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Module 1 > miRNA in Lung Disease > Flashcards

miRNA in Lung Disease Flashcards

1
Q

What are the 3 sub-groups of non-coding RNAs

A
  1. House-keeping RNAs
    1. Constitutively expressed
    2. Used as baseline to compare changes in expression
      1. Ribosomal RNA, Transfer RNA, Spliceosome RNA
  2. Long ncRNAs
    1. ~ >200 nucleotides
    2. Regulatory RNAs which alter gene expression
  3. Short ncRNAs
    1. <200 nucleotides
      1. miRNAs, Piwi-associated RNA
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2
Q

What does a non-coding RNA actrually mean

A

Does not code for proteins

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3
Q

What are miRNAs? About how long are they in length? And

A

Small, single-stranded ncRNA

~ 22 nucleotides long

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4
Q

What is tha function of miRNAs

A

They bind and regulate mRNA levels

Post-transcriptional regulators of gene expression

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5
Q

Outline the biogenesis and mechanism of action of an miRNA

A
  1. RNA Polymerase II transcribes Pri-miRNA from DNA (hairpin form)
  2. Processed by Drosha/Dgcr8 to form Pre-miRNA (~ 70nt)
  3. Exported out of nucleus by exportin-5 and RAN (GTPase) into cytoplasm
  4. Processed further by DICER into mature miRNA (~22nt)
  5. One strand is loaded onto AGO to form RISC
  6. RISC then matches miRNA to mRNA to induce translational repression or mRNA decay
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6
Q

Describe the structure of mRNA (messenger RNA)

A

5’UTR - protein coding region - 3’UTR - poly A tail

UTR = untranslated region

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7
Q

What is the function of the poly A tail

A

Dictates the stability and life span of the mRNA

↑Translation = ↑deadenylation = shorter A tail

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8
Q

Where does miRNA bind to on mRNA

A

miRNA binds to the 3’UTR region of mRNA

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9
Q

Once miRNA binds to the mRNA on the 3’UTR region, how does it regulate gene expression?

A
  1. Suppression of translation by dislodging of ribosome.Therefore no translation
  2. Deadenylation - signals degradation of mRNA
  3. mRNA degradation
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10
Q

How do miRNAs target mRNA

A

miRNA needs to bind to the seed region (~6nt) and needs to be 100% complementary

The other sequences of whole RNA do not need to be 100% complementary

Therefore, miRNA can have multiple targets

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11
Q

It is important to meause the protein expression as well as _____ expression as it can tell us how the miRNA is working. If there is only a decrease in protein expression then the miRNA has prevented translation of mRNA. If there is reduction of protein and mRNA levels, then the miRNA is working by _____ the mRNA.

A

It is important to meause the protein expression as well as mRNA expression as it can tell us how the miRNA is working. If there is only a decrease in protein expression then the miRNA has prevented translation of mRNA. If there is reduction of protein and mRNA levels, then the miRNA is working by degrading the mRNA.

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12
Q

What samples can you use to collect miRNA from?

A

Lung homogenate (multiple cell types)

Sputum (mainly macrophages)

Small Airway Epithelial Cells

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13
Q

Describe the overview of how RNA nad miRNA are extracted from samples

A

Sample is taken

Tissue is degraded by enzymes

Cells are plated

The cells are then washed and scraped

The cells are lysed

RNA and miRNA are extracted

cDNA (copy DNA) formed through reverse transcription (miRNA less stable, DNA more stable)

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14
Q

How can miRNA levels be measured

A

real time qPCR

miRNA sequencing

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15
Q

Differentiate between the type of data/information collected from miRNA sequencing vs qPCR

A

Sequencing: provides array of altered miRNA

qPCR: Specific miRNA target

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16
Q

Describe what molecules we can use to modulate miRNA expression in vitro

A
  1. Endogenous miRNA
  2. miRNA mimics
  3. Antagomirs (miRNA inhibitors)
17
Q

How can you transfect miRNA into a cell

A

Combination of miRNA mimic and transfection agent

Incubation with cells

Extract miRNAs and perform RT-qPCR

18
Q

To idenfity a potntial mRNA target we can use a luciferase assay. Explain how this works

A

Transfection of a plasmid that codes for a luminescent protein and has a 3’UTR site that will bind to miRNA

miRNA binds to 3’UTR and inhibits protein expression

Therefore, less light is emitted and can confirm miRNA has bound

19
Q

How can we tell if an miRNA will have a mRNA target?

A

Use of online algorithms that can try and predict the ability of miRNA to bind to mRNA

20
Q

Describe how an miRNA plays a role in respiratory disease

A

COPD

SIRT-1 is anti-inflammatory and protects from proteolysis

miRNA 570 negatively regulates SIRT-1 levels

Oxidative stress from CS increases miRNA-570 expression

Decrease in SIRT-1

Increase in cytokines (CXCL8, IL-1, IL-6)

Increase in proteases (MMP-2, MMP-9)

21
Q

Describe how you can change miRNA expression or activity in the lab

A

Mimics - when transfected, can overexpress miRNA

Luciferase Assays - Observe binding of miRNA to 3’UTR and study its effects

Antagomir - nihibits miRNA activity

Module 1 (16 decks)

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