Modulation of intracellular calcium signaling by microRNA-34a-5p

Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca2+ influx through Ca2+ release-activated Ca2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions.


Supplementary figures and tables:
Supplementary figure 1: Schematic representation of reporter gene constructs.
Supplementary figure 2: Results of luciferase assays, showing no impact of miR-34a-5p on predicted target genes related to SOCE or calcineurin/NFAT signaling.

Supplementary figure 5: Cell counts of Jurkat cells after microRNA-34a-5p transfection.
Jurkat cells were transfected either with non-targeting control (ANC) or miR-34a-5p mimic for different periods of time. Corresponding cell counts were determined and are shown as means of three independent transfections with corresponding standard errors (SEM). Statistical evaluation was performed using student`s t-test. A normal distribution of the data was assumed. No significant differences were detected.

Supplementary table 1: List of oligonucleotide primer pairs including added enzyme restriction sites.
The amplified 3'UTR sequences of predicted miR-34a-5p target genes were cloned into pMIR-RNL-TK for luciferase gene reporter analysis. MiR-34a-5p binding sites within the 3'UTR sequences of miR-34a-5p target genes were replaced with enzyme restriction sites by overlap extension PCR and cloned into pMIR-RNL-TK. Wildtype 3'UTR-pMIR-RNL-TK reporter constructs were used for template. In primary overlap extension PCR two overlapping mutated sequences were amplified that were used for template in secondary PCR (in combination with cloning primer pairs) to generate the complete mutated 3'UTR insert.
* Oligonucelotide was used for whole amplicon amplification, no secondary PCR was required.