Lecture 7 - siRNAs - Nonn

Question 1

1. As a student, you have just discovered a novel microRNA in one of the breast cancer
   cell lines. We discussed several challenges and limitations to microRNA research in
   cancer. These challenges range from technical to conceptual.
   a. Describe two next steps you would take to learn more about this microRNA. Keep
   in mind the experimental challenges and be sure to propose how you would
   address these in your study. (4 points)
   b. Discuss how the challenges you described above would be similar or different for
   one of the other non-coding RNAs. Select one of the other non-coding RNAs we
   discussed in class. Support you answer in your discussion. (4 points)

Answer 1

Start with breast cancer miRNA
challenges: quality/source of sample
method of detection - how was miR expression profiling done? RT-qPCR biased. NGS would be good

a. Next step: determine mRNA targets.
- target range depends on transcriptional background and the abundance of miRNA.
- analyzing different transcriptomes may result in zero overlap
- test with both low and high miR levels and look at overlap.
- you will get tons of hits in silico so must try via transfection in vivo.
- use luciferase reporter to verify that mRNA is actually degraded
- then determine how these changes influence the phenotype of cells. this may differ by cell type so test multiple cell lines.

b.

Question 2

List 2 advantages to using a miRNA mimic as cancer therapy in comparison to siRNA

Answer 2

• miR have multiple mRNA targets rather than just one (siR)
• siRNAs regulate the same genes that express them. So if there is a gene mutation, the siRNA (which needs perfect binding) may not work.

Question 3

Canonical miR activity

Answer 3

causes translational repression OR mRNA degradation by RISC

Question 4

miR in cancer

Answer 4

• miRNA might act as a TSG and block an oncogene
• global downregulation of miR in tumors
• abberant expression
• expression at fragile sites in the genome

Question 5

ex of miR in cancer

Answer 5

1. OncomiR-1: upregulated in lymphomas
   - 3 miR families
   - inhibit inhibitors of proliferation and angiogenesis and silence apoptosis
2. miR-200 regulates EMT via p53

Question 6

miR as cancer therapies

Answer 6

1. miR mimics for ones that are lost in cancer

2. Sponge: has targets inside that miR bind to instead of the actual mRNA

Question 7

lncRNAs

Answer 7

long non-coding RNAs: found near protein coding genes

• decoy for TFs
• scaffold for proteins
• Guides for proteins
• enhancers
• regulation can be in cis or trans
• ex: HOTAIR: highly expressed in cancer, inhibits HoxD in development, transcribed with HOXC.

Question 8

ciRNAs

Answer 8

circular RNAs

Question 9

pseudogenes

Answer 9

pseudogene: competes for binding of miR
ex: PTENp1: pseudo for pten, lower expression in cancer. Normally, keeps PTEN on by having miR go to PTENp1 instead.

Question 10

piRNAs

Answer 10

piwi-interacting RNAs
- linked to a P transposable element
- used to silence trans element and promote stability
- upregulated in cancer
-

Question 11

ceRNAs

Answer 11

competing endogenous RNAs

compete with mRNA for miR binding.