The Expression And Biological Function Of MicroRNA-224 In Diffuselarge B-cell Lymphoma

Part I The Expression of miR-224 was Investigated in Four DLBCL Cell Lines and the Biological Behaviors of miR-224 in DLBCL Cell Lines were also StudiedObjective:miR-224 is considered to play important functions in many cellular processes. miR-224 plays a crucial role in tumor formation, maintenance and metastasis by modulating genes expression. However, the role of miRNA-224 has not been elucidated in DLBCL. This study aims to investigate the expression of miR-224 in ABC-type DLBCL cell lines, GCB-type DLBCL cell lines compared with normal B cells. We also investigated the expression and role of miR-224 in the regulation of cell biological behaviors in DLBCL cell lines using mimic-miRA-224 to upregulated expressions of miRNA-224.Methods:The tonsils were disaggregated by collagenase and trypsogen and separated by Ficoll. Sample purity was verified by fluorescence-activated cell sorting and normal CD 19+B lymphocyte cells were obtained by flow cytometry. The expressions of miR-224 were evaluated in two ABC-type DLBCL cell lines, two GCB-type DLBCL cell lines and normal CD 19+B lymphocyte cells by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expressions of miR-224 were evaluated in OCI-LY3, OCI-LY10, OCI-LY18 and OCI-LY19 cell lines by qRT-PCR after transfection by mimic-miRNA-224 compared with OCI-LY3/mimic-control, OCI-LY10/mimic-control,OCl-LY18/mimic-control and OCI-LY19/mimic-control. Used MTT assays to detect the changes of proliferation level of the OCI-LY3, OCI-LY10, OCI-LY18 and OCI-LY19 cells after transfection by mimic-miRNA and compared with OCI-LY3/mimic-control, OCI-LY10/mimic-control, OCI-LY18/mimic-control and OCI-LY19/mimic-control. The effect of mimic-miR-224 on invasion in OCI-LY3, OCI-LY10, OCI-LY18 and OCI-LY19 cells in vitro was detected by transwell chamber. The effect of mimic-miR-224 on apoptosis in OCI- LY3, OCI-LY10, OCI-LY18 and OCI-LY19 cells in vitro was detected by flow cytometry.Results:Separated by Ficoll, normal B cells which defined by the expression of surface CD 19 were obtained by fluorescence-activated cell sorting of tonsils, Sample purity was found to be more than 90% in all cases. Four DLBCL cell lines and normal CD 19+B-cell were first analyzed to quantitate the expression level of miR-224 used the RNAs Real-Time PCR Quantitation Assay. Results showed that the expression level of miR-224 was down-regulated in four DLBCL cell lines compared with normal CD 19+B-cell. There was no difference between the level of the ABC-type cell lines (OCI-LY3, OCI-LY10) and the GC-type DLBCL cell lines (OCI-LY18, OCI-LY19) (P> 0.05). After transfection with mimic-miR-224, the expressions of miR-224 were significantly improved by over 4.2-fold in OCI-LY10 cells,4.6-fold in OCI-LY3 cells,5.1-fold in OCI-LY18 cells and 4.8-fold OCI-LY19 cells respectively relative to their mimic-miR control. Performed by the MTT analyses, miR-224 mimic reduced proliferation of DLBCL cells. Compared with the OCI-LY3/mimic-control, OCI-LY10/mimic-control, OCI-LY18/mimic-control and OCI-LY19/mimic-control, the proliferation of the OCI-LY3/mimic-miR-224, OCI-LY10/mimic-miR-224 OCI-LY18/mimic-miR-224 and OCI-LY19/mimic-miR-224 were inhibited down to 86% (P<0.05),82%(P<0.05),74%(P<0.01);79%(P<0.01),75%(P<0.01),68%(P <0.01);83%(P<0.01),78%(P<0.01),71%(P<0.01) and 85%(P<0.01),78%(P <0.01),69%(P<0.01) of the pre-transfected levels at the 2,3, and 4 days, respectively. Performed by an in vitro Matrigel Transwell analysis, the results showed that the number of OCI-LY3/mimic-miR-224, OCI-LY10/mimic-miR-224, OCI-LY18/mimic-miR-224 and OCI-LY19/mimic-miR-224 cells passing through the Matrigel were markedly lower than the numbers of OCI-LY3/mimic-control OCI-LY10/mimic-control, OCI-LY18/mimic-control and OCI-LY19/mimic-control cells (P< 0.05). After transfection with mimic-miR-224, an increase in early apoptosis was detected in OCI-LY3/mimic-miR-224, OCI-LY10/mimic-miR-224, OCI-LY18/mimic-miR-224 and OCI-LY19/mimic-miR-224 cells (P< 0.05).Conclusion:Quantitative analysis demonstrated that the expression of miR-224 was downregulated in four DLBCL cell lines compared with normal CD19+ B lymphocytes. There was no difference between the expression of miR-224 in the ABC-type cell lines and the GCB-type DLBCL cell lines. We demonstrated that upregulated expression of miR-224 induced suppression of proliferation and invasion, as well as increased apoptosis in DLBCL cell lines after transfection with mimic-miR-224. The study showed that the change of miR-224 expression could affect the biological behaviors of DLBCL cells. miR-224 may play an important role in the development and progression of DLBCL and contribute to DLBCL pathogenesis.PartⅡ Computational Prediction, Bioinformatic Screening and Verification of the Target Gene Regulated by miR-224 in DLBCLObjective:microRNAs (miRNAs) are endogenous and short noncoding RNAs that are involved in the regulation of thousands of gene targets. miRNAs are likely to be master regulators of many important biological processes. Therefore, it is of particular important to reliably predict potential miR-224 targets which might be involved in DLBCL for elucidating the biological functions and processes of miR-224. In this study, the target genes of miR-224 was predicted and screened through multiple software programs and verificated by the methods of molecular biology. It may provide the new approach for the tumorigenesis, treatment and prognostic biomarkers of DLBCL.Methods:We used the bioinformatics softwares of TargetScan 5.1, miRDB, miRWalk, RNA22 and miRanda to predict the possible target genes of miR-224. Based on the characteristic of immunochemotherapy and bioinformatics scores, possible candidate of the target gene was selected. Expression vector of target gene 3’UTR was constructed, and then target gene was verified by dual-luciferase reporter assay system. The recombinant plasmid OCI-LY3/mimic-miR-224, OCI-LY3/miR-control、OCI-LY10/mimic-miR-224, OCI-LY10/miR-control,OCI-LY18/micmic-miR-224, OCI-LY18/mimic-control, OCI-LY19/mimic-miR-224,OCI-LY19/miR-control were transfected into OCI-LY3, OCI-LY10,OCI-LY18 and OCI-LY19 cells, and the change of the expression of CD59 mRNA and protein in transfected cells was identified by RT-PCR, Werstemblot and flow cytometry.Results:CD59 is the target gene of miR-224 predicted accutely by bioinformatics algorithms of TargetScan 5.1, miRDB, miRWalk, RNA22 and miRanda and verificated by dual-luciferase reporter assay. Upon transfection with mimic-miR-224, endogenous protein levels were all significantly down regulated relative to the effect of the miR-control detected by Western blot and flow cytometry. However, there was not any significant inhibition of CD59 at the mRNA level, as measured by qRT-PCR.Conclusion:CD59 is the target gene of miR-224 in DLBCL. miR-224 post-transcriptionally regulates CD59 expression mainly by binding site in CD593’UTR.PartⅢ Expression of miR-224 in Diffuse Large B Cell Lymphoma and its Clinical SignificanceObjective:Deregulated expression of miR-224 has been verified in DLBCL cell lines. The change of miR-224 expression could affect the biological behaviors of DLBCL cells. miR-224 may contributes to the lymphomagenesis of DLBCL. In this study, we want to investigate the expression of miR-224 in DLBCL and its relationship with clinical pathological features and prognosis.Methods:Real-time PCR was used to detect the expression of miR-224 in 168 DLBCL tissues and 25 normal lymphoid tissues.Results:The expression of miR-224 in DLBCL tissues (0.97±0.33) was significantly lower than that in normal lymphoid tissues (1.87±0.43, P<0.05). There were no significant correlations between the expression of miR-224 and age (P=0.4342), gender (P=0.6129) tumors stage (P=0.2498), IPI (P=0.3547) and lactate dehydrogenase (P=0.3985) (P>0.05). Among the 168 patients the CR rate was 59.3%, the PR rate was 19.2% and the SD+PD rate was 21.5%. The miR-224 expression level was 0.60±0.12 in the SD+PD group,0.84±0.09 in the PR group, and 1.18±0.10 in the CR group. Compared with that of the NC group (1.87±0.43), these differences were all statistically significant (all P values<0.05). Using the median expression of miR-224 as the threshold, we separated DLBCL patients into a low expression group and a high expression group. The five-year progression-free survival and overall survival were significantly lower along with the low expression level of miR-224 than with high expression level of miR-224.Conclusions:The expression absence of miR-224 may play an important role in the development and progression of DLBCL. The expression of miR-224 can predict response and outcome of DLBCL patients treated with R-CHOP.PartIV Increased CD59 protein expression is associated with the outcome of patients with diffuse large B-cell lymphoma treated with R-CHOPObjective:Deregulated expression of miR-224 has been verified in DLBCL patients compared with normal lymphoid tissues. Low expression of miRNA-224 predicts poor clinical outcomes in DLBCL treated with R-CHOP. In this study, we want to investigate the expression and prognostic values of CD59 expression, which is the target gene of miR-224, in patients with DLBCL who underwent rituximab-CHOP.Methods:The immunohistochemical expressions of CD59 in 186 well-characterized DLBCL patients were evaluated using tissue microarrays, and then were related to known tumor and patient-related variables and to survival.Results:About 151 patients (81.2%) showed IHC-positive CD59. By the Muris algorithm,68 patients (36.6%) were classified as the GCB subtype and 83 patients (44.6%) as the non-GCB subtype. About 35 patients (18.8%) showed IHC-negative CD59.18 patients out of 35 patients (9.7%) were classified as the GCB subtype and 17 patients (9.1%) as the non-GCB subtype. There were no statistically significant differences in DLBCL subclassification between moderate positivity and high positivity groups (P>0.05). We also observed the associations of the CD59 expression with age, gender, stage, lactate dehydrogenase and IP1 score, but no statistical significances were reached. CD59 was expressed in low expression group in 95 complete remissions (75.4%) and in high expression group only in 31eases (24.6%) (P< 0.05). In patients with complete remission (n=126), CD59 was expressed in 24.1% of the tumor cells on average (95% confidence interval [CI],9-32), whereas patients without complete remission, CD59 was expressed in 54.2% of tumor cells (95% CI,38-71, P< 0.05). Expression of protein CD59 was the highest in patients with non-response (NR), and was higher in patients with PR than in patients with CR (P<0.05). All factors associated with failure to achieve complete remission in the univariate models were involved in multivariate analysis, which showed that CD59 expression in>50% of tumor cells (P=0.001), B symptoms (P=0.002), and high IPI (P=0.005) were the independent predictive factors for treatment resistance. Using the median staining mean density of CD59 by IHC as the staining threshold, we separated patients into a low expression group and a high expression group. The cumulative survival rate of the patients with high expression of CD59 was significantly lower compared with patients with low expression of CD59 (P<0.05). Patients with low expression of CD59 lived longer compared with patients with high expression of CD59.Conclusions:Our findings indicate that the CD59 level at onset is an independent predictor of the prognosis of DLBCL patients treated with R-CHOP. These results may enable us conveniently to identify DLBCL cases with poor clinical outcome, and then guide us to adopt more intensive treatment for those patients.