mid sem report

7/18/2019 Mid Sem Report

http://slidepdf.com/reader/full/mid-sem-report 1/27

Mid-Semester Report

on the Masters’ thesis titled

UNDERSTANDING RNA BINDING PROTEIN AND MICRORNANETOR!S

by

NIS"ANT C"AUD"AR#

Entry No - 2010BB50023

Under the supervision of

Dr$ Rit% !%lshreshtha Assistant Professor, DBEB, IIT DELHI

Department of Biocemica! En"ineerin" an# Biotecno!o"yIn#ian Instit$te of Tecno!o"y- De!i

Ne% De!i & 110 01'

April &'()

1



Table of Contents

1. ABSTRACT

………………………………………………………………………………………

………….. 3

2. INTRODUCTION

………………………………………………………………………………………

…. 4

3.

OBJECTIVES……………………………………………………………………

…………………………... 6

4. ITERATURE REVIE!

………………………………………………………………………………....."

4.1 RNA B#n$#n% &'ote#ns

…………………………………………………………………………"

4.2 (#)'o RNAs

………………………………………………………………………………

……...*

4.3 Inte'+la, of RB& an$ -#RNAs

……………………………………………………………11

4.4 Ta'%et -#RNAs an$ RB&s

………………………………………………………………………..11

. (ATERIAS AND (ET/ODS

……………………………………………………………………... 13

.1 Cell #nes

………………………………………………………………………………

…….... 13

.2 Cell &assa%#n%

………………………………………………………………………………

… 13

2



.3 RNA E0t'a)t#on

……………………………………………………………………………….

.13

.4 Ste- oo+ RT &CR

……………………………………………………………………….. .14

. Ste- oo+ an$ &CR +'#-e's fo'

-#RNAs ...................................................1

. Se-# ant#tat#e &CR an$ 5el Ele)t'o+o'es#s

……………………………….....1

.6 Inse't )lon#n% #n

e)to'…………………………………………………………………………….

1

6. RESUT AND DISCUSSION

………………………………………………………………………….1"

6.1 &'#-e' Des#%n an$ Test#n%

………………………………………………………………..1"

6.2 Real7T#-e &CR $ata

…………………………………………………………………........1*6.3 -#' 1*3b )lon#n% fo'

oe'e0+'ess#on……………………………………………………….28

". 9UTURE COURSE

………………………………………………………………………………………

. 22

:. RE9ERENCES………………………………………………………………………………………

….... 23

3



1. ABSTRACT

Micro RNAs (miRNAs) are small, non-coding RNA molecules which are

involved in transcriptional regulation of genes !he" do so #" #inding

to the micro RNA recognition site on the 3$ %!R of the target transcript

RNA #inding proteins (R&'s) are involved in a variet" of functions

including post-transcriptional regulation of genes !he" have also #een

shown to aect the messenger RNA at the 3$ %!R &oth R&'s and

miRNAs are nown to #e e" pla"ers mediated #" *"po+ia *"po+ic

conditions present in cancer cells have #een shown to aect the fate of

the cell and drive towards tumor aggressiveness ince #oth R&'s and

miRNAs have #een shown to #e aected #" *"po+ia and more

importantl" since the site of action of #oth these pla"ers is the same

(3$%!R of mRNA), it is highl" liel" that the" might function in tandemor #e involved in each others regulation !he aim of this stud" is to

uncover such interactions !heir involvement might also add a new

dimension to the development of targeted therapeutics in cancer

#iolog"



2. INTRODUCTION

*"po+ia has emerged as an important factor in tumor #iolog" .t has

#een identi/ed as a driver of angiogenesis, tumor aggressiveness,

cancer stem cells etc .t is also a negative prognostic factor for several

cancers, primaril" #reast cancer, glio#lastoma, cervical cancer etc

*"po+ia-induci#le genes regulate several #iological processes,

including cell proliferation, angiogenesis, meta#olism, apoptosis,

immortali0ation and migration ancer cells have a variet" of

mechanisms to tae advantage of some of these responses (eg

angiogenesis induction), and to evade others (eg apoptosis) Man" of

the nown oncogenic signaling pathwa"s overlap with h"po+ia-induced

pathwa"s +pression pro/ling studies have highlighted man" of the

genes that are regulated #" h"po+ia and #" *.-14, the masterregulator of *"po+ia5induci#le genes !he" include angiogenic factors,

proliferation and cell-adhesion genes onsidering all of these things,

targeting h"po+ic responses #ecomes an important tool in cancer

therap" and development of cancer therapeutics 617 'ost-

transcriptional regulation pla"s an important role in regulation of gene

e+pression under *"po+ia 'ost-transcriptional regulation can occur

through a variet" of means that are mainl" centered on the 3$ %!R !wo

of those means are modulation through RNA #inding proteins (R&')

and a small class of non-coding RNAs called micro RNAs (miRNAs)

Micro RNAs are 18-29 nt long small RNAs which share se:uence

similarit" with 3$%!Rs of speci/c mRNAs and depending on the e+tent

of complementarit", the" either completel" degrade the transcript

9



(complete complementarit") or inhi#it translation (partial

complementarit") RNA #inding proteins #ind to dou#le stranded or

single stranded RNAs and can perform a variet" of functions including

RNA processing and modi/cation, RNA editing, #inding and recognition

features including 0inc-/nger #inding etc !he" also pla" a role in

regulation of mRNA translation #" #inding to 3$ %!R ;ne of the

mechanisms in which the" prevent translation is through interference

with Ri#osome-mRNA #inding and sta#ilit" <ulshreshtha et al were the

/rst to report that *"po+ia induces a speci/c spectrum of miRNAs, with

various roles in cell fate, in #reast and colon cancer and found that *.-

1 (*"po+ia induci#le factor 1), a master regulator of gene e+pression in

*"po+ia is the transcription factor involved in their introduction 627

Masuda et al reported implication of RNA #inding proteins, which

modulate mRNA translation, in *"po+ia 637 onsidering that #oth

miRNAs and R&'s have #een shown to #e dierentiall" regulated under

h"po+ic conditions, identif"ing their roles in each other$s regulation

#ecomes a novel facet in our understanding of these transcriptional

regulators

=



3. OBJECTIVES

Sta%e I ; I$ent#f,#n% Can$#$ate -#RNAs an$ RB&s

!he /rst o#>ective is to do a thorough literature surve" and identif"

micro RNAs which have #een shown to #e dierentiall" regulated in

*"po+ia response ?iterature surve" was also done to identif" e" R&'

pla"ers in *"po+ia !he candidate genes for the R&'s thus identi/ed

were used to identif" miRNA targets through !argetcan, which is a

we# server which predicts #iological targets of miRNAs #" searching

for the presence of sites that match the seed region of each miRNA

!hese miRNAs were then correlated with the list of *"po+ia regulated

miRNAs found #efore !he miRNAs common among the two lists were

identi/ed as candidate miRNAs for our stud"

Sta%e II ; Ve'#<)at#on of 'e%lat#on of )an$#$ate RB&s an$

-#RNAs #n /,+o0#a

!he candidate miRNAs and R&'s selected through literature review

were su#>ected to regulation studies to chec for individual

@



upregulation or downregulation in h"po+ia !his is performed using :-

R! 'R

Sta%e III ; Oe'e0+'ess#on of -#RNAs to )e)= RB& leels

an$ #)e7e'sa

;nce the individual levels of the candidate miRNAs and R&'s are

ascertained, then one$s regulation in *"po+ia would #e checed #"

overe+pressing the other ;vere+pression would #e achieved using

vector insertion and regulation would #e determined #" luciferase

assa" of 3$ %!R of mRNA and :-R! 'R

Sta%e IV 7 To )on<'- e>e)t of )an$#$ate -#RNA7RB&

#nte'a)t#on on ta'%et %enes

!he candidate miRNAs will #e overe+pressed glio#lastoma cell lines

(%8@M) and its eect on nown target genes of candidate R&' will #e

studied

4. ITERATURE REVIE!

4.1 RNA Binding Proteins (RBPs)

4.1.1 Introduction

RNA-#inding proteins (R&'s) are proteins that #ind to the dou#le or

single stranded RNA in cells and participate in forming

ri#onucleoprotein comple+es R&'s contain various structural motifs,

such as RNA recognition motif (RRM), dsRNA #inding domain, ssRNA

#inding domain, 0inc /nger domains etc A speci/c proteinBs

recognition of a speci/c RNA has evolved through the rearrangement

8

http://en.wikipedia.org/wiki/RNA

http://en.wikipedia.org/wiki/Structural_motif

http://en.wikipedia.org/wiki/RNA_recognition_motif

http://en.wikipedia.org/w/index.php?title=DsRNA_binding_domain&action=edit&redlink=1

http://en.wikipedia.org/wiki/Zinc_finger

http://en.wikipedia.org/wiki/Structural_motif

http://en.wikipedia.org/wiki/RNA_recognition_motif

http://en.wikipedia.org/w/index.php?title=DsRNA_binding_domain&action=edit&redlink=1

http://en.wikipedia.org/wiki/Zinc_finger

http://en.wikipedia.org/wiki/RNA



of these few #asic domains R&' targets can #e mRNAs as well as a

num#er of non-coding RNAs including miRNAs, small interfering RNAs

(siRNAs) as well as splicesomal small nuclear RNAs (snRNA) 67

4.1.2 RBPs and post-transcriptional control

R&'s dictate the life of the mRNA all throughout its e+istence R&'s

#ind to speci/c cis-acting elements that can #e found across the whole

messenger RNA #ut are more usuall" located in the 9C- or 3C-

untranslated regions (%!Rs) Although R&'s could in principle activate

or repress translation, most nown e+amples depict proteins that

repress translation #" #inding to the 3C-%!R !he 3$-%!R is a ma>or site

for mRNA sta#ilit" with lots of dierent mechanisms occurring in the

site ;ne of the primar" modes of R&'-mediated control of mRNA

sta#ilit" is through interference with ri#osome recruitment and

su#se:uent prevention of translation 697

4.1.3 RBPs and Hypoxia

.t has #een o#served that total RNA levels and RNA transcription was

found to reduce maredl" in the presence of *"po+ia !he primar"mode of this change is due to post transcriptional modi/cations which

occur mainl" through mRNA turnover and translational control and the

two e" modulators are RNA-#inding proteins and non-coding RNAs

Masuda et al did a comprehensive review stud" on important R&'s

D



involved in mRNA sta#ilit" and their #ehavior under *"po+ia !he"

mainl" reviewed the following R&'sE *uR, '!&, .R's, '&s, hnRN's637

4.2 (#)'o RNAs ?-#RNAs@

4.2.1 Introduction

MicroRNAs (miRNAs) are short (1D52 nucleotides) noncoding RNAs

that regulate gene e+pression at the post-transcriptional level !he /rst

miRNAs, lin- and let-@, were discovered in the nematode

Caenorhabditis elegans ince then, thousands of miRNAs have #een

found in animals, plants and even in viruses .t has #een shown that

these small RNA molecules function as e" regulators of a wide variet"

of #iological processes such as cell proliferation, cell dierentiation,

apoptosis and development !o date, there are 1881 human miRNA

entries in the miRNA registr" (miR&ase) and it is estimated that up to

3FG of human protein-coding genes ma" #e targeted #" miRNAs

Mature miRNAs are named with the HHmiR$$ pre/+ followed #" a uni:ue

identif"ing num#er assigned

se:uentiall" ?ettered suI+es are given to miRNAs with slightl"

dierent se:uences (eg, miR-19a and miR- 19#)

Fig 2: Biogenesis of miRNAs showing formation of pri-miRNA in nucleus followed by

transport to cytoplasm through exportin-5 and subsequent maturing using Dicer enzyme.[6]

MiRNA genes are generall" transcri#ed #" RNA pol"merase .. into long

(J1 <#) primar" transcripts (pri-miRNAs), which contain a 9$ @-meth"l

guanosine cap, a 3$ pol"(A) tail and a local hairpin structure !he stem5

1F



loop structure is cropped #" the nuclear en0"me Krosha and cofactor'asha into a L@F-nucleotide precursor miRNA (pre- miRNA) !he pre-

miRNA is then moved to c"toplasm #" e+portin-9, where it is further

processed into a L22-nucleotide miRNA duple+ #" the en0"me Kicer

!he miRNA duple+ is unwound and one strand (the mature miRNA) is

assem#led

4.2.2 iR!" and post-transcriptional control

Mature miRNAs regulate their gene targets through imperfect

complementar" #ase-pairing to the 3Buntranslated regions (%!Rs) of

their target mRNAs, leading to translational repression andor mRNA

degradation !he miRNA duple+ is unwound and one strand (the

mature miRNA) is assem#led with argonaute protein into the eector

comple+ termed miRNA-containing ri#onucleoprotein (miRN') comple+

to mediate translational repression andor mRNA degradation of target

mRNA !he /rst 258 nucleotides (the HHseed$$ se:uence) at the 9$ region

of miRNA are essential for interaction with the miRNA #inding site in

the 3$ %!R of target mRNA Most microRNAs pair imperfectl" with sites

in the 3$-%!R of their target mRNAs, and can act #" #oth decreasing

11



target mRNA levels through cleavage or #" directl" inhi#iting

translation Reported mechanisms of translational repression #"

microRNAs include interference of recognition of the 9$ cap of the

mRNA to prevent translation initiation, or desta#ili0ation of mRNA via

deaden"lation of the pol"-A tail, and recruitment of the ri#osome

inhi#itor" protein e.= 617

4.2.3 #icroR!" and Hypoxia

A functional lin #etween h"po+ia and the microRNA e+pression pro/le

was esta#lished #" <ulshreshtha et al 627 .n the stud", a microarra"-

#ased screening was done to identif" all the miRNA that were over-

e+pressed in case of h"po+ic tumors as compared to normo+ic tumors

.t was also found that the *"po+ia .nduci#le actors (*.) pla"ed a

critical role in the transcription of most of these miRNAs A strong

correlation was also seen with the miRNA e+pression pro/les of

dierent cancers in general with the h"po+ic e+pression pro/le, clearl"

indicating a relation #etween the three, ie, h"po+ia, tumor growth and

miRNA e+pression

4.3 Interplay of RBPs and microRNAs in 3’UR

tudies have shown that the ?A (*uR) class of RNA #inding proteins

are involved in tandem with miRNAs when it comes to post

transcriptional regulation or e+ample, in response to cellular stress,

*uR is dephosphor"lated and translocates, which relieves cationic

amino acid transporter 1 (A!1) mRNA from miR-122-mediated

repression *uR is also re:uired for let-@-mediated repression of MO

mRNA 6@7 .t was also demonstrated that com#ination of *uR and mir-

313 promotes more sta#ilit" of *uR targeted transcripts lie ;PD,

A and R as compared to sta#ilit" #" *uR alone !he same

stud" also found out that *uR also regulated a#out 8FG of the genes

12



regulated #" miR-313 687 rom these studies we can see that there is

de/nitel" some amount of interpla" #etween RNA #inding proteins

involved in transcriptional regulation and micro RNAs !his interaction

however, has not #een studied under the eect of *"po+ia

4.4 arget miRNAs and RBPs

After going through literature, a list of R&'s and miRNAs was created

#ased on their dierential regulation under h"po+ia in cancer cells 687

6D7 After this individual R&'s were searched on !argetscan

(httpEwwwtargetscanorg) and their respective putative micro RNA

targets were identi/ed rom this list of target miRNAs for each R&',

onl" those were selected which were shown to #e dierentiall"

regulated under h"po+ia !he following ta#le contains a list of R&'s

and their possi#le target miRNAs which are nown to #e dierentiall"

regulated under h"po+ia

RNA b#n$#n% +'ote#n -#RNA +'e$#)te$ to ta'%et RB&

Re%late$ b,

/,+o0#a

Re%late$ b, /,+o0#a

*uR (also called

?A?1)

miR-1D1 (%npu#lished data from la#)

'!&'1 miR-2F=, miR-D3, miR-1D3#

'!&'2 miR-19F

!!'A miR-129a

.3A miR-322, miR-2, miR-D@

'&1 miR-2F=, miR-22

miR-29, let-@, miR-8D

13

http://www.targetscan.org/

http://www.targetscan.org/



'&2 miR-322, miR-D@, miR-2F9, miR-D3, miR-

3@3, miR-21F

miR-29, miR-2

'&3 miR-21, miR-1F@, miR-D@, miR-D3, miR-3@3,

miR-1F@

miR-29, miR-2

'& miR-2F3, miR-192, miR-D3

miR-29

!.A1 miR-199

miR-29

&ased on this, &TB&1 was chosen as candidate R&' and -#R71*3b

was chosen as candidate micro RNAs for further stud"

. (ATERIAS AND (ET/ODS

!ell "ines

1



Qe are using %8@M cell lines !hese cells are grown in KMM

(.nvitrogen) with 1FG & and 'entrep

!ell Passage

Qhen a culture dish is full" conuent, either a stoc is made or the cell

passaging is done to create more cultures from the same or

passaging, the media is /rst removed from the plate .n a 1F cm

culture dish, we add 19 ml of tr"psin and incu#ate this at 3@S for 3

minutes Meanwhile, 1F ml of fresh media is poured in 2 or 3 new

plates depending on the conuenc" of the original plate After 2-3

minutes of incu#ation, 2FF to 9FF Tl of the tr"psin containing the

detached cells are added to the fresh media plates !hese are then

sealed with paraIn strips and put awa" in a ;2 incu#ator

RNA #$traction

RNA was e+tracted from the cells using !ri0ol method or ermentasU

RNA 'uri/cation <it !he concentration and purit" of RNA e+tracted

was measured using Nanodrop and the RNA was su#se:uentl" stored

at -8FF

c%NA &ynt'esis

cKNA s"nthesis was performed using erso cKNA s"nthesis it from

!hermo/sherU !his it uses oligod! primers which can #e used to

form cKNA from all messenger RNAs (mRNAs) owing to presence of

pol"A signal in 3$ %!R of all mRNAs ;ligod! primers cannot #e used

for cKNA s"nthesis of micro RNAs #ecause of much smaller length

(L29 nt) and non availa#ilit" of pol"A signal !herefore for micro RNAs

speci/c stem loop primers were designed which has #een discussed in

ne+t section

19



&tem "oop R P!R

ig 3E tem ?oop R! 'R

tem-loop R! primers #ind to the 3B portion of miRNA molecules,

initiating reverse transcription of the miRNA !hen, the R! product is

ampli/ed using a miRNA speci/c forward primer and a universal

reverse primer !he speci/cit" of stem-loop R! primers to individual

miRNA is conferred #" a si+-nucleotide e+tension at the 3B endV this

e+tension is a reverse complement of the last si+ nucleotides at the 3B

end of the miRNA orward primers are speci/c to the miRNA se:uence

#ut e+clude the last si+ nucleotides at the 3B end of the miRNA A 9B

e+tension of 95@ nucleotides is added to each forward primer to

increase the melting temperature Real !ime-'R ena#les #oth

detection and :uanti/cation !he :uantit" can #e either an a#solute

num#er of copies or a relative amount when normali0ed to KNA input

or additional normali0ing genes !his method is used to :uantif" the

miRNAs in the control and infected samples

1=



No !arget !m

(S)Name e:uence

198

%niv R' !A!A!

2 miR-1D3# 938 1D3#-!AA!!AAA

3 mir-1D3#1D3#-tem

?oop

!!A!A!A!A!A!!A!A!A

A

mir-1D3# 98 loning ' AA())ATT((A(((A)TAA()ATT((TT(A)

9 mir-1D3# 9D loning R' AT()AATT(())(AAA(T)A)AA(A(A(A(

Ste- oo+ &CR an$ )lon#n% +'#-e's fo' -#)'o RNAsE

&emi*+antati,e P!R and -el #lectrop'oresis

emi-Wuantative 'R was performed on micro RNA and gene transcript

cKNAs radient 'R function was utili0ed on thermal c"cler to /nd

optimum melting temperature of the primers or running micro RNA

'R product, a 2-3G agarose gel was used owing to resolution of small

fragments (L9F-@9 nucleotides) and low range (minimum #and of 29

#p) ermentasU ladder was utili0ed or running gene transcript cKNA

product, a 1-19G agarose gel was suIcient in providing optimal

resolution of 19F-2FF #p #and of desired amplicon Medium range, 1FF

#p ladder was used to identif" the #ands thus o#tained

!onstr+ction of microRNA e$pression ,ector

miRNA cloning primers were designed and standardi0ed and the

optimum !m was identi/ed for further use After this, a 'R was

performed in 1FF Tl total volume to amplif" the insert !his was run on

1@



a 1G agarose gel and the speci/c #and was cut and eluted from the

gel After checing the purit" of the eluent, a dou#le digestion was

performed on the insert and also on the vector (p&A&) with en0"mes

coR1 and &am*1 !he samples were ept overnight at 3@F for

digestion ;nce the digestion was complete a ligation was performed

on the digested insert and the vector in a total volume of 1F Tl *ere

too the sample was ept overnight at 3@F @ Tl of the ligated product

was added to 2FF Tl of competent cells and this mi+ture was spread on

a ?A plate with Ampicillin resistance !he plate was ept at 3@F

overnight !he ne+t da" 8 individual colonies were piced up from this

plate and grown on ?& media with Ampicillin resistance !hese were

then ept in a shaing incu#ator at 3@F overnight !he ne+t da"

plasmid e+traction was performed for all the 8 colonies that were

grown After this a dou#le digestion was performed on the puri/ed

plasmid with the en0"mes &am*1 and coR1 and the digested product

was run on a 1G Agarose gel to chec for insert addition which would

indicate the success of the cloning e+periment

18



6. RESUTS AND DISCUSSSION

6.1 &'#-e' Des#%n an$ Test#n%

&TB&1

(ig E '!&'1 primer set gel, 1-2E A'K* samples, 3E 1FF #p fermentas ladder(Mid range), -

@E '!&'1 primers at dierent !ms(91F , 92F , 93F , 9@F ))

rom the a#ove /gure , it can #e o#served that the 'R product for

'!&'1 was ampli/ed at the desired si0e of amplicon (L2FF #p) in the

@th well !he !m for this was 9@o and this temperature was used for

e+pression anal"sis

1D



-#R71*3b

(ig 9E miR-1D3# primer gel with KM;E 1E %= sample at 2 h Normo+ia, 2E Negative control, 3-

8E miR-1D3# sample at 2 h Normo+ia with !m gradient D-93 F , ?adderE ?ow range - 29 #p

ladder)

rom the a#ove /gure 9, it can #e o#served that the 'R product for

miR-1D3# was ampli/ed at the desired amplicon si0e (F #p) in all thegradient temperatures chosen ;wing to the high intensit" of #and at

!m X 91F , we chose this temperature as melting temperature for :-

R! 'R stud" of this micro RNA

2F



6.2 Real T#-e &CR $ata

%= 1D3#

F

1

2

3

-

9

1*3b 'e%lat#on #n U:"(5 n$e' /,+o0#a

Nor

*"p

.t was o#served that mir-1D3# was appro+imatel" fold upregulated in

lio#lastoma (%8@M) under h"po+ic conditions

21



A%, '!&'1

F

F2

F-

F=

F8

1

12

&TB&1 'e%3lat#on #n U:"(5 3n$e' /,+o0#a

Nor

*"p

.t was o#served that '!&'1 was appro+imatel" 2 fold downregulated in

lio#lastoma (%8@M) under h"po+ic conditions

6.3 -#R 1*3b )lon#n% fo' oe'e0+'ess#on

Clon#n% +'#-e' test#n%

22



(ig =E mir1D3# overe+pression primer testingE 1E Mid range fermentas ladder 1FF #p, 2-@E

radient 'R from 99-=F F )

rom the a#ove /gure =, it can #e o#served that the 'R product for

overe+pression of mir1D3# was ampli/ed at the desired si0e ofamplicon (LFF #p) in all the wells reatest intensit" is seen in the @th

well which had a !m of =FF , and this temperature was chosen for

cloning studies

Clon#n% e'#<)at#on

23



(ig @E loning con/rmation after plasmid e+traction and restriction digestion 1E Mid-range

fermentas ladder 1FF #p, 2-DE 8 individual colonies piced up with plasmid e+traction and

restriction digestion performed on each, 1FE *igh range fermentas ladder 1#)

rom the a#ove /gure @, it can #e o#served that none of the colonies

which were piced up contained the desired insert rom /gure = we

con/rmed that the desired insert si0e is FF #p which is not seen in

an" of the wells !he #right #ands in each well indicate the plasmid

#ac#one

2



". 9UTURE COURSE

Qe have compiled a list of microRNAs and R&'s that are dierentiall"

e+pressed under h"po+ia Qe chose miR-1D3# and '!&'1 miRNAEtarget

pair for further anal"sis in #reast cancer and glio#lastoma (%8@M) cell

lines An inverse correlation of the pair was o#served in %8@M cells

under h"po+ia in accordance with the h"pothesis 'resentl", we are

woring on cloning miR-1D3# in p&A& plasmid with purom"cin

resistance for overe+pression of the microRNA ;nce this is achieved

we will chec the eect of this overe+pression on the e+pression levels

of our candidate R&' u#se:uentl" we will con/rm whether the

microRNA is directl" targeting the R&' #" performing 3$ %!R luciferase

assa" or this we will clone the 3$%!R of '!&'1 transcript in front of

luciferase en0"me .f miR-1D3# directl" targets '!&' then we e+pect

the luciferase activit" to go down Qe would also perform this

e+periment #" interchanging the roles of R&' and miRNA, that is to sa"

we would also overe+press '!&'1 gene and chec levels of miR-1D3#

inall" we would loo at the levels of target genes of '!&'1 in the

presence of overe+pressed miR-1D3#

29



:. RE9ERENCES

1 *arris A? E /,+o0#a ; a =e, 'e%lato', fa)to' #n t-o' %'ot.

!at Re$ Cancer 2FF2, 2E 38-@

2 <ulshreshtha et alE A -#)'o RNA s#%nat'e of /,+o0#a

#olecular and Cellular Biology 2%%&' ol 2@ no 9E 189D-18=@

3 Masuda et alE RNA7b#n$#n% +'ote#ns #-+l#)ate$ #n /,+o0#a'es+onse. (. Cell. #ol. #ed. ol 13, No DA, 2FFDE 2@9D-2@=D

lisovic et alE RNA7b#n$#n% +'ote#ns an$ +ost7t'ans)'#+t#onal

%ene 'e%lat#on )*B+ ,etters(lsevier) 2FF8, :2 (1)E 1D@@5

1D8=

9 0osta , e#auer E T'anslat#onal )ont'ol b, 37UTR7b#n$#n%

+'ote#ns Brie )unct. enoics 2%13E 12(1)E98-=9

= illies Y<, ?orimer .AE Re%lat#on of +2"#+1 b, -#RNA 221F222

#n %l#oblasto-a Cell Cycle 2FF@, 6(1=)E2FF9-2FFD

@ Mariee van <ouwenhove, Marti>n <edde Z Reuven AgamiE

(#)'oRNA 'e%lat#on b, RNA7b#n$#n% +'ote#ns an$ #ts

#-+l#)at#ons fo' )an)e'. !ature Re$ie/s Cancer 2F11, 11 =-

=9=

8 hivani harma, uneer erma,Madavan asudevan, u#hasisamanta, Yitendra < !haur and Ritu <ulshreshtha[, Te #nte'+la,

of /R an$ -#R73134 #n 'e%lat#on of AU '#) t'ans)'#+to-e

R!" Biology 2%13, 1FE8, 158

2=

http://en.wikipedia.org/wiki/FEBS_Letters

http://www.ncbi.nlm.nih.gov/pubmed?term=Szostak%20E%5BAuthor%5D&cauthor=true&cauthor_uid=23196851

http://www.ncbi.nlm.nih.gov/pubmed?term=Gebauer%20F%5BAuthor%5D&cauthor=true&cauthor_uid=23196851

http://en.wikipedia.org/wiki/FEBS_Letters

http://www.ncbi.nlm.nih.gov/pubmed?term=Szostak%20E%5BAuthor%5D&cauthor=true&cauthor_uid=23196851

http://www.ncbi.nlm.nih.gov/pubmed?term=Gebauer%20F%5BAuthor%5D&cauthor=true&cauthor_uid=23196851



D uomin hen, Piao#o ?i, Oong-feng Yia, ar" A 'ia00a and Oaguang

Pi /,+o0#a7'e%late$ -#)'oRNAs #n -an )an)e'. "cta

Pharacologica +inica' 2%13, 3E 33=531

mid sem report

Documents