Objective: Increased fatty acids in the circulation and their accumulation in non-adipose tissues play a significant role in the development of obesity related metabolic and inflammatory disorders such as insulin resistance, diabetes and atherosclerosis. While fat tissue has the ability to store excess fatty acids, uptake of excess fatty acids to other tissues burdens intracellular metabolic organelles such as mitochondria and endoplasmic reticulum (ER), leading to stress response and lipotoxic cell death. Unfolded protein response (UPR) is a key adaptation of the ER to stress. It is still not completely clear how lipids engage the UPR and how UPR manages both the adaptive and destructive consequences under its control. Increasing evidence point to the importance of miRNA regulation of the UPR as well as UPR’s role in miRNA biogenesis. In order to understand how lipids engage the UPR, we set forth to identify microRNAs regulated by lipotoxic ER stress in macrophages.Methods: We stressed the mouse macrophage cell line (RAW 264.7) with a saturated fatty acid, 500μM palmitate, reflecting the levels found in the circulation of obese patients. We analyzed the microRNAome profiles of this cell line using QRT-PCR based miScript miRNA PCR array which contained all known mouse microRNAs in miRBase release16 and performed pathway analysis for potential targets.Results: 227 microRNAs showed altered expression levels; 43 microRNAs above 2 fold difference and 13 microRNAs 3-24 fold difference. Pathway analysis enriched the target mRNAs of these lipotoxic ER stress associated miRNAs.Conclusion: When exposed to high concentrations of saturated fatty acids that can induce ER stress, macrophages display a dynamic range of changes in their microRNAome profiles. Our findings reflect the consequences of lipotoxic stress on circulating monocytes and tissue-associated macrophages in obesity. Further studies are needed to deliniate which UPR arm is reponsible for the microRNA changes reported here.
A class of non-coding miRNAs has attracted a lot of interest in the field of forensic sciences. miRNA has a relatively small size thus it is quite stable to the external environmental pressures and factors, which makes it very useful in forensic examination when used as a bio marker. At present, many of the specific miRNAs of body fluids can be distinguished by measuring their level of expression. Fresh blood samples and saliva samples were taken from 3 males and 3 females. RNA was extracted using TRIzol method and cDNA synthesis was done. RT reactions were made using SYBR Green master mix for RT-qPCR for quantification and detection of miRNAs. The designed primers were runs against the sample in qPCR and result was seen and compiled. Two miRNA were detected and expressed in both blood and saliva but they failed to differentiate between blood and saliva due to the non-specific primer binding according the methodology used in this study. Hsa-miR20a and Hsa-miR583 have been found to be non-specific for the blood and saliva. No stark difference could be observed on the basis of which we can say that Hsa-miR20a and HsamiR583 were specific for blood and saliva due to non-specific primer binding. Further research work is required in this domain of body fluids identification and differentiation using miRNA markers.
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