MiR429: Potential regulator of PAK2 in Inflammatory Monocytes

Abstract

PurposeRheumatoid Arthritis is an inflammatory disease that leads to the eventual deterioration of joints. Monocytes are key players in its pathogenesis and migrate to the sites of inflammation. Micro (mi)RNAs regulate mRNAs and the synthesis of proteins and have the ability to silence specific mRNAs by preventing translation or aiding their degradation. Previous results from our lab have shown that specific microRNAs are elevated in monocytes from patients with Rheumatoid Arthritis. Using in silico analysis, we have also determined potential gene targets for these miRNAs in monocytes. miR429 is a differentially expressed miRNA in monocytes in response to inflammatory stimuli and has PAK2 as a theoretical gene target. miR429 has been shown to inhibit cancer cell migration by reducing the expression of its target genes. PAK2, which is highly expressed in leukocytes, including monocytes, is known to regulate inflammation. Therefore, we hypothesized that miR429 regulates inflammation by binding to PAK2 and mediating its degradation.Methods & ResultsIn order to assess the binding between miR429 and PAK2 we cloned the 3′ untranslated regions (UTR) of PAK2 into the pmirGLO dual luciferase vector. The vector was propagated, purified, and treated with the restriction enzymes SacI and SaclI, and cleavage was confirmed by PCR analysis. THP‐1 monocytes were grown and used as a source of mRNA. cDNA was prepared and primers specific to the miR429 predicted binding site on the 3′ UTR of PAK2 were used to produce a PCR product of 238 base pairs. Another double digest involving the same enzymes was performed on the PCR product, and the product was ligated into the vector and confirmed by gel electrophoresis. Sanger sequencing was used to determine the sequence of the resulting DNA, ensuring that the plasmid contains the 3′ UTR of PAK2 with the predicted miR429 binding site.ConclusionThe 3′ UTR of PAK2 was cloned into the pmirGLO vector for the assessment of miR429 binding.Support or Funding InformationThis work was supported by the Albion College Biology Department, Foundation for Undergraduate Research, Scholarship, and Creative Activity (FURSCA), Hewlett‐Mellon Fund for Faculty Development at Albion College, and the University of Michigan Division of Rheumatology.