| Objective: This study aimed to investigate the detailed functions and underlying mechanisms of miR-518a-5p/CCR6 in diffuse large B cell lymphoma(DLBCL).This study also explored the effect of CCR6 on the immune microenvironment of DLBCL,and constructed targeting oligodeoxynucleotides as DLBCL immune adjuvants by bioinformatics and biological experiments,in order to provide a new way for the treatment of DLBCL.Methods:1.A web-based search in the Gene Expression Omnibus(GEO)database and Array Express database was performed.Through multiple bioinformatics tools,CCR6 was explored to be correlated with DLBCL.Then CCR6 expression levels were tested both in DLBCL cell lines and specimens.The correlation between CCR6 expression and clinicopathological features in DLBCL was also performed.2.Through bioinformatics analysis and quantitative real-time polymerase chain reaction(qRT-PCR)validation,CCR6 mRNA’s targeted miRNA was obtained.Dual luciferase assay was used to verify their targeted relationship.Through transfecting SU-DHL-2 and SU-DHL-6cells with CCR6 small interfering RNA or miR-518a-5p mimic,the expression levels of miR-518a-5p and CCR6 were tested.3.Using CCK8,Transwell assays,and flow cytometry,the detailed functions of CCR6 and its mRNA’s targeted miRNA in DLBCL were detected.Co-transfecting SU-DHL-2 and SUDHL-6 cells with CCR6 small interfering RNA(siRNA)and the miR-518a-5p inhibitor,the relationship between CCR6 and miR-518a-5p was explored through CCK8,Transwell assays,and flow cytometry.Main factors in the JAK2-STAT6 signaling pathway were tested in DLBCL cells transfected with miR-518a-5p mimic and CCR6 siRNA,respectively.4.To detect the mechanism contributed to the abnormal expression of CCR6,several databases were searched,including c Bio Portal(http://www.cbioportal.org/),Cistrome Data Browser(http://cistrome.org/db/)and the UCSC Genome Browser database(http://genome.ucsc.edu/).Then,CCR6’s expression levels and the transcriptional regulation of histone acetylation were determined by treating SU-DHL-2 and SU-DHL-6 cell lines with C646,a histone acetyltransferase inhibitor.Next,methylation levels were detected both in DLBCL tissues and cells through methylation-specific PCR.Pyrophosphate sequencing was performed to validate the relationship between miR-518a-5p expression and miR-518a-5p’s promoter methylation in SU-DHL-2 and SU-DHL-6 cells,respectively.Additionally,the levels of miR-518a-5p in SU-DHL-2 and SU-DHL-6 cells were measured when exposed to azacitidine for different time periods(0,12,24,and 48h)or concentrations(0,2,4,and 6μmol/L).5.The expression levels of CCR6 in CD4+CD25+ regulatory T cells and CD4+CD25-T cells were detected by flow cytometry.Both CCR6 and CCL20 expression levels were tested in DLBCL specimens.Since we could not obtain the conserve sequences of CCR6 mRNA3’untranslated region(3’UTR),we designed oligodeoxynucleotides(ODNs)targeting the conserve sequences of both the human and mouse CCL20 mRNA 3’UTR,tested their inhibitory effects on CCL20.Results:1.Through multiple bioinformatics tools,CCR6 was explored to be correlated with DLBCL.Then CCR6 was found to be markedly overexpressed in DLBCL cell lines and specimens.Interestingly,CCR6 was considered as an effective diagnosis biomarker to differentiate between reactive lymph node hyperplasia and DLBCL,with an area under the curve(AUC)of0.917.High-expression of CCR6 was observed to be correlated with the presentation of B symptoms(p=0.047)and worse international prognostic index(IPI)scores(p=0.029).2.Through bioinformatics analysis,miR-518a-5p was obtained as the CCR6 mRNA’s targeted miRNA.The negative correlation existed between CCR6 and miR-518a-5p in DLBCL.Dual luciferase assay revealed that miR-518a-5p mimic transfection significantly declined the relative luciferase activity of CCR6,indicating the regulatory relationship between miR-518a-5p and CCR6.3.Both up-regulated miR-518a-5p and down-regulated CCR6 resulted in decreased proliferation ability,decreased colony amounts,inhibited invasion capability and a remarkable rise in cell apoptosis in vitro.Significant changes in the G1 and S cycles only appeared in cells by transferring CCR6 siRNA.Furthermore,the decreased proliferation ability,diminished colony amounts and inhibited invasion capability of DLBCL cells induced by the CCR6 inhibitor was partially reversed by co-transfected with miR-518a-5p siRNA.Cell apoptosis analysis through flow cytometry exhibited that co-transfection with the CCR6 and miR-518a-5p inhibitor resulted in a remarkable fall in cell apoptosis rates compared with the CCR6 inhibitor group.Further experiment verified that JAK2 and STAT6 levels were reduced in DLBCL cells transfected with miR-518a-5p mimic or CCR6 small interfering RNA.4.Mechanistically,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 timeand concentration-dependent manner.We also 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.5.The expression of CCR6 in CD4+ CD25+ regulatory T cells was significantly higher than that in CD4+ CD25-T cells.Both CCR6 and CCL20 were found to be markedly overexpressed in DLBCL specimens.Since we could not obtain the conserve sequences of CCR6 mRNA3’UTR,we designed ODNs targeting the conserve sequences of both the human and mouse CCL20 mRNA 3’UTR.Conclusion:The miR-518a-5p-CCR6 feedback loop plays a critical role in promoting aggressive biological behaviors of DLBCL.The abnormal expression of CCR6 is mainly mediated by epigenetic modification through transcriptional and post-transcriptional activation,which then activates the JAK2-STAT6 signaling pathway and promotes DLBCL progression.Oligodeoxynucleotides of CCL20,the CCR6’s receptor,may be served as DLBCL immune adjuvants and provide new directions for DLBCL treatment. |
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