Investigation of SOCS5 as a gene target of miR‐9 in inflammatory monocytes

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammatory joint destruction. The inflammation is caused in part by monocyte migration into the joints and subsequent macrophage differentiation. MicroRNA (miRNA) are short, single‐stranded, noncoding RNA that bind to mRNA to regulate gene expression. Our lab has previously found that miR‐9 was upregulated in monocytes isolated from patients with RA. SOCS5 is part of the suppressor of cytokine signaling (SOCS) family which downregulates cytokine expression such as epidermal growth factor receptor and interleukin‐6. We hypothesize that SOCS5 is a gene target of miR‐9 and propose a luciferase assay to confirm binding.Using microRNA.org, SOCS5 was determined as a target gene of miR‐9 in silico with a mirSVR predicted binding score of −2.17. microRNA.org uses a combination of miRanda, an algorithm to predict miRNA binding sites, and mirSVR, a support vector regression that provides a predicted binding score. PCR primers that amplify the miR‐9 3′ UTR binding site of SOCS5 were designed using primer‐BLAST. U937 monocytes were grown, mRNA was isolated, and cDNA was produced. The PCR product and pmirGLO vector were digested with Sac1 and Sal1 restriction enzymes and then ligated. The ligated vector was transformed into E. coli cells. Plasmid vector was isolated from single colonies. We determined the ligation and transformation were successful by performing PCR using sequencing primers that amplify the multiple cloning site. Gel electrophoresis was used to separate and view the ligated plasmid PCR product. The SOCS5 insert was calculated to be 576 bp, whereas the non‐ligated plasmid came out to be 380 bp. The ligated plasmid was sequenced to confirm ligation.In conclusion, we have determined that SOCS5 is expressed by U937 monocytes. We have also successfully cloned the 3′ UTR miR‐9 binding site of SOCS5 into the pmirGLO vector for use in luciferase assays to assess binding between miR‐9 and SOCS5.Support or Funding InformationAlbion College Biology Department and The Foundation for Undergraduate Research, Scholarship, and Creative Activity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.