miR-150再表达通过靶向c-Myb诱导EBV阳性Burkitt淋巴瘤分化
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摘要
研究背景
Burkitt淋巴瘤是一种高度侵袭性B细胞性淋巴瘤,根据发病区域及临床表现可分为三种类型：地方性、散发性和免疫缺陷相关性；根据EB病毒感染情况还可分为EBV阳性和EBV阴性Burkitt淋巴瘤。至于Burkitt淋巴瘤的起源,有研究报道,EBV阳性的肿瘤细胞可能起源于中心母细胞,而EBV阴性的肿瘤细胞可能起源于后中心细胞或记忆B细胞。近年来,虽然在肿瘤治疗方面做了许多努力,如抑制肿瘤细胞快速增殖及高强度短期的联合化疗,但还是会有明显的副作用,如细胞毒性和肿瘤溶解综合征。因此,迫切需要寻找更有效的治疗方法。
肿瘤细胞的发生是正常细胞在各种内外环境因素影响下细胞分化发生紊乱的结果,而细胞分化紊乱是一个多基因、多步骤的过程,单独改变某种基因的表达很难对细胞分化产生实质性的影响。miRNAs(miRNAs)是一类普遍存在于动植物中的长度约为22个核苷酸的内源性非编码小分子RNA,迄今为止,大约有2000多种miRNAs被发现鉴定,它们参与细胞增殖、分化、凋亡、和胚胎发育等一系列重要的生命过程。一种miRNA可以调控数百种靶基因,而且它所调控的基因中相当一部分是转录因子,后者本身就扮演一个调控者的角色。因此,改变一种miRNA的表达就会对细胞产生剧烈的效应。种种研究迹象表明,miRNAs具有强烈的调控细胞分化的作用,调整其表达能诱导肿瘤细胞向正常细胞分化,很有可能成为诱导分化治疗的新型诱导分化剂。
诱导分化治疗又称为肿瘤细胞的再分化(redifferentiation),是在某些诱导分化剂的作用下诱导肿瘤细胞向正常细胞方向分化,改变肿瘤细胞的增殖、浸润、侵袭、转移等恶性特征,达到治疗肿瘤的目的。诱导分化将会成为肿瘤治疗的一个新的发展方向。值得一提的是,淋巴造血系统肿瘤是研究诱导分化的较好的模式肿瘤,因为该系统肿瘤的发生是由于淋巴造血细胞分化紊乱或分化阻滞的结果,在淋巴造血细胞发育分化的不同阶段都可以产生相对应类型的肿瘤,不同分化阶段的淋巴造血细胞都有其特定的免疫表型及基因特征,便于鉴别和检测。
在淋巴造血细胞发生过程中,miRNAs的动态表达模式提示其在决定造血细胞分化过程中发挥重要的作用,其中最引人注目的是miR-150,该基因位于19q13.33,成熟的miR-150由22个核酸组成,它随着B细胞发育的成熟表达量逐步升高,是B淋巴细胞分化的关键因子。有研究发现,与正常B细胞相比,不同亚类的弥漫性大B细胞淋巴瘤(DLBCL)肿瘤细胞miR-150普遍低表达。同时也有研究发现,与正常NK/T细胞相比,在NK/T细胞淋巴瘤细胞株和临床标本中,miR-150低表达或表达缺失。然而在不同类型的淋巴瘤细胞miR-150表达情况怎样?在Burkitt淋巴瘤细胞中纠正miR-150的表达后,能否诱导细胞向成熟浆细胞方向分化了?
新近研究表明,在老鼠动物模型内,miR-150通过负性调节c-Myb表达来控制pro-B向pre-B细胞分化。c-myb是一种原癌基因,编码核DNA结合蛋白,行使转录活化和抑制功能。随着造血细胞的成熟分化,其表达逐渐下降,与miR-150的表达模式刚好相反。那么在人类Burkitt淋巴瘤中,miR-150和c-Myb的关系又如何了?需要进一步探究。
目的
1.观测hsa-miR-150和c-Myb在人类正常不同分化阶段B淋巴细胞和不同淋巴瘤细胞系表达谱。
2.构建Burkitt淋巴瘤细胞株稳定表达hsa-miR-150细胞亚系Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150。
3.探究miR-150再表达诱导Burkitt淋巴瘤细胞向末端浆细胞方向分化的可能性及其机制。
方法
1. hsa-miR-150和c-Myb在人类正常不同分化阶段B淋巴细胞亚群和不同淋巴瘤细胞系表达模式
采用流式细胞技术从扁桃体炎患者中的扁桃体内分选出不同分化阶段B淋巴细胞群,包括naive细胞,中心母细胞,中心细胞和记忆B细胞；运用real-time PCR检测hsa-miR-150和c-Myb在正常不同分化阶段B淋巴细胞群和各株淋巴瘤细胞中的表达水平；利用Western Blot在蛋白水平检测正常B细胞各株淋巴瘤细胞c-Myb的表达水平。
2.稳定表达hsa-miR-150的Burkitt淋巴瘤细胞亚系Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150的构建及其生物学特性
含目的基因(hsa-miR-150)的慢病毒表达载体购自上海吉凯公司；将含有重组慢病毒质粒(实验组,对照组)的病毒上清分别转染Burkitt淋巴瘤细胞亚系Raji, Daudi, Ramos和BJAB细胞系,并命名为Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150细胞,实验组和对照组均带有GFP标签,对照组分别命名为Raji-GFP, Daudi-GFP,Ramos-GFP和BJAB-GFP细胞。利用流式细胞技术根据GFP的表达筛选出阳性细胞。对新构建的细胞亚系,通过real-time PCR检测实验组和对照组miR-150的表达；流式细胞技术检测细胞增殖和凋亡,MTT法检测细胞增殖。
3. hsa-miR-150再表达可诱导EBV阳性的Burkitt淋巴瘤再分化
通过流式细胞技术检测生发中心标记物CD10, B细胞标记物CD19及浆细胞标记物CD38和CD138在各细胞亚系实验组和对照组中的表达情况,并检测与细胞大小及细胞内细胞器多少相关的流式物理参数FS和SS的情况。
运用real-time PCR, Western Blot和免疫共聚焦检测各细胞亚系实验组和对照组中与B细胞分化相关基因BCL6, PRDM1及bcl-2的表达。
4. hsa-miR-150再表达通过调整c-Myb的表达发挥作用
运用real-time PCR, Western Blot和免疫共聚焦检测各细胞亚系实验组和对照组中c-Myb的表达。在Raji和Daudi细胞中干扰c-Myb的表达,再通过real-time PCR和Western Blot检测与B细胞分化相关基因BCL-6,PRDM1及bcl-2的表达。在Daudi-miR-150及Raji-miR-150中抑制miR-150的表达,运用Western Blot检测c-Myb的表达。
5.初步探讨在Burkitt淋巴瘤中hsa-miR-150表达紊乱的机制
运用real-time PCR和Western Blot检测Burkitt淋巴瘤Raji和Daudi及相应各细胞亚系实验组和对照组中c-Myc的表达。在Daudi细胞中抑制c-Myc的表达后检测miR-150的表达。
统计学处理
采用统计软件SPSS13.0对实验结果进行统计学处理和分析。计量资料以均数±标准差(x±SD)表示。Real-time PCR结果用单向方差分析(one-way ANOVA),流式检测结果、MTT分析结果用两独立样本t检验。按P＜0.05,差异具有统计学意义,P＜0.01具有显著性差异。
结果
1. hsa-miR-150和c-Myb在人类正常不同分化阶段B淋巴细胞群和不同淋巴瘤细胞系表达模式
(1)人扁桃体不同分化阶段B淋巴细胞群的分选
根据以下免疫表型分选相应细胞群：B细胞(CD19+), Naive B细胞(CD19+,IgD+,CD38-),中心母细胞(CD19+,IgD-,CD38+,CD77+),中心细胞(CD19+,IgD-,CD38+,CD77-),及记忆B细胞(CD19+,IgD-,CD38-)。
(2) hsa-miR-150和c-Myb在正常不同分化阶段B淋巴细胞群中的表达
real-time PCR结果显示,miR-150和c-Myb的表达在不同分化阶段呈现动态性的变化,二者在四群细胞中的表达模式基本互补,即miR-150在NaiveB细胞和记忆B细胞中高表达,而在中心母细胞和中心细胞中低表达；c-Myb在Naive B细胞和记忆B细胞中低表达,而在中心母细胞和中心细胞中高表达。
(3) hsa-miR-150和c-Myb在不同淋巴瘤细胞系表达
real-time PCR结果显示,与正常成熟的B细胞相比,miR-150在各株淋巴瘤细胞株中包括Raji, Daudi, Ramos, BJAB, KM3, KARPAS-299, Jurkat及L428均低表达,而c-Myb均普遍高表达；Western Blot进一步显示c-Myb在这些淋巴瘤细胞株中高表达。
2.稳定表达hsa-miR-150的Burkitt淋巴瘤细胞亚系Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150的构建及其生物学特性
(1)表达hsa-miR-150慢病毒的设计和病毒颗粒的合成
本实验运用了表达hsa-miR-150慢病毒载体(PP-GFP)及对照组慢病毒空载体(只带GFP),启动子均为CMV。载体的设计由上海吉凯生物公司承担。
(2)稳定表达hsa-miR-150细胞亚系的构建
将载有hsa-miR-150的慢病毒载体转染Burkitt淋巴瘤细胞株Raji, Daudi, Ramos和BJAB细胞株,同时将空载体转染这些细胞。运用流式细胞技术筛选出GFP+的细胞,real-time PCR检测转染效率,结果显示,转染后,实验组miR-150的表达与正常中心母细胞相近,而对照组变化不大。
(3)各细胞亚系增殖和凋亡的观测
流式细胞技术检测Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150与对照组Raji-GFP, Daudi-GFP, Ramos-GFP和BJAB-GFP的增殖和凋亡情况；MTT进一步检测了Raji-miR-150, Daudi-miR-150及其对照组的增殖情况。结果显示,与对照组相比,Daudi-miR-150和Raji-miR-150的增殖显著减慢,而其余各组没有明显变化；与对照组相比,Daudi-miR-150的凋亡显著增加,而其余各组没有明显变化。
3. hsa-miR-150再表达诱导EBV阳性的Burkitt淋巴瘤向浆细胞方向分化
(1)各细胞亚系与B细胞分化相关指标的观测
real-time PCR和Western Blot检测了BCL6, PRDM1及bcl-2在各细胞亚系中的表达,结果显示,与对照组相比,BCL6在Raji-miR-150和Daudi-miR-150中表达明显降低,而PRDMl及bcl-2在Raji-miR-150和Daudi-miR-150中表达明显增强；三者在Ramos-miR-150及BJAB-miR-150中的表达没有明显变化。免疫共聚焦在Raji-miR-150细胞中进一步验证了上述结果。
(2)各细胞亚系表面标记物的观测
流式细胞技术检测了CD19, CD10, CD38及CD138的表达,结果显示,与对照组相比,在Raji-miR-150中CD10的表达明显减弱,CD38的表达明显增强,具有统计学意义(t=-160.66,P＜0.001；t=-51.19,P＜0.001)；在Daudi-miR-150中,CD19和CD10的表达明显减弱(t=-350.67,P＜0.001),并出现了CD138的表达(t=-186.75,P＜0.001),结果具有统计学意义；而在Ramos-miR-150及BJAB-miR-150中,与对照组相比,这些表面标记物没有明显变化。
(3)各细胞亚系细胞大小和细胞内细胞器的观测
利用流式细胞技术检测了与细胞大小和细胞内细胞器多少有关的物理参数FS及SS,结果显示,与对照组相比,Raji-miR-150细胞明显增大且细胞内细胞器明显增多(t=65.57,p＜0.001),在Daudi-miR-150中,细胞内细胞器也明显增多(t=86.77；p＜0.001),结果具有统计学意义。
4. hsa-miR-150再表达通过调整c-Myb的表达发挥作用
(1) c-Myb在各细胞亚系中的表达情况
real-time PCR和Western Blot检测了c-Myb在各细胞亚系中的表达,结果显示,与对照组相比,尽管在蛋白水平上,c-Myb的表达在Raji-miR-150及Daudi-miR-150均降低,但在mRNA水平上,c-Myb的表达分别是降低及没有明显变化。运用荧光共聚焦实验在Raji-miR-150细胞中进一步得到了验证。
(2)在Daudi-miR-150及Raji-miR-150中抑制miR-150的表达后c-Myb的表达检测
在Daudi-miR-150及Raji-miR-150中通过竞争性抑制剂抑制miR-150的表达后,利用Western Blot在蛋白水平上检测了c-Myb的表达,结果显示,与对照组相比,c-Myb的表达明显升高。
(3)在Raji及Daudi细胞中干扰c-Myb的表达后相关分化指标的观测
利用干扰片段干扰了Raji及Daudi细胞中c-Myb的表达,real-time PCR和Western Blot检测了干扰效果,结果显示,c-Myb的表达明显降低；之后,real-time PCR和Western Blot检测了bcl-2, BCL6及PRDM1的表达,结果显示,与对照组相比,在基因水平上,BCL6的表达明显减弱,bcl-2及PRDM1的表达明显升高；在蛋白水平上,BCL6的表达明显减弱,bcl-2的表达明显升高,在Daudi细胞中出现了PRDM1的表达。
5.初步探讨Burkitt淋巴瘤hsa-miR-150表达紊乱的机制
(1) c-Myc在Raji, Daudi及相应细胞亚系细胞中表达
Western Blot检测了c-Myc在Raji, Daudi及相应细胞亚系细胞中表达,结果显示,c-Myc在Raji, Daudi细胞均高表达,与Raji, Daudi细胞及对照组相比,Raji-miR-150及Daudi-miR-150细胞c-Myc的表达明显降低。
(2)干扰c-Myc表达后miR-150的表达
在Daudi细胞中干扰掉c-Myc表达,real-time PCR检测了干扰效率,结果显示,c-Myc表达明显下降；real-time PCR检测了miR-150的表达,结果显示,miR-150的表达明显升高。
结论
1. hsa-miR-150与c-Myb在人类不同分化阶段B细胞淋巴群中的表达呈阶段性及动态性,且表达模式相反；在人类淋巴瘤细胞株中,hsa-miR-150低表达,c-Myb高表达,二者的表达模式也相反。
2. hsa-miR-150再表达对EBV阳性和EBV阴性的Burkitt淋巴瘤的生物行为具有不同的影响。
3. hsa-miR-150再表达可以诱导EBV阳性的Burkitt淋巴瘤Raji和Daudi细胞系向浆细胞方向分化,而对EBV阴性的Burkitt淋巴瘤Ramos及BJAB没有影响。
4. hsa-miR-150通过调节其靶基因c-Myb的表达发挥作用。
5. c-Myc的表达紊乱可能导致了hsa-miR-150在Burkitt淋巴瘤中的表达紊乱。
创新之处
1.揭示了hsa-miR-150与c-Myb在人类不同分化阶段B细胞淋巴群呈阶段性及动态性表达。
2.提出了hsa-miR-150再表达可诱导EBV阳性的Burkitt淋巴瘤向浆细胞方向分化,而对EBV阴性的Burkitt淋巴瘤没有影响。
3.验证了在EBV阳性的Burkitt淋巴瘤中,hsa-miR-150通过调节其靶基因c-Myb的表达发挥作用。
Background
Burkitt lymphoma (BL) is a highly aggressive B-cell malignancy identified and described for the first time by Dennis Burkitt in1958. There are3major subgroups of BL that differ in geographic distribution and clinical manifestation:endemic, sporadic and immunodeficiency-associated. According the Epstein-Barr virus (EBV) infection status, BLs are divided into2subgroups:EBV-positive and EBV-negative BL. BLs have a tendency to morphologically resemble germinal center (GC) cells, and to immunophenotypically express characteristic GC cell markers such as CD10and BCL6, and coexpress B-cell markers CD19and CD20, which suggests follicle center B-cell origin for this lymphoma. In recent years, efforts have focused on improving therapy for this the fastest growing human tumor while minimizing treatment-associated toxicity. Outcome with intensive chemotherapy has improved and is now excellent in children, but the prognosis is poor in elderly adults. In addition, there are significant associated toxicities, such as frequent myelosuppression, mucositis, neuropathy, and complication of tumor lysis syndrome. Therefore, it is imperative and important to pursuit more effective therapies.
MicroRNAs (miRNAs), a novel class of small noncoding RNAs of-19-26nt, exert multiple cellular functions and play critical roles in cellular proliferation, apoptosis, cellular differentiation and tumorigenesis. MiRNAs expression profiling studies have found that in hematopoiesis, certain miRNAs are expressed in a stage-specific fashion in the lymphoid hematopoietic system. Therefore, deregulations of their expression may result in the development of hematopoietic malignancies.
MiR-150has attracted our great attention in hematopoiesis among a large number of miRNAs in recent years, which has been studied in B, T, and NK/T cells. There are strong evidences that miR-150is preferentially expressed in mature lymphocytes, but not their progenitors. Ectopic miR-150expression of murine hematopoietic stem cells impairs the transition of pro-B to pre-B stage. In addition, miR-150is found to target c-Myb, which is highly expressed in lymphocytes progenitors, down-regulated as maturation, and again increased after activation of the mature cells. It has been shown that expression of miR-150was consistently down-regulated in diffuse large B-cell lymphoma (DLBCL) cell lines compared with centroblasts. Furthermore, aberrantly low expression of miR150is identified in Sezary syndrome and NK/T-cell lymphoma. Re-expression of miR-150in NK/T-cell lymphoma cells increases the incidence of apoptosis and reduces cell proliferation, suggesting that miR-150functions as a tumor suppressor.
The discovery of miRNAs has added an entirely new dimension to antitumor therapeutic approaches. Depending on miRNA function and status in tumor, miRNAs are generally classified as tumor suppressors or oncomiRs. Therefore, from the therapeutic point of view, tumor suppressor-miRNAs can be induced by ectopic expression, and those oncogenic-miRNAs can be inhibited. Re-expressing lost miRNA in a cell can deliver a dramatic effect, because miRNAs regulate a vast number of genes and pathways. The ectopic expression of miR-223, miR15/miR-16, let7, mir-342, miR-29a and miR-142-3p in acute myeloid leukemia could stimulate myeloid differentiation of leukemia cells. Therefore, differentiation induction by miRNAs is proposed to be a potentially attractive strategy for leukemia. Recently, our group found that CD99can trigger Hodgkin/Reed-Sternberg cells redifferentiation by upregulating miR-9-modulated PRDM1/BLIMP1. Here, we found that Burkitt lymphoma cell lines exhibited an extremely low expression of miR-150. Consequently, testing whether transfection of miR-150into BL cell lines can shift the regulators expression profiles toward that of the terminal B-cell is one purpose in our works. Although in immature hematopoietic system of mouse model, miR-150functions by targeting c-Myb, it is remain unknown whether there is the similar relationship miR-150with c-Myb in BL, which is another one question we'll explore in our works.
Objective
1. Explosive expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines
2. Constructing cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150with stable expression of miR-150
3. Explosive the possibility and potential mechanism that re-expression of miR-150induces Burkitt lymphomas differentiation
Method
1. Expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines
Selection of B cell subpopulations by FACS according the corresponding surface markers:B lymphocytes CD19+, naive cells CD19+CD38-IgD+, centroblasts CD19+CD38+IgD-, centrocytes CD19+CD38+IgD-, memory cells CD19+CD38-IgD-. Analysis of miR-150and c-Myb in B cell subpopulations from tonsils and in human peripheral lymphoma cell lines by real-time PCR. Analysis of c-Myb in human peripheral lymphoma cell lines by Western Blot.
2. Construction of cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150stably expressing miR-150and analysis of their biological characteristics
Stable expression lentiviral vectors for miR-150were provided by the Shanghai GeneChem (China), and the lentiviral supernatant was synthesized by GeneChem. Raji, Daudi, Ramos and BJAB were transfected by lentiviral supernatant (experimental group and control group), and the cell sublines were namely respectively Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150, all of which were tagged by GFP including control, and then sorted for GFP expression on a FACS aria. Transfection efficancy was confirmed by real-time PCR. The proliferation and apoptosis rate were examined by flow cytometry and MTT test in cell sublines.
3. Re-expression of miR-150induces EBV-positive Burkitt lymphomas differentiation
Analysis of germinal center marker CD10, B lymphocyte marker CD19and plasma cell markers CD38and CD138in cell sublines by flow cytometry. Analysis of BCL6, PRDM1and bcl-2in cell sublines by real-time PCR, Western Blot and confocal.
4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas
Analysis of c-Myb in cell sublines by real-time, Western Blot and confocal. Transient transfections with inhibition of miR-150in Daudi-miR-150and Raji-miR-150as well as c-Myb in Raji and Daudi cell lines were performed. Then BCL6, PRDM1and bcl-2were analyzed by real-time and Western Blot in c-Myb RANi Raji and Daudi cell lines. Analysis of c-Myb in miR-150inhibition Daudi-miR-150and Raji-miR-150cells by Western Blot.
5. Explosive of underlying mechanism by which miR-50was dysregulated in Burkitt lymphomas
Analysis of c-Myc in Burkitt lymphomas and cell sublines by real-time PCR and Western Blot. Analysis of miR-150in c-Myc RNAi Daudi cell line by real-time PCR.
Statistical analysis
All images such as western Blot and flow cytomety are representative of at least three independent experiments. QRT-PCR assays were performed in triplicate for each experiment. The data shown are presented as the mean±standard derivation (SD) for three independent experiments. The SPSS software13.0was used for the statistical analysis. Differences are considered statistically significant at p<0.05, assessed using the one-way ANOVA for assays with qRT-PCR, and two-tailed independent Student's t test for MTT assays and flow cytometry.
Results
1. Expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines
(1). Selection of B cell subpopulations by FACS
Five B cell subpopulations were selected according the surface markers:B lymphocytes CD19+, naive cells CD19+CD38-IgD+, centroblasts CD19+CD38+IgD-, centrocytes CD19+CD38+IgD-, memory cells CD19+CD38-IgD-.
(2). Expression profiles of miR-150and c-Myb in B cell subpopulations from tonsils
We employed qRT-PCR to analyze expression pattern of miR-150in these four B cell subsets. The result showed that the expression pattern of miR-150of four B cell subpopulations was dynamic. Additionally, we determined the expression levels of c-Myb in the four B cell subsets. A reciprocal correlation of expression pattern of miR-150and c-Myb was found.
(3). Expression profiles of miR-150and c-Myb in human peripheral lymphoma cell lines
We detected the levels of expression of miR-150in human peripheral lymphoma cell lines i.e. Daudi, Raji, Ramos, BJAB, KM3, L428, KARPAS-299and Jurkat. The result showed that miR-150expression was significantly reduced in all cell lines with respect to normal B cell. What's more, we analyzed the expression of c-Myb in lymphoma cell lines in the mRNA levels and in the protein levels. The results demonstrated that the most of these lymphoma cells strongly expressed c-Myb.
2. Construction of cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150stably expressing miR-150and analysis of their biological characteristics
(1).The design and synthesize of lentiviral vector for miR-150
Lentiviral vector for miR-150(PP-GFP) and control lentiviral vector in this experiment were used, both of which were provided by the Shanghai GeneChem (China).
(2).Construction of cell sublines with stable expression of miR-150
Lentiviral vectors constitutively expressing pre-miR-150labeled with GFP were stably transfected into Daudi, Raji, Ramos and BJAB along with a control vector labeled with GFP. GFP+cells were selected by FACS. The efficiency of transfection was measured by qRT-PCR. The result showed that transfected cells expressed activity of miR-150in the vicinity of physiological levels.
(3).Examination of proliferation and apoptosis in cell sublines
We monitored weekly the GFP expression in the mixed cultures with GFP-miR-150positive (GFP-miR-150+) cells and GFP negative (GFP-) cells by flow cytometer. Our results showed that there was a significant reduction in%GFP-miR-150+compared with GFP-in Daudi-miR-150and Raji-miR-150but not in BJAB-miR-150and Ramos-miR-150, then, the results were confirmed by MTT test. We found that re-expression of miR-150resulted in a strong increase in the early apoptotic fraction and late apoptotic cells in Daudi-miR-150but not in Raji-miR-150, Ramos-miR-150and BJAB with respect to GFP+control.
3. Re-expression of miR-150induces EBV-positive Burkitt lymphoma differentiation
(1). Analysis of BCL6, PRDM1and bcl-2in cell sublines
Our results showed that re-expression of miR-150resulted in a consistent down-regulation of BCL6in the protein and mRNA levels compared with controls in Daudi-miR-150, Raji-miR-150. On the other hand, re-expression of miR-150induced PRDM1and bcl-2expression in the protein and mRNA levels in Daudi-miR-150and Raji-miR-150. The results were further confirmed by confocal analysis in Raji-miR-150. No obvious alterations were observed in Ramos-miR-150and BJAB-miR-150about expression of BCL6, PRDM1and bcl-2compared with controls.
(2). Analysis of CD10, CD19, CD38and CD138in cell sublines
Our results indicated that there was a significant increase in the percentage of CD19-CD10-CD138+cells in Daudi-miR-150, and CD10-CD38++cells in Raji-miR-150compared with empty vectors (GFP). No changes were observed in Ramos-miR-150and BJAB-miR-150about immunophenotypic profiles compared with GFP.
(3).Test of cell size and intracellular organelles in cell sublines
By analyzing SS and FS, the optical physical parameters of flow cytometry, we found that intracellular organelles were significantly increased in both Daudi-miR-150and Raji-miR-150compared with GFP. Furthermore, Raji-miR-150displayed increased cellular size.
4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas
(1). Analysis of c-Myb in cell sublines
The results demonstrated that although c-Myb protein was down-regulated in Daudi-miR-150and Raji-miR-150cells compared with controls, there was different expression profiling in these cells in the mRNA levels in comparison to controls:no obvious alteration and significant downregulation, respectively. The confocal analysis also displayed that c-Myb protein was reduced in Raji-miR-150compared with GFP. The miR-150-mediated effect on c-Myb in Ramos-miR-150and BJAB-miR-150could not be found.
(2). Analysis of c-Myb in miR-150inhibition Daudi-miR-150and Raji-miR-150cells
The results displayed that the expression of c-Myb protein increased again compared with controls when miR-150expression was inhibited in Daudi-miR-150and Raji-miR-150, determined by Western Blot.
(3).Analysis of BCL6, PRDM1and bcl-2in c-Myb RNAi Raji and Daudi cell lines
The c-Myb siRNA Raji and Daudi cells displayed downregulation of BCL6and upregulation of bcl-2in the levels of mRNA and protein compared with controls. While PRDM1mRNA was induced in c-Myb siRNA Raji and Daudi cells, Western blot analysis showed that PRMD1protein levels only were detectable in c-Myb siRNA Daudi cells, and flow cytometry analysis showed that there were no changes of phenotypes in c-Myb siRNA Raji and Daudi cells compared with controls.
5. Explosive of underlying mechanism by which miR-50was dysregulated in Burkitt lymphomas
(1). Analysis of c-Myc in Burkitt lymphomas and cell sublines
The results revealed that they strongly expressed c-Myc protein. Additionally, re-expression of miR-150led to downregulation of c-Myc protein in Daudi-miR-150and Raji-miR-150compared to controls, whereas had little effect on c-Myc transcripts.
(2). Analysis of miR-150in c-Myc RNAi Daudi cell line
We knocked down expression of c-Myc in Daudi cell line. The efficacy of knockdown was examined by real-time PCR. The result showed that miR-150was induced in low-status c-Myc.
Conclusion
1. The expression pattern of miR-150and c-Myb in human four B cell subpopulations was dynamic. And a reciprocal correlation of expression pattern of miR-150and c-Myb was found, which also can be observed in human peripheral lymphoma cells, that is, miR-150was lowly expressed or absent and c-Myb was highly expressed.
2. Re-expression of miR-150has different influence on biological characteristics in EBV-positive and EBV-negative Burkitt lymphomas.
3. Re-expression of miR-150can induce EBV-positive Burkitt lymphomas Raji and Daudi differentiation toward plasma cell, but no effect on EBV-negative Burkitt lymphoma Ramos and BJAB.
4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas.
5. Dysregulated c-Myc contributes to repressed miR-150.
Innovation points
1. The dynamic expression pattern of miR-150and c-Myb in human four B cell subpopulations.
2. Re-expression of miR-150has different influence in EBV-positive and EBV-negative Burkitt lymphomas.
3. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas.
Burkitt淋巴瘤是一种高度侵袭性B细胞性淋巴瘤,根据发病区域及临床表现可分为三种类型：地方性、散发性和免疫缺陷相关性；根据EB病毒感染情况还可分为EBV阳性和EBV阴性Burkitt淋巴瘤。至于Burkitt淋巴瘤的起源,有研究报道,EBV阳性的肿瘤细胞可能起源于中心母细胞,而EBV阴性的肿瘤细胞可能起源于后中心细胞或记忆B细胞。近年来,虽然在肿瘤治疗方面做了许多努力,如抑制肿瘤细胞快速增殖及高强度短期的联合化疗,但还是会有明显的副作用,如细胞毒性和肿瘤溶解综合征。因此,迫切需要寻找更有效的治疗方法。
肿瘤细胞的发生是正常细胞在各种内外环境因素影响下细胞分化发生紊乱的结果,而细胞分化紊乱是一个多基因、多步骤的过程,单独改变某种基因的表达很难对细胞分化产生实质性的影响。miRNAs(miRNAs)是一类普遍存在于动植物中的长度约为22个核苷酸的内源性非编码小分子RNA,迄今为止,大约有2000多种miRNAs被发现鉴定,它们参与细胞增殖、分化、凋亡、和胚胎发育等一系列重要的生命过程。一种miRNA可以调控数百种靶基因,而且它所调控的基因中相当一部分是转录因子,后者本身就扮演一个调控者的角色。因此,改变一种miRNA的表达就会对细胞产生剧烈的效应。种种研究迹象表明,miRNAs具有强烈的调控细胞分化的作用,调整其表达能诱导肿瘤细胞向正常细胞分化,很有可能成为诱导分化治疗的新型诱导分化剂。
诱导分化治疗又称为肿瘤细胞的再分化(redifferentiation),是在某些诱导分化剂的作用下诱导肿瘤细胞向正常细胞方向分化,改变肿瘤细胞的增殖、浸润、侵袭、转移等恶性特征,达到治疗肿瘤的目的。诱导分化将会成为肿瘤治疗的一个新的发展方向。值得一提的是,淋巴造血系统肿瘤是研究诱导分化的较好的模式肿瘤,因为该系统肿瘤的发生是由于淋巴造血细胞分化紊乱或分化阻滞的结果,在淋巴造血细胞发育分化的不同阶段都可以产生相对应类型的肿瘤,不同分化阶段的淋巴造血细胞都有其特定的免疫表型及基因特征,便于鉴别和检测。
在淋巴造血细胞发生过程中,miRNAs的动态表达模式提示其在决定造血细胞分化过程中发挥重要的作用,其中最引人注目的是miR-150,该基因位于19q13.33,成熟的miR-150由22个核酸组成,它随着B细胞发育的成熟表达量逐步升高,是B淋巴细胞分化的关键因子。有研究发现,与正常B细胞相比,不同亚类的弥漫性大B细胞淋巴瘤(DLBCL)肿瘤细胞miR-150普遍低表达。同时也有研究发现,与正常NK/T细胞相比,在NK/T细胞淋巴瘤细胞株和临床标本中,miR-150低表达或表达缺失。然而在不同类型的淋巴瘤细胞miR-150表达情况怎样?在Burkitt淋巴瘤细胞中纠正miR-150的表达后,能否诱导细胞向成熟浆细胞方向分化了?
新近研究表明,在老鼠动物模型内,miR-150通过负性调节c-Myb表达来控制pro-B向pre-B细胞分化。c-myb是一种原癌基因,编码核DNA结合蛋白,行使转录活化和抑制功能。随着造血细胞的成熟分化,其表达逐渐下降,与miR-150的表达模式刚好相反。那么在人类Burkitt淋巴瘤中,miR-150和c-Myb的关系又如何了?需要进一步探究。
目的
1.观测hsa-miR-150和c-Myb在人类正常不同分化阶段B淋巴细胞和不同淋巴瘤细胞系表达谱。
2.构建Burkitt淋巴瘤细胞株稳定表达hsa-miR-150细胞亚系Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150。
3.探究miR-150再表达诱导Burkitt淋巴瘤细胞向末端浆细胞方向分化的可能性及其机制。
方法
1. hsa-miR-150和c-Myb在人类正常不同分化阶段B淋巴细胞亚群和不同淋巴瘤细胞系表达模式
采用流式细胞技术从扁桃体炎患者中的扁桃体内分选出不同分化阶段B淋巴细胞群,包括naive细胞,中心母细胞,中心细胞和记忆B细胞；运用real-time PCR检测hsa-miR-150和c-Myb在正常不同分化阶段B淋巴细胞群和各株淋巴瘤细胞中的表达水平；利用Western Blot在蛋白水平检测正常B细胞各株淋巴瘤细胞c-Myb的表达水平。
2.稳定表达hsa-miR-150的Burkitt淋巴瘤细胞亚系Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150的构建及其生物学特性
含目的基因(hsa-miR-150)的慢病毒表达载体购自上海吉凯公司；将含有重组慢病毒质粒(实验组,对照组)的病毒上清分别转染Burkitt淋巴瘤细胞亚系Raji, Daudi, Ramos和BJAB细胞系,并命名为Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150细胞,实验组和对照组均带有GFP标签,对照组分别命名为Raji-GFP, Daudi-GFP,Ramos-GFP和BJAB-GFP细胞。利用流式细胞技术根据GFP的表达筛选出阳性细胞。对新构建的细胞亚系,通过real-time PCR检测实验组和对照组miR-150的表达；流式细胞技术检测细胞增殖和凋亡,MTT法检测细胞增殖。
3. hsa-miR-150再表达可诱导EBV阳性的Burkitt淋巴瘤再分化
通过流式细胞技术检测生发中心标记物CD10, B细胞标记物CD19及浆细胞标记物CD38和CD138在各细胞亚系实验组和对照组中的表达情况,并检测与细胞大小及细胞内细胞器多少相关的流式物理参数FS和SS的情况。
运用real-time PCR, Western Blot和免疫共聚焦检测各细胞亚系实验组和对照组中与B细胞分化相关基因BCL6, PRDM1及bcl-2的表达。
4. hsa-miR-150再表达通过调整c-Myb的表达发挥作用
运用real-time PCR, Western Blot和免疫共聚焦检测各细胞亚系实验组和对照组中c-Myb的表达。在Raji和Daudi细胞中干扰c-Myb的表达,再通过real-time PCR和Western Blot检测与B细胞分化相关基因BCL-6,PRDM1及bcl-2的表达。在Daudi-miR-150及Raji-miR-150中抑制miR-150的表达,运用Western Blot检测c-Myb的表达。
5.初步探讨在Burkitt淋巴瘤中hsa-miR-150表达紊乱的机制
运用real-time PCR和Western Blot检测Burkitt淋巴瘤Raji和Daudi及相应各细胞亚系实验组和对照组中c-Myc的表达。在Daudi细胞中抑制c-Myc的表达后检测miR-150的表达。
统计学处理
采用统计软件SPSS13.0对实验结果进行统计学处理和分析。计量资料以均数±标准差(x±SD)表示。Real-time PCR结果用单向方差分析(one-way ANOVA),流式检测结果、MTT分析结果用两独立样本t检验。按P＜0.05,差异具有统计学意义,P＜0.01具有显著性差异。
结果
1. hsa-miR-150和c-Myb在人类正常不同分化阶段B淋巴细胞群和不同淋巴瘤细胞系表达模式
(1)人扁桃体不同分化阶段B淋巴细胞群的分选
根据以下免疫表型分选相应细胞群：B细胞(CD19+), Naive B细胞(CD19+,IgD+,CD38-),中心母细胞(CD19+,IgD-,CD38+,CD77+),中心细胞(CD19+,IgD-,CD38+,CD77-),及记忆B细胞(CD19+,IgD-,CD38-)。
(2) hsa-miR-150和c-Myb在正常不同分化阶段B淋巴细胞群中的表达
real-time PCR结果显示,miR-150和c-Myb的表达在不同分化阶段呈现动态性的变化,二者在四群细胞中的表达模式基本互补,即miR-150在NaiveB细胞和记忆B细胞中高表达,而在中心母细胞和中心细胞中低表达；c-Myb在Naive B细胞和记忆B细胞中低表达,而在中心母细胞和中心细胞中高表达。
(3) hsa-miR-150和c-Myb在不同淋巴瘤细胞系表达
real-time PCR结果显示,与正常成熟的B细胞相比,miR-150在各株淋巴瘤细胞株中包括Raji, Daudi, Ramos, BJAB, KM3, KARPAS-299, Jurkat及L428均低表达,而c-Myb均普遍高表达；Western Blot进一步显示c-Myb在这些淋巴瘤细胞株中高表达。
2.稳定表达hsa-miR-150的Burkitt淋巴瘤细胞亚系Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150的构建及其生物学特性
(1)表达hsa-miR-150慢病毒的设计和病毒颗粒的合成
本实验运用了表达hsa-miR-150慢病毒载体(PP-GFP)及对照组慢病毒空载体(只带GFP),启动子均为CMV。载体的设计由上海吉凯生物公司承担。
(2)稳定表达hsa-miR-150细胞亚系的构建
将载有hsa-miR-150的慢病毒载体转染Burkitt淋巴瘤细胞株Raji, Daudi, Ramos和BJAB细胞株,同时将空载体转染这些细胞。运用流式细胞技术筛选出GFP+的细胞,real-time PCR检测转染效率,结果显示,转染后,实验组miR-150的表达与正常中心母细胞相近,而对照组变化不大。
(3)各细胞亚系增殖和凋亡的观测
流式细胞技术检测Raji-miR-150, Daudi-miR-150, Ramos-miR-150和BJAB-miR-150与对照组Raji-GFP, Daudi-GFP, Ramos-GFP和BJAB-GFP的增殖和凋亡情况；MTT进一步检测了Raji-miR-150, Daudi-miR-150及其对照组的增殖情况。结果显示,与对照组相比,Daudi-miR-150和Raji-miR-150的增殖显著减慢,而其余各组没有明显变化；与对照组相比,Daudi-miR-150的凋亡显著增加,而其余各组没有明显变化。
3. hsa-miR-150再表达诱导EBV阳性的Burkitt淋巴瘤向浆细胞方向分化
(1)各细胞亚系与B细胞分化相关指标的观测
real-time PCR和Western Blot检测了BCL6, PRDM1及bcl-2在各细胞亚系中的表达,结果显示,与对照组相比,BCL6在Raji-miR-150和Daudi-miR-150中表达明显降低,而PRDMl及bcl-2在Raji-miR-150和Daudi-miR-150中表达明显增强；三者在Ramos-miR-150及BJAB-miR-150中的表达没有明显变化。免疫共聚焦在Raji-miR-150细胞中进一步验证了上述结果。
(2)各细胞亚系表面标记物的观测
流式细胞技术检测了CD19, CD10, CD38及CD138的表达,结果显示,与对照组相比,在Raji-miR-150中CD10的表达明显减弱,CD38的表达明显增强,具有统计学意义(t=-160.66,P＜0.001；t=-51.19,P＜0.001)；在Daudi-miR-150中,CD19和CD10的表达明显减弱(t=-350.67,P＜0.001),并出现了CD138的表达(t=-186.75,P＜0.001),结果具有统计学意义；而在Ramos-miR-150及BJAB-miR-150中,与对照组相比,这些表面标记物没有明显变化。
(3)各细胞亚系细胞大小和细胞内细胞器的观测
利用流式细胞技术检测了与细胞大小和细胞内细胞器多少有关的物理参数FS及SS,结果显示,与对照组相比,Raji-miR-150细胞明显增大且细胞内细胞器明显增多(t=65.57,p＜0.001),在Daudi-miR-150中,细胞内细胞器也明显增多(t=86.77；p＜0.001),结果具有统计学意义。
4. hsa-miR-150再表达通过调整c-Myb的表达发挥作用
(1) c-Myb在各细胞亚系中的表达情况
real-time PCR和Western Blot检测了c-Myb在各细胞亚系中的表达,结果显示,与对照组相比,尽管在蛋白水平上,c-Myb的表达在Raji-miR-150及Daudi-miR-150均降低,但在mRNA水平上,c-Myb的表达分别是降低及没有明显变化。运用荧光共聚焦实验在Raji-miR-150细胞中进一步得到了验证。
(2)在Daudi-miR-150及Raji-miR-150中抑制miR-150的表达后c-Myb的表达检测
在Daudi-miR-150及Raji-miR-150中通过竞争性抑制剂抑制miR-150的表达后,利用Western Blot在蛋白水平上检测了c-Myb的表达,结果显示,与对照组相比,c-Myb的表达明显升高。
(3)在Raji及Daudi细胞中干扰c-Myb的表达后相关分化指标的观测
利用干扰片段干扰了Raji及Daudi细胞中c-Myb的表达,real-time PCR和Western Blot检测了干扰效果,结果显示,c-Myb的表达明显降低；之后,real-time PCR和Western Blot检测了bcl-2, BCL6及PRDM1的表达,结果显示,与对照组相比,在基因水平上,BCL6的表达明显减弱,bcl-2及PRDM1的表达明显升高；在蛋白水平上,BCL6的表达明显减弱,bcl-2的表达明显升高,在Daudi细胞中出现了PRDM1的表达。
5.初步探讨Burkitt淋巴瘤hsa-miR-150表达紊乱的机制
(1) c-Myc在Raji, Daudi及相应细胞亚系细胞中表达
Western Blot检测了c-Myc在Raji, Daudi及相应细胞亚系细胞中表达,结果显示,c-Myc在Raji, Daudi细胞均高表达,与Raji, Daudi细胞及对照组相比,Raji-miR-150及Daudi-miR-150细胞c-Myc的表达明显降低。
(2)干扰c-Myc表达后miR-150的表达
在Daudi细胞中干扰掉c-Myc表达,real-time PCR检测了干扰效率,结果显示,c-Myc表达明显下降；real-time PCR检测了miR-150的表达,结果显示,miR-150的表达明显升高。
结论
1. hsa-miR-150与c-Myb在人类不同分化阶段B细胞淋巴群中的表达呈阶段性及动态性,且表达模式相反；在人类淋巴瘤细胞株中,hsa-miR-150低表达,c-Myb高表达,二者的表达模式也相反。
2. hsa-miR-150再表达对EBV阳性和EBV阴性的Burkitt淋巴瘤的生物行为具有不同的影响。
3. hsa-miR-150再表达可以诱导EBV阳性的Burkitt淋巴瘤Raji和Daudi细胞系向浆细胞方向分化,而对EBV阴性的Burkitt淋巴瘤Ramos及BJAB没有影响。
4. hsa-miR-150通过调节其靶基因c-Myb的表达发挥作用。
5. c-Myc的表达紊乱可能导致了hsa-miR-150在Burkitt淋巴瘤中的表达紊乱。
创新之处
1.揭示了hsa-miR-150与c-Myb在人类不同分化阶段B细胞淋巴群呈阶段性及动态性表达。
2.提出了hsa-miR-150再表达可诱导EBV阳性的Burkitt淋巴瘤向浆细胞方向分化,而对EBV阴性的Burkitt淋巴瘤没有影响。
3.验证了在EBV阳性的Burkitt淋巴瘤中,hsa-miR-150通过调节其靶基因c-Myb的表达发挥作用。
Background
Burkitt lymphoma (BL) is a highly aggressive B-cell malignancy identified and described for the first time by Dennis Burkitt in1958. There are3major subgroups of BL that differ in geographic distribution and clinical manifestation:endemic, sporadic and immunodeficiency-associated. According the Epstein-Barr virus (EBV) infection status, BLs are divided into2subgroups:EBV-positive and EBV-negative BL. BLs have a tendency to morphologically resemble germinal center (GC) cells, and to immunophenotypically express characteristic GC cell markers such as CD10and BCL6, and coexpress B-cell markers CD19and CD20, which suggests follicle center B-cell origin for this lymphoma. In recent years, efforts have focused on improving therapy for this the fastest growing human tumor while minimizing treatment-associated toxicity. Outcome with intensive chemotherapy has improved and is now excellent in children, but the prognosis is poor in elderly adults. In addition, there are significant associated toxicities, such as frequent myelosuppression, mucositis, neuropathy, and complication of tumor lysis syndrome. Therefore, it is imperative and important to pursuit more effective therapies.
MicroRNAs (miRNAs), a novel class of small noncoding RNAs of-19-26nt, exert multiple cellular functions and play critical roles in cellular proliferation, apoptosis, cellular differentiation and tumorigenesis. MiRNAs expression profiling studies have found that in hematopoiesis, certain miRNAs are expressed in a stage-specific fashion in the lymphoid hematopoietic system. Therefore, deregulations of their expression may result in the development of hematopoietic malignancies.
MiR-150has attracted our great attention in hematopoiesis among a large number of miRNAs in recent years, which has been studied in B, T, and NK/T cells. There are strong evidences that miR-150is preferentially expressed in mature lymphocytes, but not their progenitors. Ectopic miR-150expression of murine hematopoietic stem cells impairs the transition of pro-B to pre-B stage. In addition, miR-150is found to target c-Myb, which is highly expressed in lymphocytes progenitors, down-regulated as maturation, and again increased after activation of the mature cells. It has been shown that expression of miR-150was consistently down-regulated in diffuse large B-cell lymphoma (DLBCL) cell lines compared with centroblasts. Furthermore, aberrantly low expression of miR150is identified in Sezary syndrome and NK/T-cell lymphoma. Re-expression of miR-150in NK/T-cell lymphoma cells increases the incidence of apoptosis and reduces cell proliferation, suggesting that miR-150functions as a tumor suppressor.
The discovery of miRNAs has added an entirely new dimension to antitumor therapeutic approaches. Depending on miRNA function and status in tumor, miRNAs are generally classified as tumor suppressors or oncomiRs. Therefore, from the therapeutic point of view, tumor suppressor-miRNAs can be induced by ectopic expression, and those oncogenic-miRNAs can be inhibited. Re-expressing lost miRNA in a cell can deliver a dramatic effect, because miRNAs regulate a vast number of genes and pathways. The ectopic expression of miR-223, miR15/miR-16, let7, mir-342, miR-29a and miR-142-3p in acute myeloid leukemia could stimulate myeloid differentiation of leukemia cells. Therefore, differentiation induction by miRNAs is proposed to be a potentially attractive strategy for leukemia. Recently, our group found that CD99can trigger Hodgkin/Reed-Sternberg cells redifferentiation by upregulating miR-9-modulated PRDM1/BLIMP1. Here, we found that Burkitt lymphoma cell lines exhibited an extremely low expression of miR-150. Consequently, testing whether transfection of miR-150into BL cell lines can shift the regulators expression profiles toward that of the terminal B-cell is one purpose in our works. Although in immature hematopoietic system of mouse model, miR-150functions by targeting c-Myb, it is remain unknown whether there is the similar relationship miR-150with c-Myb in BL, which is another one question we'll explore in our works.
Objective
1. Explosive expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines
2. Constructing cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150with stable expression of miR-150
3. Explosive the possibility and potential mechanism that re-expression of miR-150induces Burkitt lymphomas differentiation
Method
1. Expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines
Selection of B cell subpopulations by FACS according the corresponding surface markers:B lymphocytes CD19+, naive cells CD19+CD38-IgD+, centroblasts CD19+CD38+IgD-, centrocytes CD19+CD38+IgD-, memory cells CD19+CD38-IgD-. Analysis of miR-150and c-Myb in B cell subpopulations from tonsils and in human peripheral lymphoma cell lines by real-time PCR. Analysis of c-Myb in human peripheral lymphoma cell lines by Western Blot.
2. Construction of cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150stably expressing miR-150and analysis of their biological characteristics
Stable expression lentiviral vectors for miR-150were provided by the Shanghai GeneChem (China), and the lentiviral supernatant was synthesized by GeneChem. Raji, Daudi, Ramos and BJAB were transfected by lentiviral supernatant (experimental group and control group), and the cell sublines were namely respectively Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150, all of which were tagged by GFP including control, and then sorted for GFP expression on a FACS aria. Transfection efficancy was confirmed by real-time PCR. The proliferation and apoptosis rate were examined by flow cytometry and MTT test in cell sublines.
3. Re-expression of miR-150induces EBV-positive Burkitt lymphomas differentiation
Analysis of germinal center marker CD10, B lymphocyte marker CD19and plasma cell markers CD38and CD138in cell sublines by flow cytometry. Analysis of BCL6, PRDM1and bcl-2in cell sublines by real-time PCR, Western Blot and confocal.
4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas
Analysis of c-Myb in cell sublines by real-time, Western Blot and confocal. Transient transfections with inhibition of miR-150in Daudi-miR-150and Raji-miR-150as well as c-Myb in Raji and Daudi cell lines were performed. Then BCL6, PRDM1and bcl-2were analyzed by real-time and Western Blot in c-Myb RANi Raji and Daudi cell lines. Analysis of c-Myb in miR-150inhibition Daudi-miR-150and Raji-miR-150cells by Western Blot.
5. Explosive of underlying mechanism by which miR-50was dysregulated in Burkitt lymphomas
Analysis of c-Myc in Burkitt lymphomas and cell sublines by real-time PCR and Western Blot. Analysis of miR-150in c-Myc RNAi Daudi cell line by real-time PCR.
Statistical analysis
All images such as western Blot and flow cytomety are representative of at least three independent experiments. QRT-PCR assays were performed in triplicate for each experiment. The data shown are presented as the mean±standard derivation (SD) for three independent experiments. The SPSS software13.0was used for the statistical analysis. Differences are considered statistically significant at p<0.05, assessed using the one-way ANOVA for assays with qRT-PCR, and two-tailed independent Student's t test for MTT assays and flow cytometry.
Results
1. Expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines
(1). Selection of B cell subpopulations by FACS
Five B cell subpopulations were selected according the surface markers:B lymphocytes CD19+, naive cells CD19+CD38-IgD+, centroblasts CD19+CD38+IgD-, centrocytes CD19+CD38+IgD-, memory cells CD19+CD38-IgD-.
(2). Expression profiles of miR-150and c-Myb in B cell subpopulations from tonsils
We employed qRT-PCR to analyze expression pattern of miR-150in these four B cell subsets. The result showed that the expression pattern of miR-150of four B cell subpopulations was dynamic. Additionally, we determined the expression levels of c-Myb in the four B cell subsets. A reciprocal correlation of expression pattern of miR-150and c-Myb was found.
(3). Expression profiles of miR-150and c-Myb in human peripheral lymphoma cell lines
We detected the levels of expression of miR-150in human peripheral lymphoma cell lines i.e. Daudi, Raji, Ramos, BJAB, KM3, L428, KARPAS-299and Jurkat. The result showed that miR-150expression was significantly reduced in all cell lines with respect to normal B cell. What's more, we analyzed the expression of c-Myb in lymphoma cell lines in the mRNA levels and in the protein levels. The results demonstrated that the most of these lymphoma cells strongly expressed c-Myb.
2. Construction of cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150stably expressing miR-150and analysis of their biological characteristics
(1).The design and synthesize of lentiviral vector for miR-150
Lentiviral vector for miR-150(PP-GFP) and control lentiviral vector in this experiment were used, both of which were provided by the Shanghai GeneChem (China).
(2).Construction of cell sublines with stable expression of miR-150
Lentiviral vectors constitutively expressing pre-miR-150labeled with GFP were stably transfected into Daudi, Raji, Ramos and BJAB along with a control vector labeled with GFP. GFP+cells were selected by FACS. The efficiency of transfection was measured by qRT-PCR. The result showed that transfected cells expressed activity of miR-150in the vicinity of physiological levels.
(3).Examination of proliferation and apoptosis in cell sublines
We monitored weekly the GFP expression in the mixed cultures with GFP-miR-150positive (GFP-miR-150+) cells and GFP negative (GFP-) cells by flow cytometer. Our results showed that there was a significant reduction in%GFP-miR-150+compared with GFP-in Daudi-miR-150and Raji-miR-150but not in BJAB-miR-150and Ramos-miR-150, then, the results were confirmed by MTT test. We found that re-expression of miR-150resulted in a strong increase in the early apoptotic fraction and late apoptotic cells in Daudi-miR-150but not in Raji-miR-150, Ramos-miR-150and BJAB with respect to GFP+control.
3. Re-expression of miR-150induces EBV-positive Burkitt lymphoma differentiation
(1). Analysis of BCL6, PRDM1and bcl-2in cell sublines
Our results showed that re-expression of miR-150resulted in a consistent down-regulation of BCL6in the protein and mRNA levels compared with controls in Daudi-miR-150, Raji-miR-150. On the other hand, re-expression of miR-150induced PRDM1and bcl-2expression in the protein and mRNA levels in Daudi-miR-150and Raji-miR-150. The results were further confirmed by confocal analysis in Raji-miR-150. No obvious alterations were observed in Ramos-miR-150and BJAB-miR-150about expression of BCL6, PRDM1and bcl-2compared with controls.
(2). Analysis of CD10, CD19, CD38and CD138in cell sublines
Our results indicated that there was a significant increase in the percentage of CD19-CD10-CD138+cells in Daudi-miR-150, and CD10-CD38++cells in Raji-miR-150compared with empty vectors (GFP). No changes were observed in Ramos-miR-150and BJAB-miR-150about immunophenotypic profiles compared with GFP.
(3).Test of cell size and intracellular organelles in cell sublines
By analyzing SS and FS, the optical physical parameters of flow cytometry, we found that intracellular organelles were significantly increased in both Daudi-miR-150and Raji-miR-150compared with GFP. Furthermore, Raji-miR-150displayed increased cellular size.
4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas
(1). Analysis of c-Myb in cell sublines
The results demonstrated that although c-Myb protein was down-regulated in Daudi-miR-150and Raji-miR-150cells compared with controls, there was different expression profiling in these cells in the mRNA levels in comparison to controls:no obvious alteration and significant downregulation, respectively. The confocal analysis also displayed that c-Myb protein was reduced in Raji-miR-150compared with GFP. The miR-150-mediated effect on c-Myb in Ramos-miR-150and BJAB-miR-150could not be found.
(2). Analysis of c-Myb in miR-150inhibition Daudi-miR-150and Raji-miR-150cells
The results displayed that the expression of c-Myb protein increased again compared with controls when miR-150expression was inhibited in Daudi-miR-150and Raji-miR-150, determined by Western Blot.
(3).Analysis of BCL6, PRDM1and bcl-2in c-Myb RNAi Raji and Daudi cell lines
The c-Myb siRNA Raji and Daudi cells displayed downregulation of BCL6and upregulation of bcl-2in the levels of mRNA and protein compared with controls. While PRDM1mRNA was induced in c-Myb siRNA Raji and Daudi cells, Western blot analysis showed that PRMD1protein levels only were detectable in c-Myb siRNA Daudi cells, and flow cytometry analysis showed that there were no changes of phenotypes in c-Myb siRNA Raji and Daudi cells compared with controls.
5. Explosive of underlying mechanism by which miR-50was dysregulated in Burkitt lymphomas
(1). Analysis of c-Myc in Burkitt lymphomas and cell sublines
The results revealed that they strongly expressed c-Myc protein. Additionally, re-expression of miR-150led to downregulation of c-Myc protein in Daudi-miR-150and Raji-miR-150compared to controls, whereas had little effect on c-Myc transcripts.
(2). Analysis of miR-150in c-Myc RNAi Daudi cell line
We knocked down expression of c-Myc in Daudi cell line. The efficacy of knockdown was examined by real-time PCR. The result showed that miR-150was induced in low-status c-Myc.
Conclusion
1. The expression pattern of miR-150and c-Myb in human four B cell subpopulations was dynamic. And a reciprocal correlation of expression pattern of miR-150and c-Myb was found, which also can be observed in human peripheral lymphoma cells, that is, miR-150was lowly expressed or absent and c-Myb was highly expressed.
2. Re-expression of miR-150has different influence on biological characteristics in EBV-positive and EBV-negative Burkitt lymphomas.
3. Re-expression of miR-150can induce EBV-positive Burkitt lymphomas Raji and Daudi differentiation toward plasma cell, but no effect on EBV-negative Burkitt lymphoma Ramos and BJAB.
4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas.
5. Dysregulated c-Myc contributes to repressed miR-150.
Innovation points
1. The dynamic expression pattern of miR-150and c-Myb in human four B cell subpopulations.
2. Re-expression of miR-150has different influence in EBV-positive and EBV-negative Burkitt lymphomas.
3. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas.
引文
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[1]. Mishra P J, Merlino G. MicroRNA reexpression as differentiation therapy in cancer[J]. J Clin Invest,2009,119(8):2119-2123.
[2]. Fazi F, Rosa A, Fatica A, et al. A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis[J]. Cell,2005,123(5):819-831.
[3]. Ramkissoon S H, Mainwaring L A, Ogasawara Y, et al. Hematopoietic-specific microRNA expression in human cells[J]. Leuk Res,2006,30(5):643-647.
[4]. Georgantas R R, Hildreth R, Morisot S, et al. CD34+hematopoietic stem-progenitor cell microRNA expression and function:a circuit diagram of differentiation control[J]. Proc Natl Acad Sci U S A,2007,104(8):2750-2755.
[5]. Wu H, Neilson J R, Kumar P, et al. miRNA profiling of naive, effector and memory CD8 T cells[J], PLoS One,2007,2(10):e1020.
[6]. Vasilatou D, Papageorgiou S, Pappa V, et al. The role of microRNAs in normal and malignant hematopoiesis[J]. Eur J Haematol,2010,84(1):1-16.
[7]. Zhou B, Wang S, Mayr C, et al. miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely[J]. Proc Natl Acad Sci U S A,2007,104(17):7080-7085.
[8]. Lu J, Guo S, Ebert B L, et al. MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors [J]. Dev Cell,2008,14(6):843-853.
[9]. Tan L P, Wang M, Robertus J L, et al. miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes[J]. Lab Invest,2009,89(6):708-716.
[10]. Xiao C, Calado D P, Galler G, et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb[J]. Cell,2007,131(1):146-159.
[11]. Malumbres R, Sarosiek K A, Cubedo E, et al. Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[J]. Blood,2009,113(16):3754-3764.
[12]. Bezman N A, Chakraborty T, Bender T, et al. miR-150 regulates the development of NK and iNKT cells[J]. J Exp Med,2011,208(13):2717-2731.
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[9]. Tan L P, Wang M, Robertus J L, et al. miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes[J]. Lab Invest,2009,89(6):708-716.
[10]. Xiao C, Calado D P, Galler G, et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb[J]. Cell,2007,131(1):146-159.
[11]. Malumbres R, Sarosiek K A, Cubedo E, et al. Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[J]. Blood,2009,113(16):3754-3764.
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