Abstract
The inflammation-associated cytokine interleukin-6 (IL-6) can contribute to tumor growth and resistance to therapy by the activation of survival mechanisms. In several human cancers, IL-6-activated survival signaling involves the signal transducers and activators of transcription (Stat) factors or protein kinase cascades. microRNAs (miRNAs) are endogenous regulators of gene expression that are altered in expression in many cancers. However, the effect of inflammatory cytokines on miRNA expression and the role of miRNA in modulating IL-6-mediated cell survival are unknown. We investigated the involvement of miRNA in malignant cholangiocytes stably transfected to overexpress IL-6, which enhances tumor growth in vivo by inhibition of apoptosis. We provide evidence that (i) miRNA expression both in vitro and in vivo is altered by overexpression of IL-6; (ii) selective miRNAs including let-7a are up-regulated and contribute to the survival effects of enforced IL-6 activity; and (iii) let-7a contributes to the constitutively increased phosphorylation of Stat-3 by a mechanism involving the neurofibromatosis 2 (NF2) gene. These findings reveal a novel mechanism by which IL-6 mediates tumor cell survival that may be therapeutically targeted and emphasize the presence of complex interrelationships between deregulated expression of miRNA and transcription factors in human cancers.
Highlights
Comes and resistance to chemotherapy [1]
Alterations in miRNA Expression—To identify miRNA that may contribute to survival signaling and chemoresistance, we first assessed the effect of IL-6 on miRNA expression
The role of cytokines as stimulators of miRNA expression have not been explored, and here, we demonstrate a role for persistent IL-6 stimulation on altered miRNA expression in a human cancer
Summary
Cell Lines and Cultures—Mz-1 and KMCH-1 human malignant cholangiocytes and their respective IL-6-overexpressing stable transfectants, Mz-IL-6 and KM-IL-6, were obtained and. Sense inhibitor, or controls were added to 1 ϫ 106 cells sus- NF2-MUT-luc firefly luciferase reporter vectors, which contain pended in 80 l of Nucleofector solution ToTALLY RNA isolation kit (Ambion), and cDNA was gener- was directly ligated into the HindIII and SpeI cloning sites ated by reverse transcription using 1 g of total RNA and the of the pMIR-REPORT luciferase expression vector (Ambion). Mz-IL-6 cells were co-transfected with 1 g of pMIRNF2-UTR or pMIR-NF2-MUT-UTR construct and 1 g of pRL-TK Renilla luciferase expression construct without (empty vector control) or with anti-let-7a inhibitor using TransIT-siQUEST transfection reagent (Mirus, Madison, WI). The difference between two groups was analyzed using a double-sided Student’s t test, and the null hypothesis was rejected at the 0.05 level
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