Expression And Clinical Significance Of MiR-149-5p And CCL18 In Diffuse Large B Cell Lymphoma

OBJECTIVE:to investigate the expression and clinical sigificance of microRNA-149-5p(miR-149-5p)and C-C motif chemokine ligand 18(CCL18)in diffuse large B cell lymphoma(DLBCL).METHODS:(1)DLBCL miRNA expression datas were obtained from TCGA and GEO databases,related articles were also searched.The expression of miR-149-5p in 107 DLBCL tissues and 105 negative control lymph tissues(non-neoplastic lymph tissues)were detected by PCR.Relationship between miR-149-5p expression and clinicopathological parameters(including gender,age,subtype,Ann Arbor stage,ECOG score,extranodal involvement,IPI,B symptom),immunohistochemical markers(including Bcl-2,Bcl-6,CD 10,Mum-1,Ki-67)and prognosis of DLBCL were analyzed.(2)Target genes of miR-149-5p were predicted by mirwalk database,in combination of genes that up-regulated in DLBCL from GEPIA website,to filter out potential target genes of miR-149-5p.These genes were used to perform function and pathway enrichment analysis by DAVID.(3)Immunohistochemistry was applied to detect the expression level of CCL18 in 119 DLBCL tissues and 105 non-neoplastic lymph tissues.The relationship between CCL18 expression and DLBCL clinicopathological parameters,immunohistochemical markers and prognosis were also assessed.Gene expression data,clinical and prognositc information of DLBCL were obtained from TCGA,GEO database and current articles,in combination with immunohistochemistry results,meta analysis was conducted.to analyze the relationship between CCL18 and DLBCL clinicopathological parameters and prognosis.Then we detected serum CCL18 levels in 37 DLBCL patients and 40 healthy adults by enzyme linked immunosorbent assay and evaluated the diagnostic value of serum CCL18 levels in DLBCL.The correlation between miR-149-5p and CCL18 in DLBCL was also analyzed.RESULTS:(1)The expression of miR-149-5p in DLBCL was’significantly lower than that in non-neoplastic lymph tissues(P=0.038).No distinct miR-149-5p levels were found in different genders,ages,Ann Arobr stages,subtypes,ECOG scores,IPIs in DLBCL.The overall survival time in miR-149-5p low expression group tended to be shorter than that in miR-149-5p high expression group(P=0.062).(2)There were 3737 genes predicted by 6 or more prediction websites in mirwalk datases.A total of 4196 genes that up-regulated in DLBCL with log2 FC>2 were filtered out.In combination,427 potential target genes of miR-149-5p were finally selected.These genes were enriched in biological process such as regulation of DNA replication,cellular response to DNA damage,humoral immunity,cell cycle,B cell differenciation,p53-mediated signal transduction pathways and metabolic pathway,amino acid synthesis,Epstein-Barr virus infection,B cell receptor signaling pathway.CCL18 was chosen as target genes of miR-.149-5p for further study.(3)Immunohistochemistry results suggested that expression of CCL18 in DLBCL was higher than non-neoplastic lymph tissues(P<0.001),CCL18 expression levels were positively correlated with age(r=0.200,P=0.029),subtype(r=0.214,P=0.020),extranodal sites(r=0.333,P<0.001),IPI(r=0.249,P=0.006)and hemoglobin(r=0.241,P=0.008),.while no significant correlation were found between CCL18 and gender,Ann Arbor stage,ECOG score,B symptom,serum LDH levels.CCL18 levels were positively correlated with Ki-67(r=0.200,P=0.029),while negatively correlated with Bcl-6(r=-0.282,P=0.002).The area under the ROC curve of CCL18 in diagnose DLBCL was 0.859(P<0.001),while in diagnose DLBCL non-GCB subtype was 0.655(P=0.014).Results of single factor analysis for overall survival time revealed that DLBCL patients with CCL18 high expression(P=0.026),Ann Arbor stage Ⅲ-Ⅳ(P=0.038),ECOG score ≥2(P=0.001),extranodal involvement sites ≥2(P=0.008),B symptoms(P=0.027),IPI>2(P=0.001),high LDH levels in serum(P=0.010)had a poorer prognosis,while Cox’s proportional hazard regression model suggested that IPI was an independent risk factor of the prognosis.29 studies from TCGA and GEO databases combined with immunohistochemistry results were included to conduct meta-analysis.Results of meta-analysis suggested that the expression of CCL18 in DLBCL tissues was significantly higher than that in non-neoplastic lymph tissues(P<0.001),CCL18 in age>60 years group was higher than that in age<60 years group(P<0.001),higher in non-GCB subtype than GCB subtype(P<0.001),furthermore,CCL18 high expression group indicated a shorter overall survival time(P=0.003),while no significant difference of CCL18 expression was found in distinct genders(P=0.357),IPIs(P=0.066),ECOG scores(P=0.291),Ann Arbor stages(P=0.094),extranodal involvement sites(P=0.788).sROC curve was used to evaluate the diagnostic value of CCL18 in DLBCL and DLBCL non-GCB subtype,and the results showed that the area under the curve for diagnosis of DLBCL was 0.918(P=0.016),besides,for diagnosis of DLBCL non-GCB subtype was 0.823(P=0.012).DLBCL patients had significant higher levels of CCL18 in serum than healthy adults(P=0.038).The area under the ROC curve of serum CCL18 levels in diagnosis of DLBCL was 0.637(P=0.038).In DLBCL,there was a significant negative correlation between CCL18 and miR-149-5p(r=-0.206,P=0.333).CONCLUSIONS:In DLBCL,miR-149-5p expression is down-regulated,while CCL18 is highly expressed.Both of them have impact on the occurrence of DLBCL,but their relationship with DLBCL progression still needs further study.CCL18 high expression indicates a poorer prognosis.miR-149-5p may target CCL18 to affect the formation of DLBCL.