Epigenetic regulation of miR-518a-5p-CCR6 feedback loop promotes both proliferation and invasion in diffuse large B cell lymphoma

Abstract

To investigate the detailed functions and underlying mechanisms of miR-518a-5p/CCR6 in diffuse large B cell lymphoma (DLBCL) is needed. In this study, CCR6 expression levels were tested both in DLBCL cell lines and specimens. Through bioinformatics analysis and quantitative real-time PCR (qRT-PCR) validation, CCR6’s targeted miRNA was obtained. Dual luciferase assay was used to verify their targeted relationship. Futhermore, using qRT-PCR, western blot, CCK8, Transwell assays, flow cytometry, pyrosequencing, chromatin immunoprecipitation, and azacitidine/C646 treatment, the detailed functions and underlying mechanisms of CCR6 and its targeted miRNA in DLBCL were detected. We found that negative correlation existed between CCR6 and miR-518a-5p in DLBCL. Both up-regulated miR-518a-5p and down-regulated CCR6 inhibited cell proliferation and invasion in vitro. Experiment then verified the regulatory relationship between miR-518a-5p and CCR6. JAK2 and STAT6 levels were reduced in DLBCL cells transfected with miR-518a-5p mimic or CCR6 small interfering RNA. Interestingly, we showed for the first time that a hyper-methylated condition existed at the promoter region of miR-518a-5p and azacitidine changed levels of miR-518a-5p in a time- and concentration-dependent manner. Finally, we found an enriched histone H3 on lysine 27 acetylation existed in the promoter of CCR6, whose expression could also be changed via C646 in a time- and concentration-dependent manner. The above results suggest that miR-518a-5p-CCR6 feedback loop plays a critical role in DLBCL development. The overexpression of CCR6 is mainly mediated by epigenetic modification through transcriptional and post-transcriptional activation, which provides new directions for DLBCL treatment.