36



vii


4.2.1 EGFR Gen



______________________
___
37



4.2.2 Tp53 Gene

___________




_________

37



4.2.3 Kras Gene








_
__ 38



4.2.4

ERBB Gene _________

___________________
______3
9



4.3
Target

Prediction of miRNAs in Humans





_

3
9



4.4

Results of miRanda


__________

40



4.5

miRanda Predicted Targets for EGFR Gene


41




4.6 miRanda predicted Targets for p53 Gene


___________

43



4.7 miRanda predicted Targets for Kras Gene_________________________44



4.8 miRanda predicted Targets for ERBB Gene _______________________47



4.9 MiRNAs Targeting Multiple Genes
______________________________49



4.10 Summarizing Results of miRanda ______________________________50



4.11 Verification of Results via RNAhybrid __________________________53



4.12 Multiple Sequence Alignment _________________________________55



4.13 Designing miRNA __________________________________________55




Conclusion & Future Enhancements








(
5
6)


Bibliography










(
56
-
6
4)









viii






LIST OF FIGURES


Fig
1.1:

R
ates for lung cancer compared with other common types of cancer.

_____________3

Fig
1.2:

Comparison of Incident and Mortality
Rates of Lung Cancer and other Cancers.

____
4

Fig

2.1
:
Contribution of Lung Cancer to Worldwide mortality rates for 2010.
___________
_

8

Fig

2.2
:

Common symptom associated with the diagnosis of lung cancer

________________
_

9

Fig 2
.3
:

30 Years Lag Time between Peak Smoking and Peak Lung Cancer from 1920
-
2005.
_______________________________________
_____________________________

12

Fig 2.4
:

A

complex pathway that leads to the development of non small cell lung cancer

_ 14

Fig 2.5
:

Five

year survival rates for different classes of lung cancer. _______________
__ 16

Fig 2.6
:

Different locations of miRNA in genome.






______ ______

18

Fig 2.7
:

Multiple steps involved in the maturation of

a

miRNA inside the nucleus and cytoplasm.






__________________________________________ 19

Fig 2.8
:

Various steps involved in RNA interference therapy
.





21






ix

LIST OF TABLES

Table
4
.1
:
All known genes involved in the development of lung cancer





35

Table 4.2
:

4.2
miRNA Targets for EGFR Genes






______

4
2

Table 4.3
:

miRNA Targets for Tp53 Gene




________________________

4
4

Table 4.4
:

miRNA Targets for Kras Gene




__________________

45

Table 4.5
:

miRNA Targets for ERBB Gene

____________________________________47

Table 4.6
:

miRNAs Targeting
Multiple Genes

______________________________

49

Table 4.7
:

Scores and Positions of miRNA Targets



__________________

5
1


Table 4.8
:
Comparison of miRanda and RNAhybrid Results
________________________

53
















x


LIST OF ABBREVIATIONS



A:
Adenine

A
A
T
:

Anterioraxillary T
horacotomy



ALT
:

A
nterior
olimited T
horactomy

BRAF:
Serine/threonine
-
protein kinase B
-
Raf

BAT3 and MSH5
:

HLA
-
B associated transcript 3 and
mutS homolog 5

C: Cytosine

C
-
MET:

MNNG HOS Transforming gene

CHRNA3 and CHRNA5:

Cholinergic
Receptor, Nicotinic, A
lpha 3 and

Cholinergic Receptor,
Nicotinic, A
lpha 5

CLPTM1L:
Cleft Lip and Palate Transmembrane Protein 1
-
Like P
rotein

DLEC1
:
Deleted in Lung and Esophageal C
ancer 1

EGFR:
Epidermal Growth Factor

EML4
-
ALK:
Echinoderm
microtubule
-
associated protein
-
like 4 (EML4) and anaplastic
lymphoma kinase (ALK)

EPHA3
:
E
ph
-
like tyrosine kinase

ERBB:
Erythroblastic Leukemia Viral Oncogene Homolog

ERCC6
:
excision repair cross
-
complementing rodent repair deficiency, complementation grou
p6

G
: Guanine

GSTM1
:
glutathione S
-
transferase M1


xi

HMOX1:
Heme Oxygenase (Decycling) 1

ITGA9:

I
integrin, A
lpha 9


KDR
:
Kinase insert Domain R
eceptor (a type III receptor tyrosine kinase)

KLC1
:
kinesin Light C
hain 1

KRAS
:
v
-
Ki
-
ras2 Kirsten rat sarcoma viral
Oncogene

H
omolog

LINUX:

Linu
s' UNIX

LKB1:
Serine/threonine kinase 11


miRNAs:
Micro RNA

mRNA:
Messenger RNA


MALAT1:
metastasis associated lung adenocarcinoma transcript 1 (non
-
protein coding)

MAP3K8
:
Mitogen
-
Activated Protein Kinase

8

MET
:
Met
Proto
-
Oncogene (Hepatocyte Growth Factor R
eceptor)

MYCL1
:
v
-
Myc Myelo Cytomatosis viral O
ncogene homolog 1, lung carcinoma derived (avian)

MPO:
M
yelo

P
er

O
xidase

NKX2
-
1:

Homeobox P
rotein Nkx
-
2.1

NTRK:
SLIT and NTRK
-
like family, member 6


NSCLC:
Non Small
Cell Lung Cancer

PARK2:
Parkinson Protein 2, E3 Ubiquitin Protein Ligase (P
arkin)


PIK3CA:
Phosphoinositide
-
3
-
kinase, Catalytic Alpha Polypeptide

PLT:
Posterolateral T
horacotomy

RNA:
Ribose Nucleic Acid

RNase III
: Ribonuclease III

SCLC:
Small Cell Lung
Cancer


xii


SOX2
: SRY (sex determining region Y)
-
box 2, also known as

SOX2

STK11
: Serine/Threonine kinase 11

T
: Thymine

TERT
:
Telomerase Reverse T
ranscriptase

Tp53:

Tumor protein 53

TSG11
:
Tumor suppressor gene on chromosome 11

U
: U
racil






















xiii



ACKNOWLEDGMENTS




T
he foremost

and heartiest

thank
s to
Allah (SWT); the creator who created me a human
being and
the Sovereign and the All Knowing, who showered such a knowledge upon me;
the
Guider who guided me well
and broaden my understanding to diff
erentiate between right and
wrong; the Source of Peace an Provider due to Whom I was able to perform my task even in the
time of severe depression due to loss of my loving Father; the loving and the Opener, Who
opened the door leading to success generally
throughout my life and specifically during my
research. I am not even a particle without His soft and kind always upon my head.

I pay my paramount and doubtless gratitude for my parents, who always sacrificed their own
needs for mine and tried their level
best to provide me full convenience at the cost of their own
rest

since my birth. My Grand Mother, and my parents are always the source of prayers fo me.
Countless words of thanks to my sisters and the only brother they provided a very friendly and
peacefu
l environment that ensured the accomplishment of my goal.

.
I
show

thanks to my supervisor
Dr. Sumbul Khalid
,

Assistant Professor, International Islamic
University Islamabad who taught us to face the up
-
coming challenges open heartedly and guided
me to complete the manuscript in time.


I shall be failing my duty if I do not put forth heartiest gratitude to my who
le class who
cooperated with me and showed complete friendly attitude during this study period in
International Islamic University Islamabad. All my friends Ammara Khalid, Anisa Zia, Atiya
Mehmood, Javeria Irum, Shehla Ab
b
asi
,Shekoh Waraich, Maimoona Ali,
Mehwish Huma and
Faiza Naeem are really source of energy, strength and courage.


xiv

Las
t but not the least
my deepest and cordial thanks to all those sacred souls

especially my Papa

watching me out there
and not able to celebrate my success with me. My celebra
tion is
incomplete without them. May their souls rest in peace and May I follow the path taught by them
throughout my life. Ameen

I pray to Allah (SWT) that may
H
e bestow me with true success in all fields in both worlds and
shower His blessed knowledge up
on me for the betterment of all Muslims and whole Mankind.

Ameen





Pakeeza Akram













xv


ABSTRACT



















The silent epidemic of lung cancer that has been claiming innumerable precious human
lives across the globe persistently for the last
many decades needs to be addressed seriously and
unconventionally. The complexity of disease, involvement of multigenic factors and complicated
signaling and molecular pathways, lack of affectivity of screening techniques and limitations of
available treat
ment options and consistent high death tolls and low survival rates are harsh
realities pertaining to lung cancer. The advent of miRNAs and their acknowledged role in post
transcriptional gene expression and their requirement of partial complementarity to
bind to their
targets make them highly probable to be used as therapeutic agents to control gene expression in
cancer. In this research work targets for human miRNAs have been identified in four frequently
mutated genes i.e. EGFR, ERBB, Kras and Tp53 whic
h lead to the development of lung cancer
by using computer aided tools that predict targets on the basis of sequence complementarity and
minimum free energy of the hybridized complex. . Out of 721 human miRNAs six were
identified to have targets in the nor
mal and mutated forms of these genes. The target miRNA
strand can be designed by analyzing the conserved sequences of these six identified miRNAs,
namely: miR
-
939, miR
-
93, miR
-
765, miR
-
1273, miR
-
887 and miR
-
1285 and may be applied as
a therapeutic agent to

address the four commonly mutated genes involved in lung cancer.