Regulatory effects of miR-۲۱۸ on RIC-۳ gene expression

Background and Aim: RIC-۳ (Resistant to Inhibitor of cholinesterase) has an essential role in maturation of diverse ligand gated ion channels such as nAChRs (nicotinic acetylcholine receptors), in particular the homomeric α۷ nicotinic receptor. RIC-۳ causes the proper assembly at the ER and enhances the expression of functional α۷ subunit of nAChRs on the cell surface. As nAChRs are activated by Ach, loss of these receptors especially in hippocampus and neocortex results in Alzheimer’s disease. A critical event in the process of AD is the aggregation of Amyloid-β peptide which interacts with α۷ nAChRs and has been observed to reduce neuronal survival. Meanwhile, the level of miRNAs have been witnessed to alter in AD and a great number of the physiological processes underlying AD are influenced by miRs. Particularly, miR-۲۱۸ deregulates in AD. The aim of this study was to evaluate the down-regulation of RIC-۳ gene expression in the presence of miR-۲۱۸.Methods: Electroporation Systems were used to transfect the microRNAs to the HEK-۲۹۳ cell line. On the other hand, using the computational algorithm of Target-scan the putative binding sites in the ۳′-UTR of RIC-۳ mRNA were identified and characterized for miR-۲۱۸ targeting. The putative miR-۲۱۸ binding site was amplified by PCR, cloned into the Xba۱ site downstream of the Luciferase reporter gene of the pGL۳-Control vector. HEK-۲۹۳ cells were co-transfected using electroporation. Luciferase activities were measured using the Dual-Luciferase Reporter Assay system ۴۸ h after transfection. Western blot analysis was used to analyze the changes in protein levels. Statistical analysis was performed using PRISM version۶. P<۰.۰۵ was considered to indicate a statistically significant result.Results: Luciferase assay test results confirmed the repression of RIC-۳ expression in the presence of miR-۲۱۸ at %۳۲. It is also worth mentioning that Western blotting test revealed that RIC-۳ expression levels reduce with high levels of miR-۲۱۸ in the cells.Conclusion: The present work shows that miR-۲۱۸ severally decreased the level of RIC-۳ gene expression. As it has been proven that RIC-۳ protein is responsible for correct assembly of nAChRs, controlling the expression of the RIC-۳ gene might play an important role in modulating AD.

کلیدواژه ها:

Alzheimer’s ، RIC-۳ protein ، nAChRs ، miR-۲۱۸