摘要
背景与目的：最近几年,一类新的小分子RNA,microRNA(亦称miR),引起了科学研究者的极大关注。近来的研究发现,microRNAs的异常表达在血液系统恶性肿瘤的发病中起到重要作用,其在慢性淋巴细胞白血病的发病及预后中的作用尤其受到重视。在其他恶性淋巴增殖性疾病中,microRNAs与疾病的关系也得到了初步的研究。有作者报道可以根据microRNA表达谱的不同,对疾病进行鉴别诊断。本研究旨在通过实时定量PCR的方法检测miR-29c、miR-150、miR-223、miR-155及miR-146a在我国慢性淋巴细胞白血病、套细胞淋巴瘤及脾边缘区淋巴瘤患者CD19+B淋巴细胞中的表达情况,并与正常人进行比较,观察不同疾病之间及不同疾病与正常人之间有无差异；同时对这5个microRNA在慢性淋巴细胞白血病中是否具有预后意义进行分析。
方法：慢性淋巴细胞白血病53例,套细胞淋巴瘤(Ⅳ期患者)13例,脾边缘区淋巴瘤(Ⅳ期患者)9例,正常人标本12例。标本主要为外周血(78例),少部分为骨髓(9例)。常规分离单个核细胞,并行CD19磁珠分选。应用mirVanaTM miRNA Isolation Kit提取CD19+细胞的总RNA,然后应用Taqman microRNA RT Kit及miR-29c、miR-150、miR-223、miR-155、miR-146a及内对照RNU48逆转录引物将每例标本的RNA(每个miR应用10ng)逆转录为cDNA,采用TaqMan探针法进行每例标本5个microRNA及内对照的相对实时定量PCR检测。校正器选用Namalwa细胞系,PCR结果采用2-△△Ct法进行分析。应用SPSS 16.0软件进行统计学分析。慢性淋巴细胞白血病疾病不同分期间及各不同诊断之间microRNAs平均水平的比较应用单因素方差分析,并进一步用最小显著差法进行两两比较；慢性淋巴细胞白血病各不同预后分组间microRNAs平均水平的比较,采用两独立样本t检验。在慢性淋巴细胞白血病患者,采用ROC曲线方法,根据IgVH的突变状态寻找microRNAs的适宜切点,应用Kaplan-Meier方法绘制生存曲线,组间的中位生存期比较采用log-rank检验。检验水准α均取0.05。
结果：
一、miR-29c、miR-150、miR-223、miR-155及miR-146a在慢性淋巴细胞白血病中的表达及其预后意义：
1.慢性淋巴细胞白血病不同Rai分期及Binet分期之间miR-29c、miR-150、miR-223的表达均有显著性差异(P<0.05),均随疾病进展下调；β2微球蛋白升高组患者miR-223的表达水平显著下调(P=0.045)。
2.miR-29c、miR-223在疾病进展组患者与未进展组患者及死亡组患者与未死亡组患者之间的表达有显著性差异(P<0.05)。
3.IgVH突变组患者与未突变组患者间miR-223、miR-155的表达有显著性差异(P<0.05)。
4.miR-223在13q-阳性组患者的表达显著高于13q-阴性组(P=0.044)。
5.CD38阳性组患者与阴性组患者之间miR-29c、miR-150、miR-223、miR-155和miR-146a的表达无统计学差异(P>0.05)。
6.所有患者中位随访时间12个月(4-92月)。IgVH突变组患者的中位疾病无进展生存期为90个月,高于IgVH未突变组的7个月(P=0.000)；IgVH突变组患者的中位总生存期为92个月,高于IgVH未突变组的36个月(P=0.015)；根据IgVH突变状态采用ROC曲线方法,将miR-29c、miR-223的表达分为阳性组及阴性组。miR-29c阳性组患者的中位疾病无进展生存期为36个月,高于miR-29c阴性组的9个月(P=0.003),miR-29c阳性组患者与阴性组患者的中位总生存期尚无统计学差异(P=0.088)；miR-223阳性组患者的中位疾病无进展生存期为48个月,高于miR-223阴性组的9个月(P=0.001),miR-223阳性组患者截至随访结束,尚无1例死亡。
二、不同慢性淋巴增殖性疾病及正常人之间miR-29c、miR-150、miR-223、miR-155及miR-146a表达水平比较：
1.miR-29c、miR-150及miR-223在套细胞淋巴瘤的表达较在慢性淋巴细胞白血病及脾边缘区淋巴瘤显著下调(P<0.05),而其在慢性淋巴细胞白血病与脾边缘区淋巴瘤之间表达无统计学差异(P>0.05)。
2.miR-155在慢性淋巴细胞白血病的表达显著高于套细胞淋巴瘤及脾边缘区淋巴瘤(P<0.05)；脾边缘区淋巴瘤中miR-146a的表达显著高于慢性淋巴细胞白血病及套细胞淋巴瘤(P<0.05)。
3.脾边缘区淋巴瘤患者中miR-29c的表达水平较正常人显著升高(P=0.035),套细胞淋巴瘤患者中miR-150的表达较正常人显著下调(P=0.004),而miR-223的表达在慢性淋巴细胞白血病、套细胞淋巴瘤及脾边缘区淋巴瘤则显著低于正常人(P<0.05)。
结论：
1.miR-29c、miR-150、miR-223、miR-155及miR-146a在慢性淋巴细胞白血病、套细胞淋巴瘤及脾边缘区淋巴瘤之间表达水平有显著性差异。
2.慢性淋巴细胞白血病、套细胞淋巴瘤及脾边缘区淋巴瘤患者的miR-223表达显著低于其在正常人B淋巴细胞中的表达,提示其可能在慢性淋巴增殖性疾病的发病机制中起重要作用。
3.miR-29c、miR-223在慢性淋巴细胞白血病患者中与疾病肿瘤负荷明显相关,并随疾病进展显著下调,且与患者预后相关,是慢性淋巴细胞白血病新的较为可靠的预后指标。
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.
引文
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