Significance Of MicroRNA-146b-5p In Difeuse Large B-cell Lymphoma And Its Relation To Risk Assessment

Objective: To investigate the expression of miR-146b-5p in diffuse large B celllymphoma (DLBCL), and its relationship to risk assessment.Methods:(1)62cases of nodal DLBCL with follow-up data were collected fromShanxi Cancer Hospital, and were studied by using immunohistochemical EnVisionmethod for CD3, CD10, CD20, Bcl-6and MUM1. The DLBCL were classified intogerminal center B cell-like (GCB) and non-germinal center B cell-1ike (non-GCB)subtypes according to Hans’algorithm. Agilent Human miRNA Microarray16.0wasused to select the miRNAs on24cases paraffin-embedded tissue of DLBCL. A TaqManreal-time polymerase chain reaction (real time PCR) method was performed on62casesnodal DLBCL to validate the the expression levels of miR-146b-5p.11cases of reactivelymph node were elected as control.(2) Hsa-miR-146b-5p precursor fragment wasamplified from the human genome using PCR method, chemical synthesis method forsynthesis of miR-146b-5p analogues and cloned into pGC-LV lentiviral expressionvector respectively. After double enzyme digestion and confirmed by sequencing, theconstructed plasmid and two helper plasmids, pHelper1.0and pHelper2.0, weretransducted into293T cells by Lipofectamine2000. The titers of the lentiviral vectorswas determined by hole diluted titer assay.(3) Real time PCR method was performed on7human DLBCL cell lines such as DB、SUDHL-4、OCI-LY1、OCI-LY3、OCI-LY8、OCI-LY10、RJ-LY1and RJ-LY2to detect the the expression level of miR-146b-5p.Results：(1)In62cases of DLBCL,35.5%of cases were GCB and64.5%werenon-GCB subtypes, the expression of miR-146b-5p in GCB was3.2times as much asnon-GCB subtypes (P=0.006). We found that the expression of miR-146b-5p wasup-regulated in DLBCL, and expression level of miR-146b-5p was5.4times as much asreactivated lymph node. In62cases of DLBCL,43.5%of cases were recurrence-freeand56.5%were recurrence. The expression of miR-146b-5p was remarkably up-regulated in recurrence-free group compared with recurrence group (P=0.004).Moreover, high expression levels of miR-146b-5p in DLBCL were found to beassociated with longer relapse-free survival times (P=0.005), but not overallsurvival.Multivariate COX proportional hazard regression analysis revealed that theywere independent poor prognosis factors, including the low expression of miR-146b-5p(P=0.004) and IPI≥3(P=0.011) in62cases of DLBCL.(2) overexpression and inhibitionof miR-146b-5p of Lentiviral vector were successfully constructed, packing oflentivirus was validated by the expression of fluorescence protein and real-timequantitative PCR after screening. Titer was1.0×109-1.5×109TU/mL.(3) Thedifferential expression of miR-146b-5p in seven human DLBCL cell lines weresignificantly, and the relative expression of OCI-LY8(GCB) was highest, RJ-LY1(non-GCB) was the lowest.Conclusions：The expression of miR-146b-5p was up-regulated in recurrence-freegroup, and its higher expression levels in DLBCL were associated with improvedrelapse-free survival times. These results suggested that miR-146b-5p might be one ofthe new marker for the risk assessment; Both paraffin samples and cell experimentsshow that the expression of miR-146b-5p was significantly higher in GCB than thenon-GCB type, suggesting that miR-146b-5p can be used to distinguish two kinds ofimmune subtypes; In addition, lentiviral vectors with miR-146b-5p overexpression andinhibition were successfully constructed, which lay the foundation for the subsequentfunctional experiments.