| Background and aims:In the last few years, a new class of RNAs, called microRNAs (or miR), has been induced great interest to researchers. Some reports showed that deregulation of the normal microRNA expression profile could play a critical role in the pathogenesis of hematologic malignancies, recent reports have highlighted the importance of microRNAs in chronic lymphocytic leukemia (CLL) biology and prognosis. There were few reports on the role of microRNAs expression in other malignant lymphoproliferative disorders. Zhang et al reported that some malignancies derived from mature B cells could be distinguished from each other with using of microRNAs expression. In this study we investigated the different expression of miR-29c, miR-150, miR-223, miR-155 and miR-146a in the CD19+B cells of CLL, mantle cell lymphoma (MCL) and splenic marginal zone lymphoma (SMZL) by quantitative real-time PCR, and compared the expression of these microRNAs with normal B cells. At the same time, we analyzed the prognosis value of these microRNAs in CLL.Methods:we collected peripheral blood samples (78 cases) and bone marrow samples (9 cases) from CLL patients, MCL patients and SMZL patients (these patients were in leukemic phase), We also collected peripheral blood samples (12 cases) from healthy person and donor. Mononuclear cells were isolated from peripheral blood and bone marrow, B cells were purified with a CD19+magnetic-bead system according to the manufacturer's instructions. Total RNA was extracted from purified CD19+cells using mirVanaTM miRNA Isolation Kit,10 ng total RNA were reverse-transcribed using the microRNA reverse transcription kit and a specific reverse transcription stem-loop primer according to the manufacturer's protocol. microRNAs expression were measured using the TaqMan microRNA quantitative PCR. RNU48 expression were measured as the endogenous control under the same conditions for all our samples. The expression of each microRNA relative to RNU48 was determined using the cycle threshold (Ct) method. microRNA levels were expressed in folds change of the target miR expression in the calibrator B lymphoid cell line Namalwa. All analyses were performed with SPSS 16.0 software. Correlation between microRNAs expression and Binet or Rai stage in CLL, and microRNAs expression among different diagnosis was assessed by One-Way analysis of Variance, microRNAs expression in different prognostic group was assessed by independent Samples Test. We analyzed receiver operating characteristic (ROC) curves to determine the miR-29c, miR-150, miR-223, miR-155 and miR-146a expression cutoff values that best distinguished mutated and unmutated cases. PFS and OS distributions were plotted using Kaplan-Meier estimates and were compared using the log-rank test for different subgroups. An effect was considered to be statistically significant at P less than 0.05.Results:1. microRNAs expression and it's prognosis value in chronic lymphocytic leukemia. (1) The level of miR-29c% miR-150,miR-223 decreased significantly with progression from Rai 0,1 to 4 and Binet stage A to C (P<0.05) in CLL patients. Moreover, patients with higherβ2-microglobulin expressed significantly lower level of miR-223 (P=0.045). (2) The expression of miR-29c,miR-223 were significantly different between disease progression group and unprogression group, alive patients and dead patients (P<0.05). (3) The levels of miR-223 and miR-155 were significantly different in IgVH unmutated patients compared with mutated patients (P<0.05). (4) In 13q-negative patients performed with FISH, the expression of miR-223 decreased significantly (P=0.044). (5) The microRNAs expression was no statistical difference between CD38 positive patients and CD38 negative patients. (6) The median follow-up in CLL patients was 12 months (range,4-92 months). The median PFS in the IgVH mutated patients was 90 months, it was significantly longer than unmutated patients (P =0.000). Moreover, the median OS in the IgVH mutated patients was 92 months, it was also significantly longer than unmutated patients (P=0.015).We used ROC curve analysis to define the miR-29c and miR-223 cutoffs according to the IgVH mutational status and divided the patients into the positive and negative subgroups for miR-29c and miR-223 respectively. And using these cutoffs, miR-29c and miR-223 were predictors of PFS. The median PFS of miR-29c+and miR-223+patient subgroups were 36 and 48 months, respectively, they were too significantly longer than the negative subgroups (miR-29c, P=0.003; miR-223, P=0.001). OS was no statistical difference between miR-29c+and miR-29c" subgroups (P=0.088). In the miR-223+subgroup there was no patient died in the end of our follow-up.2. The comparison of microRNAs expression among CLL, MCL, SMZL and normal B cells. (1) The level of miR-29c,miR-150 and miR-223 decreased significantly in MCL compared with CLL and SMZL (P<0.05), there was no statistical difference between CLL and SMZL. (2) The expression of miR-155 in CLL was significantly higher than in MCL and SMZL (P<0.05), but the level of miR-146a in SMZL was significantly higher than in CLL and MCL (P<0.05). (3) The expression of miR-29c in SMZL was higher than in normal B cells (P=0.035), the expression of miR-150 in MCL was lower than in normal B cells (P=0.004); the level of miR-223 in CLL, MCL and SMZL was all significantly lower than in normal B cells (P<0.05).conclusion:(1) There existed significantly difference in the expression of miR-29c, miR-150, miR-223, miR-155 and miR-146a among CLL, MCL and SMZL. (2) The level of miR-223 expression in CLL, MCL and SMZL was significantly lower than in normal B cells, this indicates that miR-223 might play an important role in the pathogenesis of malignant lymphoproliferative disorders. (3) miR-29c and miR-223 down-regulation was associated with higher tumor burden, disease aggressiveness, and poor prognostic factors in CLL, and they could significantly predict PFS of CLL, they are powerful prognostic factors.
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