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.

译文

: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细胞系和标本中均测试了CCR6表达水平。通过生物信息学分析和定量实时PCR(qRT-PCR)验证,获得了CCR6的靶向miRNA。使用双重荧光素酶测定法来验证它们的靶向关系。此外,使用qRT-PCR,western blot,CCK8,Transwell分析,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。上调的miR-518a-5p和下调的CCR6均在体外抑制细胞增殖和侵袭。然后,实验验证了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,我们首次证明了miR-518a-5p启动子区域存在高甲基化条件,而阿扎胞苷以时间和浓度依赖性方式改变了miR-518a-5p的水平。最后,我们发现CCR6启动子中存在赖氨酸27乙酰化富集的组蛋白H3,其表达也可通过C646以时间和浓度依赖性的方式改变。以上结果表明,miR-518a-5p-CCR6反馈回路在DLBCL的发展中起着至关重要的作用。 CCR6的过表达主要是通过转录和转录后激活的表观遗传修饰介导的,这为DLBCL治疗提供了新的方向。

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