Binding Potential between miR‐126 and Klotho to Inhibit Inflammation in U937 Monocytes

Abstract

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that features inflammation of synovial tissue in the joints. Monocytes play a key role in the pathophysiology of inflammation and have been found to be activated in patients with RA. Micro RNA (miRNA) can bind to messenger RNA (mRNA) and prevent protein translation. Previous work in our laboratory has revealed specific miRNA molecules, including miR‐126, that are overexpressed in monocytes isolated from RA patients. miR‐126 is a miRNA molecule that can be regulated to impact signaling pathways involved with RA. The KLotho gene has previously been shown to play a role in inflammatory monocytes. We hypothesized that miR‐126 binds to the 3′ UTR of KLotho mRNA, thus inhibiting the translation of inflammatory proteins.Methods and ResultsThe database on microrna.org lists potential gene targets for miRNA molecules, predicted by the program miRanda. The binding scores between the target mRNA and miRNA are powered by mirSVR. Using microrna.org, the KLotho gene was selected as a potential target gene to be bound by miR‐126 based on the two mirSVR scores of −0.0602 and −1.1269 within KLotho's 3′UTR. U937 monocytes were grown, monocyte mRNA was isolated and was used to make cDNA. Forward and reverse primers were designed for the 3′UTR of KLotho mRNA. These primers were used in the qPCR reactions along with the U937 cDNA in order to amplify the 3′UTR of the KLotho mRNA insert. A double digest was performed on both the KLotho insert and a pmirGLO dual luciferase vector using Sac1 and Sal1. The cut 3′UTR of KLotho mRNA was cloned into the cut pmirGLO vector with ligation and transformation with competent E.Coli cells. A 1% agarose gel electrophoresis was performed to analyze the presence of KLotho insert in the pmirGLO vector. The gel analyzed the qPCR products, amplifying the transformation product using sequencing primers. A band on this gel around 750 base pairs confirms the presence of the KLotho insert in the pmirGLO vector. Sequencing of the transformation product is used to further confirm successful cloning of the KLotho insert into the pmirGLO vector. After transfection of the cloned insert in vector, the presence of the insert KLotho gene in the pmirGLO vector can be confirmed by performing a Dual‐Glo□ Luciferase Assay.ConclusionThe KLotho gene is expressed in U937 monocytes. To analyze the 3′ UTR KLotho mRNA cloning into the pmirGLO vector, a 1% agarose gel was performed, and the presence of KLotho mRNA was confirmed in the pmirGLO dual luciferase vector.Support or Funding InformationAlbion College Foundation for Undergraduate Research, Scholarly, and Creative Activity, and the Albion College Biology DepartmentThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.