Resistant to Inhibitor Cholinesterase gene expression regulation using miR-۹

Background and Aim: Resistant to Inhibitor Cholinesterase (RIC-۳) is a chaperone protein responsible for proper folding and assembly of α۷ nicotinic acetylcholine receptors (nAChRs). According to previous researches on Alzheimer’s disease, the nicotinic acetylcholine receptors interact with pathogenic peptides such as Amyloid β and this interaction results in reduced neuronal survival following loss of nAChRs. Therefore, controlling the number of these receptors exposed on the surface of the neurons is crucial. Moreover, microRNAs are small regulatory RNAs. Based on studies on AD, a variety of microRNAs affect the processes underlying AD and levels of which has shown to change in the patients. One of the critical microRNAs in AD is miR-۹ so the aim of this study was to investigate the targeting of RIC-۳ gene with miR-۹ and therefore evaluate the down regulation of the gene expression due to the presence of this microRNA.Methods: Transfection of the microRNAs to the HEK-۲۹۳ cell line was done using electroporation systems. Meanwhile, the putative binding sites in the ۳′-UTR of RIC-۳ mRNA were identified and characterized for miR-۹ targeting by using the computational algorithm of Target-scan. The putative miR-۹ 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 of RIC-۳. Statistical analysis was performed using PRISM version ۶. P<۰.۰۵ was considered to indicate a statistically significant result.Results: The results of Luciferase test demonstrated that the presence of miR-۹ represses the RIC-۳ gene expression at ۴۳ percent. Western blot results also confirmed the reduction of the expression of RIC-۳ in the presence of miR-۹.Conclusion: According to previous studies, RIC-۳ protein plays an essential role in maturation and assembly of nAChRs. Furthermore, decrease in the number of nAChRs on the neurons’ surface because of Amyloid β aggregation is an important characteristic in AD. Therefore, the current study worked on targeting and down regulation of RIC-۳ with miR-۹.

Keywords:

Alzheimer’s disease , nAChRs , RIC-۳ , miR-۹