Role Of MicroRNA In The Regulation Of Store Operated Calcium Entry (SOCE) In Murine CD4~+ T Cells

To investigate how microRNA?miRNA?affected Store Operated Calcium Entry?SOCE?activity,we firstly found out that two compounds with different chemical structures,Epigallocatechin-3-gallate?EGCG?and Urolithin A?UA?,could up-regulate the expression of specific miRNAs.Then some experiments were run to explore how miRNA changes would affect the expression of calcium release-activated calcium channel?CRAC?associated proteins?STIM1/2 and ORAI1?,SOCE activity in murine CD4⁺T cells and so on.Meanwhile,the intracellular mitochondrial membrane potential and calcium level were also detected.Secondly,Dicer-deficient(Dicer^?/?)mice were used as a model lacking mature miRNAs in CD4⁺T cells to elaborate SOCE activity in absence of miRNAs.The main results are as follows:1.EGCG up-regulates miR-15b expression thus attenuating SOCE activity in murine CD4⁺T cells?1?EGCG could promote CD4⁺T cell apoptosis and inhibit cell proliferation by using flow cytometry assay.?2?EGCG?10,20 and 50?M?was able to significantly reduce the slope and peak of SOCE by using calcium imaging assay.?3?10?M EGCG significantly decreased Stim2 and Orai1 transcript levels and protein abundance in CD4⁺T cells by using the mRNA qPCR and Western blot assays.?4?EGCG?5,10,20 and 50?M?was able to significantly increase the expression of miRNA-15b by using miRNA qPCR assay.?5?miRNA-15b overexpression decreased Stim2 and Orai1 both at mRNA transcript and protein levels,and thus attenuating SOCE activity in CD4⁺T cells.?6?10?M EGCG could significantly reduce the mitochondrial membrane potential and promote the occurrence of calcium overload,but ultimately reduce the intracellular calcium level in CD4⁺T cells by using flow cytometry assay.2.UA up-regulates miR-10a-5p expression thus attenuating SOCE activity in murine CD4⁺T cells?1?UA could promote CD4⁺T cell apoptosis and inhibit cell proliferation by using flow cytometry assay.?2?UA?10,20 and 50?M?was able to significantly decrease SOCE activity by using calcium imaging assay.?3?10?M UA significantly reduced Orai1 and Stim1/2 both at mRNA transcript and protein levels in CD4⁺T cells by using the mRNA qPCR and Western blot assays.?4?UA?5,10,20 and 50?M?was able to significantly up-regulate the expression of miRNA-10a-5p in CD4⁺T cells by using miRNA qPCR assay.?5?miRNA-10a-5p overexpression or inhibition by cell transfection decreased or increased Orai1 and Stim1/2 both at mRNA transcript and protein levels,and thus decreasing or increasing SOCE activity in CD4⁺T cells.?6?10?M UA significantly decreased mTOR,AKT and Phospho AKT protein abundance in murine CD4⁺T cells by using Western blot assay.3.Role of Dicer enzyme deficiency?lack of mature miRNAs?in the regulation of SOCE activity in murine CD4⁺T cells?1?The ORAI1 protein abundance of Dicer^?/?CD4⁺T cells was significantly decreased compared to Dicer^fl/fl CD4⁺T cells by using Western blot and flow cytometry assays.?2?The SOCE activity of Dicer^?/?CD4⁺T cells was also significantly declined compared to Dicer^fl/fl CD4⁺T cells by using calcium imaging assay.?3?The intracellular calcium level of Dicer^?/?activated CD4⁺T cells was significantly lower than Dicer^fl/fl activated CD4⁺T cells by using flow cytometry assay.In summary,miR-15b and miR-10a-5p which have Stim2 and Orai1 mRNA binding sites respectively,are involved in the down-regulation of SOCE;Dicer enzyme deficiency will also affect SOCE.EGCG and UA can be considered as an epigenetic regulator of SOCE activity in murine CD4⁺T cells.Furthermore,miRNA is a regulator of SOCE activity in CD4⁺T cells and also plays an important role in maintaining intracellular calcium homeostasis.