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.

摘要

需要研究 miR-518a-5p/CCR6 在弥漫性大 B 细胞淋巴瘤 (DLBCL) 中的详细功能和潜在机制。在这项研究中，在 DLBCL 细胞系和标本中测试了 CCR6 表达水平。通过生物信息学分析和实时定量PCR（qRT-PCR）验证，获得了CCR6的靶向miRNA。双荧光素酶测定用于验证它们的靶向关系。此外，使用 qRT-PCR、蛋白质印迹、CCK8、Transwell 测定、流式细胞术、焦磷酸测序、染色质免疫沉淀和阿扎胞苷/C646 处理，检测了 CCR6 及其靶向 miRNA 在 DLBCL 中的详细功能和潜在机制。我们发现 DLBCL 中 CCR6 和 miR-518a-5p 之间存在负相关。在体外，上调的 miR-518a-5p 和下调的 CCR6 均抑制细胞增殖和侵袭。实验随后验证了miR-518a-5p与CCR6之间的调控关系。在用 miR-518a-5p 模拟物或 CCR6 小干扰 RNA 转染的 DLBCL 细胞中，JAK2 和 STAT6 水平降低。有趣的是，我们首次发现 miR-518a-5p 的启动子区域存在高甲基化状态，阿扎胞苷以时间和浓度依赖性方式改变 miR-518a-5p 的水平。最后，我们发现 CCR6 的启动子中存在富含赖氨酸 27 乙酰化的组蛋白 H3，其表达也可以通过 C646 以时间和浓度依赖性方式改变。上述结果表明 miR-518a-5p-CCR6 反馈回路在 DLBCL 发展中起着关键作用。