1887

Abstract

The endoplasmic reticulum (ER) functions as a storehouse for intracellular calcium. STIM1, a calcium sensor, localizes mostly to the ER membrane. Following Ca2+ store depletion, STIM1 forms puncta that localize to the cortical ER and bind Orai1, a plasma membrane Ca2+ channel, to allow Ca2+ influx. This is the predominant pathway for Ca2+ influx in non-excitable cells and is referred to as Store-Operated Calcium Entry (SOCE). Mutations in STIM1 and Orai1 cause severe combined immunodeficiencies and are linked to several forms of cancers. Tight regulation of the levels of STIM1 and Orai1 is crucial for maintaining the subcellular levels of Ca2+ required for its numerous functions. We are interested in mechanisms of post-transcriptional regulation of STIM1. We have developed a system that allows us to test miRNA-mediated regulation by transfecting different cell types using a GFP and a 3'UTR transcriptional fusion and GFP with no 3'UTR as a control. mCherry expressed from the same vector is used as an internal control of transfection efficiency. The stoichiometry of GFP and mCherry levels in the experimental conditions for Stim1 3' UTR was found to be significantly different from those in the control. Knockdown of Ago2, an important mediator of the miRNA pathway, by siRNA restored GFP expression from these constructs suggesting miRNA-mediated regulation. We have identified a miRNA that regulates Stim1 expression in HEK 293 (embryonic kidney), MCF7 (non-metastatic breast cancer) but not in MDA-MB231 (metastatic breast cancer) cell lines. The mechanisms by which this occurs will be discussed.

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/content/papers/10.5339/qfarc.2014.HBPP0199
2014-11-18
2019-12-10
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