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Identification of Novel microRNAs Regulating Skeletal Muscle Regeneration in Sustained Intensive Care Unit Acquired Weakness.
Impressive work Gupta! I was wondering why were these 2 miRNAs specifically selected? Where they in the top 10 up- or down-regulated miRNAs?
Thank you Adele!
50 miRNAs were found to be differentially expressed between patients who improved their quadricep muscle mass, size and strength versus those who did not 6 months post ICU discharge. Of those 50, 45 miRs did not meet the criteria for correlation with quadricep size, fold change and expression, and did not have Taqman probes available in the murine orthologue (a criteria as at the time our lab only had a mouse muscle myoblast cell line (C2C12)). Of the remaining 5, miR-490-3p and miR-744-5p were found to differentially expressed (upregulated) between those who improved versus those who did not, and had a strong direct correlation with quadricep size.
This past year we were able to obtain a human muscle myoblast cell line (AB1167), which I have recently characterized and optimized for proliferation and differentiation. I then transfected in miR-490-3p and miR-744-5p to see what impact these miRs have on human myoblasts (the data shown in my poster), and if it was similar to the data we got in the mouse cell line – they did have similar effects where miR-490 downregulated proliferation and miR-744 downregulated differentiation in both the mouse and human cell lines.
Great work Muskan! How do you think AKT1 and Mef2D involved in ICUAW? Are these miRNAs known to act on the same processes? What other miRNAs were regulated?
Hi Yara, thank you!
AKT1 and Mef2D have been extensively studied in muscle and have been found to significantly regulate myoblast proliferation and differentiation, respectively. AKT is part of the phosphatidylinositol-3-kinase/AKT pathway, which is known to be one of the most influential pathways to induce muscle protein synthesis and muscle growth. Furthermore, a study has previously shown that patients in the ICU have increased AKT activation in their quadricep muscle, and this may represent a compensatory attempt at reparative/regenerative muscle growth . Mef2s (along with MyoD and myogenin) induce muscle specific structural and contractile genes, such as actin’s, myosin’s and troponins. Thus, the expression of these genes is essential for the proper formation, morphology, and function of skeletal muscle. If Mef2D’s expression is altered in ICUAW such that it is downregulated, it may lead to a decrease in muscle differentiation; if Mef2D is upregulated in ICUAW, it may lead to an increase in differentiation.
To investigate this further, I over-expressed the miRs up to 8 days post transfection (where we see normal/full differentiation in our control samples, scramble miR and untransfected cells) and assessed the impact the miRs have on the genes using qPCR. Interestingly, I found that Mef2D is significantly downregulated, demonstrating that the miRs may be regulating this gene. miR-744-5p was also found to downregulate AKT1 gene expression at 3 days post-transfection.
Therefore, I searched online databases to see if/how likely the miR(s) are to bind to these genes. I found that miR-490-3p has a strong prediction to bind to the 3′ UTR of Mef2D and miR-744-5p has a strong prediction to bind to the 3′ UTR of AKT1. I am currently in the process of cloning these 3′ UTRs into a luciferase plasmid to see if there is a direct correlation with the genes and the miRs of interest – this will hopefully give us some insight into the mechanisms/processes these miRs are going through as there is not much literature published on them.
There were 50 miRNAs found to be differentially expressed between patients who improved their quadricep muscle mass, size and strength versus those who did not, 6 months post ICU discharge. At the time of choosing which miRs to investigate in vitro, we had to focused on ones that had a murine orthologue for Taqman probes (as we only had a mouse myoblast cell line in the lab) – this narrowed the list to 5 miRs. Now that we have a characterized and optimized a human muscle myoblast cell line, it opens our ability to investigate many other miRs such as miR-4732, miR-4762, miR-4530 (to name a few) which are also found to be differentially expressed at 6 months post-ICU discharge and have a strong correlation with quadricep muscle mass, size and strength.
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