11. MicroRNAs in Cancer Flashcards
what organism was miRNA found in?
C. elegans
what are lin mutants?
why are they used?
lineage defective mutants
diff larval stages have diff fates so can trace development thru Lin mutants and map mutations
what is Lin4?
miRNA
Lin4L vs Lin4S
Lin4L base pairs with itself to form hairpin
Lin4S has shorter, linear sequence
there must be precursor and mature form
relationship btwn Lin4 and Lin14, how does it work?
Lin4 represses Lin14
Lin4 matches Lin14 sequence at 3’ and 5’ ends –> base pairing contributes to repression
describe miRNA across organisms
miRNA are conserved across organisms
how did we find that miRNA are conserved across organisms?
Let7 miRNA matches in many organisms
are miRNA encoding RNA?
no!! don’t encode things
what do miRNA do?
just control genes
how have miRNA evolved with mRNA?
miRNA and mRNA have co-evolved since the beginning
describe Let7 mutants in V cells
normally V cells divide finite number of times
Let7 mutants divide continuously and never differentiate like cancer
why do Let7 mutants in V cells cause continuous division?
division of V cell is controlled by Ras
Regions of Let7 match 3’ region of Ras to suppress Ras (dose-response relationship)
describe Let7 and Ras in cancer cells vs normal cells
what does this mean about the function of Let7?
in cancer cells:
-Let7 is reduced and Ras is increased
in normal cells:
- Let7 is increased and Ras is reduced
therefore, Let7 may have TUMOUR SUPPRESSIVE function –> lose Let7 in tumour
where are miRNA usually found in genome?
at fragile sites
what are fragile sites
region of genome in tumour cells where chunk of info is lost or amplified
how do we know that fragile sites are not random?
fragile sites contain fusions between oncogenes and promoters, as well as miRNA tumour suppressors/oncogenes
what do we see at fragile site if miRNA is oncogene?
would see region amplified (multiple copies) in tumour
what do we see at fragile site if miRNA is tumour suppressor?
region would be lost
is miRNA-17-92 oncogene or tumour suppressor?
oncogene
how did they find that miR-17-92 is oncogene?
found RNA from amplicon highly expressed in lymphoma
what oncogene does miR-17-92 interact with?
Myc
Describe 17-92 in Myc driven cancer
17-92 is amplified in Myc-driven cancer
what happens if you transduced 17-92 in mice with overexpressed Myc?
what can you conclude from this?
gives tumour with same profile as Burkitt’s Lymphoma
therefore 17-92 involved in lymphomas
describe survival in mice with:
A. overexpressed Myc
B. overexpressed Myc + 17-92
A. overexpressed Myc = survival
B. overexpressed Myc + 17-92 = survival drops quickly
what does miRNA depend on? why?
example with Let7/Ras
example with 17-92/Myc
miRNA depends on TARGET because it is just a regulator
If you have Let7 but no Ras –> nothing will happen
If you have 17-92 but no Myc –> nothing will happen
where are miRNA encoded?
in genome
what transcribes miRNA ?
miRNA is transcribed by Pol II
how many miRNA does 1 miRNA transcript make?
can make 1 or multiple miRNA
describe the biogenesis of miRNA (5 steps)
- primary transcript made by pol II in nucleus
- pri-miRNA is substrate for DROSHA (RNAse) which makes sequences with overhang –> produces pre-miRNA
- pre-miRNA exported to cytoplasm to meet DICER (RNAse) which makes 2 strands with overhang
- pre-miRNA captured by Argonaute which cleaves the 2 strands
- 1 strand is used as a guide to find mRNA targets with miRISC complex
pre-miRNA vs pri-miRNA
pri-miRNA = primary transcript transcribed from genome
pre-miRNA = cleaved after DROSHA
purpose of argonaute?
normally, the 2 strands cleaved from pre-miRNA would be degraded but argonaute cleaves one of the strand
why does the miRISC complex have specific scanning mechanism to find targets?
most contacts btwn miRISC complex and targets are non-prouctive
what is GW182?
piggybacks on miRISC to help find targets and enacts silencing
how did they crystallize the argonaute structure?
use traces of phenol
shape of argonaute
crescent shape with 2 lobes
describe the domains in the 2 lobes of argonaute
LOBE 1: mid domain and PIWI domain
LOBE 2: PAZ domain and N terminal domain
which 3 argonaute domains are well-conserved?
Mid, PIWI, PAZ
which argonaute domain is more variable/less conserved?
N-terminal
which domain of argonaute binds the 5’ end of miRNA?
Mid domain
what allows specificity for miRNA and argonaute interaction?
1st nucleotide at 5’ end embedded in pocket at mid domain allows specificity
which domain of argonaute binds the 3’ end of miRNA?
PAZ domain
describe the interaction of miRNA with argonaute in terms of domains
5’ end at Mid domain
3’ end at PAZ domain
the rest of miRNA is in the cleft
what is the significance of the non-5’/3’ parts of miRNA being in the cleft?
since the miRNA is in the cleft, the mRNA is also in the cleft
what does the PIWI domain of argonaute resemble?
RNAse H
what does the PIWI domain do?
cleaves mRNA when there’s an RNA-DNA/RNA-RNA hybrid
when does the PIWI domain cleave?
only cleaves when the miRNA is fully base paired with mRNA
where does the PIWI domain cleave?
cleaves exactly at the 10th nt
why does the argonaute scan mRNA in the cell?
to find complementarity btwn miRNA and mRNA
in animals, do miRNA perfectly base pair with mRNA targets?
example with Lin4 and Lin14
miRNA INCOMPLETELY base-pair with mRNA
Lin4 stops base-pairing with Lin 14 by 10th nt
since miRNA and mRNA don’t completely base-pair, what does indicate about the argonaute cleavage mechanism?
if the goal was to cleave, the co-evolution of the miRNA and mRNA would favour base-pairing in the middle
what did ppl initially think was the mechanism for miRNA-mediated silencing?
the cleavage by argonaute
what are the names of the 2 current models for miRNA-mediated silencing
- Deadenylation, Decapping, and Decay
- mRNA Translation Repression
describe the Deadenylation, Decapping, and Decay model of miRNA-mediated silencing
3 steps
- CCR-NOT is deadenylation complex that removes polyA tail of mRNA
- loss of polyA tail stimulates enzymes to remove 5’ cap
- deadenylation AND decapping = mRNA loses stability
describe the mRNA translation repression model of miRNA-mediated silencing
RISC shuts down transcription initiation and prevents ribosome assembly
why is deadenylation bad for mRNA?
polyA tail is important for initiation bc interacts with 5’ end and important for stability of 3’ end bc acts as protection
why are the 2 current models of miRNA-mediated silencing better than the cleavage/slicing model?
these mechanisms allow for better regulation and control bc they can be tuned –> if you just slice, the activity is fully gone
if not for miRNA-mediated silencing, what is the role of the slicing mechanism?
2 miRNA are made but must get rid of 1 so it can base pair with its target
what are P bodies?
biological condensates –> segregate from cytoplasm and concentrate specific enzymes
may be a storage place for mRNA with polyA tail and cap –> then upon exit, the mRNA becomes deadenylated and decayed
what is targetscan?
database with all matching sequences in transcriptome for miRNA-mRNA interactions
how are many miRNA’s organized?
as cluster/polycistron
do the pri-miRNAs have the same functions as the mature miRNA?
how do we know?
pri-miRNA may contain info for diff functions than mature miRNA
many intermediates are made as pri-miRNA is processed –> last intermediate miRNA-19 is most oncogenic
can miRNA profile predict cancer patient survival?
yes
in profiling of miRNA, what causes miRNA expression to increase or decrease?
miRNA expression varies with cell fate and state, i.e. depending on nutrients/conditions
how can we use miRNA to detect cancer
look at changes in miRNA expression and predict origin of cell
if you sequence all miRNA in tumour, can determine where it came from, which subtype of cancer it is, and find NEW subtypes of cancer
survival curve with Let7
what does this tell us about using miRNA to predict outcome?
data shows high Let7 has best outcome –> Let7 is tumour suppressor
just 1 miRNA can help us predict outcome, even if you didn’t know what it does
how can we profile miRNA?
with NGS
what are heatmaps used for?
what is clustering?
what does red/blue mean?
heatmaps are used to analyze multiple variations in gene expression and represent increases/decreases
clustering = grouping genes based on similarities
red = increased expression
blue = decreased expression
relationship between Myc and p53
Myc oncogene will activate p53
in cancer with increased Myc, what happens if 17-92 is removed?
17-92 removal –> pre-B cells will survive bc no self-tolerance and lead to autoimmunity
17-92 cannot activate Myc so p53 is inactive
in cancer with increased Myc, what happens if 17-92 is expressed?
17-92 expressed –> B cells will die
17-92 activates Myc so p53 is active
comparing 2 patients with Burkitt’s Lymphoma, 1 with high 17-92 and 1 with low 17-92, which has worst outcome after treatment?
patient with HIGH 17-92 –> tumour cells won’t die
what happens to PTEN if 17-92 is lost?
PTEN increases –> increased tumour suppressor activity
if you knockdown PTEN, what is the outcome?
very bad bc removing tumour suppressor
what is BIM?
Induces apoptosis
relationship btwn 17-92 and BIM
17-92 allows promotion of proliferation and growth as an oncogene and REPRESSES BIM
in lymphoproliferative disease with high 17-92, what happens with PTEN and BIM?
Both decrease
why do tumour cells develop oncogene addiction?
cancer causes the loss of many genes –> can maintain a pathway via synthetic lethality but there is strain in the background –> cells lose flexibility and are less adaptable
describe 17-92 oncogene addiction
tumour cell relies on 17-92 to survive with Myc overexpression
normal cell would be fine if 17-92 was removed
why is the DICER heavily oncogenic?
required for 17-92
why is 17-92 a good drug target?
tumour is addicted so will die if removed
3 ways for RNA to be used as a drug
- use siRNA with full base-pairing to target gene to slicing activity
- can mimic miRNA to replaces tumour suppressor
- inhibit miRNA with modified oligonucleotide
give an example of an miRNA mimic that can replace a tumour suppressor
normally, Let7 tumour suppressor suppresses Ras
so if Let7 can be restored in tumour cells, tumour will die
describe inhibiting oncogenic miRNA with modified oligonucleotide
oligonucleotide can base pair with miRNA and prevent miRNA function
what is the main problem with using RNA as a drug?
delivery
2 types of overcoming the issue with RNA drug delivery
- aerosol helpful for lung cancer
- lipid nanoparticles
why must RNA drugs have chemical modifications?
exogenous RNA is normally not taken up and is seen as foreign by innate immunity –> chemical modifications hide the RNA from innate immune system
what can determine whether miRNA will function or not?
depends on whether target site is in 3’ UTR
describe dynamics of 3’ UTR
UTRs change with cell state as miRNA changes with cell state
describe relationship btwn 3’ UTR and cancer
cancer can avoid control by miRNA due to the dynamics of 3’ UTR
how does 3’ UTR change in proliferating cancer cells?
3’ UTR can get shorter due to alternative polyadenylation
describe short vs long UTR
long UTR has many miRNA binding sites
short UTR has fewer miRNA binding sites
how can cancer avoid control by miRNA via UTR?
Cancer can shorten the UTR by alternative polyadenylation in proliferating cells
in retinoblastoma what happens if you shut down the 17-92 dicer? is this true in all tumours?
tumour dies
in other cases, if you shut down drosha or dicer to lose the miRNA, there is greater proliferation
