| Part I Screening and identification of miRNAs in patients withchronic lymphocytic leukemiaObjective：Emerging evidence suggests that altered microRNAs(miRNA) regulationis involved in the pathogenesis of cancers-mainly by regulating the translation ofoncogenes and tumor suppressors.Aberrant expression of miRNA has been recentlyassociated with chronic lymphocytic leukemia (CLL) outcome. In this study, weaimed to screen and identify the miRNAs expression in Chinese patients of CLL. Inaddition, we will further analyze its clinical significance in CLL.Methods: We performed miRNA expression profile analysis in six Chinese patientswith CLL, and in peripheral B cells from30healthy donors, using a platformcontaining866human miRNAs. Standard curves of U6and miRNA were made from10-fold serial dilutions of the cDNA, which were quantified using real-timequantitative PCR (RQ-PCR) with SYBR Green by ABI7300. U6was used as thereference gene, the relative expression levels of miR-15a, miR-16-1, miR-29b,miR-181a and miR-181b in CLL were analyzed by2(-ΔΔCT)method. The p53mutations and IGHV mutation status analysis in CLL patients were detected by PCRand direct sequencing.Cytogenetic abnormalities of patients were detected byinterphase FISH analysis. Flow cytometric analysis of CD38and ZAP-70were alsoperformed. Further, the correlation between miRNAs expression and CLL prognosticfactors(cytogenetics, IGHV mutation status, p53mutation status, ZAP-70protein andCD38expression level)were analysed.Results: The most frequent changes in miRNAs in CLL cells includeddown-regulation of miR-126, miR-572, miR-494, miR-923, miR-638, miR-130a,miR-181a, and miR-181b and up-regulation of miR-29a, miR-660, miR-20a, miR-106b, miR-142-5p, miR-101, miR-30b, miR-34a, miR-let-7f, miR-21, andmiR-155etc. We identified various degrees of down-regulation of miR-15a,miR-16-1, miR-29b, miR-181a and miR-181b in CLL mononuclear cells. Amongthese down-regulated miRNAs, we have identified miR-29b expression significantlycorrelated with IGHV mutational status, expression levels of miR-15a/miR-16-1wasextremely reduced in the cohort with deletion of13q14. The levels of miR-181a andmiR-181b expression were significantly lower in poor prognostic subgroups, definedby prognostic factors including unmutated IGHV status, p53and ATM deletions, andp53mutations. Furthermore, under-expression of miR-181a and miR-181b wassignificantly associated with shorter overall survival and treatment free survival inCLL patients.Conclusion: These different miRNAs may play an important role in the pathogenesisof CLL and might be applied for the assessment of prognosis in patients with CLL.Among the differently expressed miRNAs, the significant down-regulation ofmiR-181a/b was verified by qRT-PCR，and they are chosen as the targets of ourfurther study. Part II Molecular mechanism of miR-181s in apoptosismodulating in chronic lymphocytic leukemiaObjective：In chronic lymphocytic leukemia (CLL), aberrations along the p53axislead to decreased overall survival and therapy resistance, also the downstreamproto-oncogenes BCL-2, TCL-1, MCL-1are involved in this process.This study is toinvestigate the function of miR-181s in p53tumor suppressor axis.Methods: Analysing candidate targets of miR-181s of putative relevance in CLL pathogenesis by bioinformatics approach;Using dual-luciferase activity assay toverify the target genes of miR-181s; Using Western blot analysis and to elucidate themechanism of miR-181a and miR-181b on modulating drug resistance; Using in vitrodrug sensitivity assay to detect the drug resistance phenotype changes of primaryCLL cells via exogenous over expression of miR-15a,miR-16-1,miR-34a,miR-181aand miR-181b through transient transfection of the corresponding miRNA mimics.The p53mutation status analysis in40CLL patients were detected by directsequencing, cytogenetic aberrations (deletion in17p13or11q22.3) were detected byinterphase FISH analysis.Results: We demonstrated that miR-181a and miR-181b function as tumorsuppressors by inhibiting multiple anti-apoptosis proteins, including BCL-2, MCL-1,and XIAP. This was a direct effect,because miR-181a and miR-181b negativelyregulated expression of a BCL-2/MCL-1/XIAP3’ untranslated region-based reporterconstruct. Transfection of miR-34a, miR-181a and miR-181b mimics into CLL cellsfrom30patients without p53aberrations led to significant increases in apoptosiscompared to transfection with the miRNA control. However, enforced expression ofmiR-34a,miR-181a and miR-181b had no significant drug-sensitization effect in CLLcells from10p53-attenuated patients.Conclusion: MiR-181a and miR-181b may play important roles in the pathogenesisof CLL and may be useful for assessing prognosis in patients with CLL. Moreover,miR-181a/b may provide a possible therapeutic avenue and a sensitive indicator ofthe activity of the p53axis in CLL. Part III Prognostic significance of Dicer and Drosha expressionin patients with chronic lymphocytic leukemiaObjective：Dicer and Drosha are main regulators of miRNA biogenesis.To investigatethe role of Dicer and Drosha in CLL, we assessed the expression of Dicer and Droshaand their correlation with other prognostic factors, including Binet stages,immunoglobulin heavy chain variable gene (IGHV) mutation status, p53mutationstatus, ZAP-70protein and CD38expression level in CLL patients.Methods: We measured mRNA levels of Dicer and Drosha in165CLL patients,8monoclonal B-cell lymphocytosis (MBL) cases, and additionally10healthy donors asnormal controls, using real-time quantitative PCR (RQ-PCR). Standard curves ofDicer，Drosha and β-actin were made from10-fold serial dilutions of the cDNA,which were quantified using RQ-PCR with SYBR Green by Stratagene Mx3000P.The relative expression levels of Dicer and Drosha in CLL were analyzed by2(-ΔΔCT)method. The p53mutations and IGHV mutation status analysis in CLL patients weredetected by direct sequencing.Cytogenetic abnormalities of patients were detected byinterphase FISH analysis. Flow cytometric analysis of CD38and ZAP-70and IGHVsequencing were also performed.Results: We found that Dicer expression was significantly higher in patients of BinetB and C stage compared with Binet A stage(mean±SD,0.017±0.010vs.0.015±0.019; p=0.001). Dicer expression level in CD19+B cells from healthy donors wasvery similar to MBL (mean±SD,0.020±0.012vs0.019±0.09). Patients withunmutated IGHV genes had significant lower expression of dicer than patients withIGHV mutations (p<0.0001). The lower expression level of Dicer was alsosignificantly associated with higher level of CD38and ZAP-70(p=0.0304andp=0.0075, respectively).Lower Dicer level was found in patients with unfavorablecytogenetic aberrations (deletion in17p13or11q22.3) in contrast to higher level ingood risk cytogenetics (deletion in13q14as the sole abnormality). Furthermore, thelower expression of Dicer in CLL shows a strong association with shorter overall survival (OS)(132months vs.“not reached”, p=0.0046)as well as with reducedtreatment free survival (TFS)(24months vs.110months, p=0.0006). By contrast, nodifferences in the expression of Drosha among these groups of patients wereobserved.Conclusions: Our findings indicate that levels of Dicer mRNA in CLL cells haveassociations with outcomes in patients with CLL. |
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