oa miRNA Mediated Regulation of hOrai1 Expression

Orai1, a Calcium channel functions with STIM1, a Calcium sensor to mediate Calcium influx. Orai1 is at the plasma membrane and STIM1 localizes in a diffuse manner to the ER membrane under resting conditions. Following Ca++ store depletion, STIM1 forms puncta that localize to the cortical ER and binds Orai1 to allow Ca++ influx. This is the predominant pathway for Ca++ 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 members of Ca++ signaling pathways is crucial for maintaining the subcellular levels of Ca++ required for its numerous functions.

We are interested in the mechanisms that regulate some of the key players of the SOCE pathway. We have developed a system that allows us to test whether these genes are regulated by miRNAs in different cell types at different stages of the cell cycle. We are using normal human cell lines vs. human cancer cell lines to elucidate different mechanisms of regulation of these genes.

We used a GFP and the 3'UTR of hOrai1 as a transcriptional fusion to transfect HEK 293 cells and GFP without any 3'UTR as a control. As an internal control of transfection efficiency we expressed mCherry from the same vector in both conditions. We found the stoichiometry of GFP and mCherry levels in the experimental conditions for hOrai1 3' UTR to be significantly different from those in the control indicating a miRNA-mediated regulation of hOrai. We also found the ratio between GFP and mCherry to be constant throughout the cell cycle in the control and variable in the GFP hOrai1 3'UTR transfected cells suggesting a cell-cycle dependent regulation of hOrai1. We are taking a similar approach to determine whether hSTIM1 is regulated by miRNAs.