Background/Purpose: Terminal uridylyl transferase 7 (TUT7), also known as Zcchc6, is a zinc finger domain-containing protein responsible for terminal uridylation of miRNA, implicated in pre-miRNA maturation and mature miRNA degradation. Dysregulated microRNA (miRNA) expression in human rheumatoid arthritis synovial fibroblast (RASFs) has been observed when compared to non-diseased SFs (NLSFs). Differential expression of miRNA is found to be associated with dysregulated miRNA biogenesis and degrading enzyme activity or stoichiometric differences in the amount of these proteins inside the cells. However, the role of TUT7 remains unexplored in RASFs.

Methods: Protein levels of miRNA biogenesis and degrading enzymes were compared by Western blotting in whole cell extracts prepared from RASFs and NLSFs. RASFs were treated with TNF-α for 24 h were used to evaluate changes in gene expression of miRNA biogenesis and degrading enzymes. RNA sequencing was performed in RASFs transfected with TUT7 siRNA or negative control (NC) siRNA in presence or absence of TNF-α (20 ng/ml) using an Ion Proton™ System. Effects of the loss of TUT7 were examined by gene enrichment of differentially expressed genes (DEGs) in presence or absence of TNF-α, followed by Gene Ontology analysis to determine key pathways modulated. Culture supernatants from siRNA-transfected samples were tested by ELISA to determine cytokine or chemokine production. Statistical value of p < 0.05 was considered significant.

Results: Western blot analysis of miRNA biogenesis machinery confirmed increased expression of Ago1 and Ago2 and decreased expression of TUT7 in RASFs compared to NLSFs (N=3 p< 0.05). We assessed the effects of TUT7 ablation by siRNA on TNF-α-induced synovial inflammation in human RASFs by RNA sequencing. RNA Sequencing analysis revealed that, out of 20,814 genes in the array, 218 DEGs were statistically qualified candidates altered in the absence of TUT7. Gene ontology study of DEGs revealed significant effects of TUT7 knockdown on cellular response to starvation, protein processing in ER, skeletal system development, heart development, and spliceosomal tri-snRNP complex assembly. Validation of RNA-seq data by quantitative RT-PCR analysis of response to TNF-α during transient knockdown of TUT7 confirmed the upregulated expression of matrix metalloproteinases (MMP1, MMP3), chemokines (CXCL5, IL-8) and cytokine (IL-6) transcripts in human RASFs. In corroboration of the mRNA data, ELISA results from TUT7 siRNA-transfected RASFs supernatants showed the significant upregulation of TNF-α-induced IL-6, IL-8, CXCL5, MMP-1, and MMP-3 production (N=4; p< 0.05).

Conclusion: This study provides novel evidence showing that loss of endogenous TUT7 in RASFs may exacerbate their hyper-responsiveness to inflammatory cytokines such as TNF-α by modulating the miRNA degradation pathway.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-terminal-uridylyl-transferase-7-in-tnf-%ce%b1-induced-inflammation-in-rheumatoid-arthritis-synovial-fibroblasts-in-vitro/