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Question: You hypothesise that EV cargo from aged cells can play a role in changing the epigenetic landscape of stem cells. Outline an experimental design that allows you to explore the influence of EVs on non-coding RNAs. Include a hypothesis, rationale, methodology, what you will quantify and why, and how will you interpret the data in 700 words
. Hypothesis: Extracellular vesicles (EVs) derived from aged cells can play a role in reprogramming the epigenetic landscape of younger stem cells, specifically through the upregulation of non-coding RNA (ncRNA). Rationale: A hallmark of aging is the progression of cellular senescence, a state of arrested cell cycle and increased gene expression of proinflammatory cytokines. In addition, a decrease in the transcription and abundance of non-coding RNAs (ncRNAs) has been observed in aged cells. NcRNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are known to play a role in epigenetic regulation and have been linked to cellular aging. The transfer of EVs between cells may facilitate the reprogramming of younger cells’ gene expression by transmitting molecular signals from aged cells, such as altered ncRNA abundances. Methodology: To test the hypothesis, a cell culture system will be used to study the transfer of EVs from aged to young cells and the resulting reprogramming of the younger cells’ ncRNA expression. A549 cells, which are a line of human lung carcinoma cells, will be cultured from aged donors that are at least 82 years old, and HEK293T cells, a line of human embryonic kidney cells, will be cultured from young donors aged 18-25. The A549 cells will be grown in serum-free medium for 48 hours to induce senescence, and then the cells will be exposed to EVs isolated from the aged A549 cells. After 48 hours of exposure to EVs, the young HEK293T cells will be collected, and total RNA will be extracted and quantified. Illumina sequencing will be used to profile the expression of ncRNAs, such as miRNAs and lncRNAs, in the aged A549 and young HEK293T cells, both before and after EV exposure. What to quantify and why: The levels of expression of various ncRNAs, such as miRNAs and lncRNAs, will be quantified in both the aged A549 cells and the young HEK293T cells. This will allow us to identify how the abundances of ncRNAs differ between aged and young cells, as well as how EVs from aged cells can influence the nc
Jan. 16, 2023, 4:59 a.m.
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