AML1a在小鼠造血细胞增殖分化异常及恶性转化中的作用及全反式维甲酸处理NB4细胞的微小RNA表达谱分析
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摘要
第一部分AML1a在小鼠造血细胞增殖分化异常及恶性转化中的作用
细胞的增殖分化受到严格调控,转录因子协调细胞中多种基因的表达是其中重要的调控机制。造血细胞的增殖失控、分化能力的丧失将可能导致细胞的恶性转化及白血病的发生。因此,阐明转录因子的功能对于确定细胞增殖分化调控机制以及在细胞恶性转化中的作用具有重要意义。AML1也称之为PEBP2αB、CBFα2或RUNX1,是一种重要的转录调节因子。它在造血细胞中普遍表达,调节造血细胞的分化和增殖。
RUNX1/AML1通过选择性剪接产生至少三种异构体:AML1a、A.ML1b和AML1c。其中,AML1b与AML1c仅在氨基末端存在27个氨基酸残基的差异,均具有两个主要的功能结构域:DNA结合结构域和转录激活结构域,两者的功能基本相同,即我们通常所说的AML1。AML1a具有DNA结合结构域,但缺少转录激活结构域,因此通常被认为不具有转录激活功能,但由于其具有DNA结合结构域,可以与AML1b/1c竞争结合靶基因的DNA结合位点,导致靶基因不能正常转录,提示AML1a可能干扰AML1的转录调节功能。有证据表明,AML1a在急性髓系白血病(AML)中表达高于正常对照。我们的前期实验也已证实AML1a在急性白血病(AL)中的高表达,而且,AML1a可以抑制由AML1b介导的M-CSF受体(M-CSFR)启动子的转录,提示AML1a对AML1b具有拮抗作用。有学者认为转录激活区的缺失可能引起白血病的发生,但目前尚无此方面的报道。为了进一步研究AML1a在造血及白血病发病中的作用并探讨其作用机制,本文完成了下列研究工作:
1.AML1a表达载体的构建及病毒的制备
pMSCV-FLAG-AML1a-IRES-YFP质粒,将此质粒和pMSCV-IRES-YFP分别与辅助质粒pV Pack-Eco(含Env)、pV Pack-GP(含gal-pol)组合,通过磷酸钙沉淀法转染293T细胞,制备逆转录病毒。病毒感染3T3细胞,检测病毒滴度,并测定AML1a的表达情况。结果显示在感染的细胞中证实有AML1a和YFP的表达,所制备的病毒滴度满足后续实验的要求。
2.AML1a在小鼠造血细胞增殖分化异常及恶性转化中的作用
1) C57雄性小鼠的骨髓单个核细胞(BMMNC)通过逆转录病毒转导AML1a及空白对照后,置于含mSCF、mIL-3、mIL-6的IMDM培养液中进行培养,分别于感染后11天、23天和35天通过流式细胞仪检测确定各系表面标志的变化,结果显示感染AML1a的BMMNC中Sca-1及Thy1.2的表达均较对照组升高,提示AML1a可能将造血干细胞的分化阻滞于较为早期的淋巴细胞阶段。
2)将转导AML1a的C57小鼠的骨髓单个核细胞,尾静脉注射至致死剂量照射的C57雌性小鼠体内,单一转导YFP的小鼠作为对照。定期尾静脉取血,查血象及白细胞分类。血象有异常改变后,处死小鼠,观察有无肝、脾、淋巴结肿大,流式细胞术分析细胞表型变化。转导AML1a的12只C57小鼠中,有9只在移植后3-11个月相继死亡,解剖可见肝脾肿大,可伴有胸腺瘤和淋巴结肿大。病理可见骨髓、脾、肺、肾、胸腺或淋巴结可见大量白血病细胞浸润。流式分析显示白血病细胞主要有两个表型:Sca-1~+ cCD3~+和CD3~+CD4~+CD8~+,提示均为T-淋巴细胞白血病。发病小鼠脾细胞接种同类小鼠可再次发生表型相同的T-淋巴细胞白血病。以上结果表明AML1a在白血病的发生中发挥重要的作用,并且可能具备导致白血病的能力。
3)将AML1a发病小鼠脾细胞传代后取第二代发病鼠的骨髓细胞进行细胞周期测定。与对照组相比,发病小鼠骨髓中G0/G1细胞比例增高,S期细胞比例明显降低,提示AML1a将小鼠骨髓细胞阻滞于G0/G1期。此结果说明AML1a基因可能通过抑制细胞自G0/G1期向S期的转换,改变正常细胞增殖分裂的进程,导致造血细胞的恶性转化。
综上所述,本研究通过逆转录病毒转导AML1a至小鼠BMMNC中,并将其移植到致死剂量照射的小鼠体内,首次证实了AML1a的可以引起造血细胞恶性转化并导致白血病发生。初步推测AML1a可能在白血病特别是淋巴细胞白血病的发病中起重要作用。此外,我们的实验也为白血病的治疗提供了新的治疗靶点和动物模型。
第二部分全反式维甲酸处理NB4细胞的微小RNA表达谱分析
微小RNA(microRNA,miRNA)是一种非编码小分子RNA,可以作为基因的负调控因子调节多种生物进程。每一种微小RNA可以调控数百种靶基因。近来研究显示微小RNA的突变或者异常表达与人类许多肿瘤发生相关,它能够作为肿瘤抑制物或者癌基因发挥作用,因此可能有助于肿瘤的诊断和治疗。我们通过确定全反式维甲酸(ATRA)诱导急性早幼粒细胞白血病(APL)细胞系NB4细胞分化过程中微小RNA表达谱的变化,寻找可能对白血病细胞分化起作用的微小RNA。细胞形态学和流式细胞术验证ATRA处理NB4细胞的分化。利用微小RNA芯片技术,检测和分析NB4经ATRA处理不同时间点的微小RNA表达谱差异,实时定量RT-PCR技术验证微小RNA表达的变化,并对微小RNA进行靶基因预测。ATRA处理后的NB4细胞在形态上出现向粒细胞的分化,分析CD11b等髓系表面抗原的表达,ATRA处理0h及72h时CD11b的表达分别为3%和96%,进一步证实了NB4细胞的分化。基因芯片检测从576个候选的人类微小RNA中,筛选出24个差异表达微小RNA(表达上调的有21个,表达下调的有3个),其中,有16个微小RNA可以进行靶基因预测。这些miRNA的靶基因多涉及与白血病发生有关的同源盒基因家族成员。实时定量RT-PCR验证了2个显著上调微小RNA(miR-210和miR-342)表达的变化,两种微小RNA标准化后的比值分别为2.16和4.61。差异表达微小RNA可能与NB4的分化相关,同时也可能是转录后水平抑制基因表达调控方式的基础。
PartⅠThe role of AML1a in abnormal proliferation and differentiation and its significant function in malignant transformation of murine hematopoietic cells
Cell proliferation and differentiation are strictly regulated by intrinsic and extrinsic signals,in which coordination of a variety of gene expression by transcription factors is the most important mechanism.Uncontrolled proliferation and impaired differentiation of hematopoietic cells lead to leukemogenesis.Therefore,it is significant to clarify the functions of transcription factors for elucidating the mechanism of abnormal proliferation and differentiation,as well as leukemogenesis.
AML1,also called RUNX1,PEBP2αB or CBFα2,is a critical transcription factor in hematopoietic cell differentiation and proliferation.Its mutation and translocation lead to hematopoietic malignant.
From the AML1/RUNX1 gene,at least three isoforms,AML1a,AML1b and AML1c,are produced through alternative splicing.The proteins encoded by AML1b and AML1c have a RHD in the N-terminus and a transactivation domain in the C-terminus,which are often called AML1.In contrast,the protein encoded by AML1a has a RHD but lacks the transactivation domain.As such,the function of AML1 is believed to be mediated by AML1b and AML1c which are considered to have the same function.AML1a interferes with the function of AML1b/1c.It has been proved that AML1a is higher expressed in acute myeloid leukemia(AML) than in normal controls.Our previous study confirmed that AML1a expression in acute leukemia(AL) has a high level.In addition,AML1a can inhibit the transcription of M-CSFR promoter mediated by AML1b,suggesting that AML1a has the effect of antagonism to AML1b.Some scholars believe that loss of the transcription activation domain may cause leukemogenesis,but at present there is no report in this regard.In order to explore the role and mechanism of AML1a in abnormal hematopoesis and leukemogenesis,we completed the following work in this study:
1.The construction of AML1a expression vector and the production of retrovirus.
pMSCV-FLAG-AML1a-IRES-YFP was constructed.293T cells were transfected with pMSCV-IRES-YFP,pMSCV-FLAG-AML1a-IRES-YFP,the envelope-encoding plasmid pV Pack-Eco,or the packaging plasmid pV Pack-GP using a method of calcium phosphate precipitation to product retrovirus.The title of virus was measured using a bioassay with the 3T3 cell line.The expression of the fusion proteins in the tranduced cells was confirmed.The title of retrovirus can satisfy the continually experimental demand.
2.The role of AML1a in abnormal hematopoiesis and leukemogenesis.
1) Bone marrow mononuclear cells(BMMNCs) were infected with the retroviral vector MSCV expressing a FLAG-AML1a fusion protein and a yellow fluorescent protein(YFP).The cells were cultured in Iscove modified Dulbecco medium(IMDM) supplemented with murine IL-3(mIL-3),murine IL-6(mIL-6) and marine SCF (mSCF).The change of surface marker of each hematopoietic linage was analyzed by FACS in 11d,23d and 35d after transduction.The result showed that the expression of Sca-1 and Thy1.2 in AML1a transduced BMMNC was higher than that of the control,which indicating that AML1a may prone to block the differentiation of hematopoietic stem cell in the relatively early lymphocytic stage 2) BMMNCs from mice were transduced with AML1a and transplanted into lethally irradiated mice, which develop lymphoblastic leukemia after transplantation.Lethally irradiated female C57 mice received BMCs infected with the retroviral vector MSCV expressing a FLAG-AML1a fusion protein and a yellow fluorescent protein(YFP). BMCs infected with the vector expressing YFP only was used as a control,Number of YFP-positive cells in the peripheral blood as well as general health condition of the mice was monitored.9 out a total of 12 mice in the AML1a group developed leukemia at 3-11 months.Autopsy of these mice revealed spleenon(?)agly and hepatomegaly with thymoma and lymph node enlargement.Bone marrow,spleen, lung,liver,kidney and thymus were infiltrated with leukemia cells.A further analysis revealed that the BMCs in mice with leukemia mainly exhibited 2 phenotypes.One was Sca-1~+ cytoplasmic CD3~+(cCD3~+),a common type of T-lymphoblastic leukemia. The other was CD3~+CD4~+CD8~+,a type of T lymphocytic leukemia.All mice received secondary transplantation rapidly developed lymphocytic leukemia.Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis.
3) Cell cycle of BMCs from the secondary recipient of leukemia cells was examined.Representative cycle distribution of the BMCs was shown that BMCs in the AML1a group obviously were of higher percentage in G0/G1 phase and lower percentage in S phase in comparison to the control group,which indicated that cell cycle of transduced AML1a hematopoietic cells was arrested in the G0/G1 phase AML1a may alter the process of normal cell proliferation and division and lead to the malignant transformation of hemotopoietic cells.
In conclusion,we transduced AML1a into BMMNC of mice by retrovirus and transplanted these cells into lethally-irretiated mice.As a result,this study has revealed,for the first time,a new effect of AML1a in leukemogenesis.It indicates that AML1a may play a critical role in leukemogenesis,especially in the development of lymphoid leukemia.In addition,lymphoblastic leukemia model established in this study may serve as a valuable tool for future studies.These results may also have implications in the treatment of leukemia.
PartⅡAnalysis of microRNAs expression profiles of acute promyelocytic leukemia cell line NB4 treated with all-trans retinoic acid
MicroRNAs(miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators to regulate diverse biological processes. Each miRNA can control hundreds of gene targets.Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes.Thus, miRNAs might prove useful in the diagnosis and treatment of cancer.To explore the profile of microRNAs in acute promyelocytic leukemia(APL) cell line NB4 treated with all-trans retinoic acid(ATRA) and to look for the microRNAs which affect on cell differentiation.The differentiation of NB4 cell treated with ATRA was verified by cell morphology and flow cytometry.The difference of microRNAs expression profiles of NB4 cells treated with ATRA at different time was analyzed by means of microRNAs array.Real-time RT-PCR was used to testify the change of microRNAs expression,and the target genes of microRNAs were predicted.NB4 cells can be induced to terminal differentiation after 3 days exposure to ATRA and the differentiation antigen CD11b increased significantly from 3%to 96%.Using microRNAs array,24 differentially expressed microRNAs were screened from 576 human microRNAs candidates in which,21 microRNAs were up-regulated and 3 down-regulated.Further more,by microRNA-target gene prediction assay,the predicted target genes of the 16 up-regulated microRNAs were in volved in leukemogenesis related HOX genes.The expression of the two most significantly up-regulated microRNAs(miR-210 and miR-342) was confirmed by real time RT-PCR.The ratio of the two microRNAs after normalization was 2.16 and 4.61 respectively.The differentially expressed microRNAs in ATRA treated NB4 cells may be associated with the differentiation.Furthermore,the change of microRNAs expression may provide basis for the control mode of repressing gene expression at post-transcription level.
细胞的增殖分化受到严格调控,转录因子协调细胞中多种基因的表达是其中重要的调控机制。造血细胞的增殖失控、分化能力的丧失将可能导致细胞的恶性转化及白血病的发生。因此,阐明转录因子的功能对于确定细胞增殖分化调控机制以及在细胞恶性转化中的作用具有重要意义。AML1也称之为PEBP2αB、CBFα2或RUNX1,是一种重要的转录调节因子。它在造血细胞中普遍表达,调节造血细胞的分化和增殖。
RUNX1/AML1通过选择性剪接产生至少三种异构体:AML1a、A.ML1b和AML1c。其中,AML1b与AML1c仅在氨基末端存在27个氨基酸残基的差异,均具有两个主要的功能结构域:DNA结合结构域和转录激活结构域,两者的功能基本相同,即我们通常所说的AML1。AML1a具有DNA结合结构域,但缺少转录激活结构域,因此通常被认为不具有转录激活功能,但由于其具有DNA结合结构域,可以与AML1b/1c竞争结合靶基因的DNA结合位点,导致靶基因不能正常转录,提示AML1a可能干扰AML1的转录调节功能。有证据表明,AML1a在急性髓系白血病(AML)中表达高于正常对照。我们的前期实验也已证实AML1a在急性白血病(AL)中的高表达,而且,AML1a可以抑制由AML1b介导的M-CSF受体(M-CSFR)启动子的转录,提示AML1a对AML1b具有拮抗作用。有学者认为转录激活区的缺失可能引起白血病的发生,但目前尚无此方面的报道。为了进一步研究AML1a在造血及白血病发病中的作用并探讨其作用机制,本文完成了下列研究工作:
1.AML1a表达载体的构建及病毒的制备
pMSCV-FLAG-AML1a-IRES-YFP质粒,将此质粒和pMSCV-IRES-YFP分别与辅助质粒pV Pack-Eco(含Env)、pV Pack-GP(含gal-pol)组合,通过磷酸钙沉淀法转染293T细胞,制备逆转录病毒。病毒感染3T3细胞,检测病毒滴度,并测定AML1a的表达情况。结果显示在感染的细胞中证实有AML1a和YFP的表达,所制备的病毒滴度满足后续实验的要求。
2.AML1a在小鼠造血细胞增殖分化异常及恶性转化中的作用
1) C57雄性小鼠的骨髓单个核细胞(BMMNC)通过逆转录病毒转导AML1a及空白对照后,置于含mSCF、mIL-3、mIL-6的IMDM培养液中进行培养,分别于感染后11天、23天和35天通过流式细胞仪检测确定各系表面标志的变化,结果显示感染AML1a的BMMNC中Sca-1及Thy1.2的表达均较对照组升高,提示AML1a可能将造血干细胞的分化阻滞于较为早期的淋巴细胞阶段。
2)将转导AML1a的C57小鼠的骨髓单个核细胞,尾静脉注射至致死剂量照射的C57雌性小鼠体内,单一转导YFP的小鼠作为对照。定期尾静脉取血,查血象及白细胞分类。血象有异常改变后,处死小鼠,观察有无肝、脾、淋巴结肿大,流式细胞术分析细胞表型变化。转导AML1a的12只C57小鼠中,有9只在移植后3-11个月相继死亡,解剖可见肝脾肿大,可伴有胸腺瘤和淋巴结肿大。病理可见骨髓、脾、肺、肾、胸腺或淋巴结可见大量白血病细胞浸润。流式分析显示白血病细胞主要有两个表型:Sca-1~+ cCD3~+和CD3~+CD4~+CD8~+,提示均为T-淋巴细胞白血病。发病小鼠脾细胞接种同类小鼠可再次发生表型相同的T-淋巴细胞白血病。以上结果表明AML1a在白血病的发生中发挥重要的作用,并且可能具备导致白血病的能力。
3)将AML1a发病小鼠脾细胞传代后取第二代发病鼠的骨髓细胞进行细胞周期测定。与对照组相比,发病小鼠骨髓中G0/G1细胞比例增高,S期细胞比例明显降低,提示AML1a将小鼠骨髓细胞阻滞于G0/G1期。此结果说明AML1a基因可能通过抑制细胞自G0/G1期向S期的转换,改变正常细胞增殖分裂的进程,导致造血细胞的恶性转化。
综上所述,本研究通过逆转录病毒转导AML1a至小鼠BMMNC中,并将其移植到致死剂量照射的小鼠体内,首次证实了AML1a的可以引起造血细胞恶性转化并导致白血病发生。初步推测AML1a可能在白血病特别是淋巴细胞白血病的发病中起重要作用。此外,我们的实验也为白血病的治疗提供了新的治疗靶点和动物模型。
第二部分全反式维甲酸处理NB4细胞的微小RNA表达谱分析
微小RNA(microRNA,miRNA)是一种非编码小分子RNA,可以作为基因的负调控因子调节多种生物进程。每一种微小RNA可以调控数百种靶基因。近来研究显示微小RNA的突变或者异常表达与人类许多肿瘤发生相关,它能够作为肿瘤抑制物或者癌基因发挥作用,因此可能有助于肿瘤的诊断和治疗。我们通过确定全反式维甲酸(ATRA)诱导急性早幼粒细胞白血病(APL)细胞系NB4细胞分化过程中微小RNA表达谱的变化,寻找可能对白血病细胞分化起作用的微小RNA。细胞形态学和流式细胞术验证ATRA处理NB4细胞的分化。利用微小RNA芯片技术,检测和分析NB4经ATRA处理不同时间点的微小RNA表达谱差异,实时定量RT-PCR技术验证微小RNA表达的变化,并对微小RNA进行靶基因预测。ATRA处理后的NB4细胞在形态上出现向粒细胞的分化,分析CD11b等髓系表面抗原的表达,ATRA处理0h及72h时CD11b的表达分别为3%和96%,进一步证实了NB4细胞的分化。基因芯片检测从576个候选的人类微小RNA中,筛选出24个差异表达微小RNA(表达上调的有21个,表达下调的有3个),其中,有16个微小RNA可以进行靶基因预测。这些miRNA的靶基因多涉及与白血病发生有关的同源盒基因家族成员。实时定量RT-PCR验证了2个显著上调微小RNA(miR-210和miR-342)表达的变化,两种微小RNA标准化后的比值分别为2.16和4.61。差异表达微小RNA可能与NB4的分化相关,同时也可能是转录后水平抑制基因表达调控方式的基础。
PartⅠThe role of AML1a in abnormal proliferation and differentiation and its significant function in malignant transformation of murine hematopoietic cells
Cell proliferation and differentiation are strictly regulated by intrinsic and extrinsic signals,in which coordination of a variety of gene expression by transcription factors is the most important mechanism.Uncontrolled proliferation and impaired differentiation of hematopoietic cells lead to leukemogenesis.Therefore,it is significant to clarify the functions of transcription factors for elucidating the mechanism of abnormal proliferation and differentiation,as well as leukemogenesis.
AML1,also called RUNX1,PEBP2αB or CBFα2,is a critical transcription factor in hematopoietic cell differentiation and proliferation.Its mutation and translocation lead to hematopoietic malignant.
From the AML1/RUNX1 gene,at least three isoforms,AML1a,AML1b and AML1c,are produced through alternative splicing.The proteins encoded by AML1b and AML1c have a RHD in the N-terminus and a transactivation domain in the C-terminus,which are often called AML1.In contrast,the protein encoded by AML1a has a RHD but lacks the transactivation domain.As such,the function of AML1 is believed to be mediated by AML1b and AML1c which are considered to have the same function.AML1a interferes with the function of AML1b/1c.It has been proved that AML1a is higher expressed in acute myeloid leukemia(AML) than in normal controls.Our previous study confirmed that AML1a expression in acute leukemia(AL) has a high level.In addition,AML1a can inhibit the transcription of M-CSFR promoter mediated by AML1b,suggesting that AML1a has the effect of antagonism to AML1b.Some scholars believe that loss of the transcription activation domain may cause leukemogenesis,but at present there is no report in this regard.In order to explore the role and mechanism of AML1a in abnormal hematopoesis and leukemogenesis,we completed the following work in this study:
1.The construction of AML1a expression vector and the production of retrovirus.
pMSCV-FLAG-AML1a-IRES-YFP was constructed.293T cells were transfected with pMSCV-IRES-YFP,pMSCV-FLAG-AML1a-IRES-YFP,the envelope-encoding plasmid pV Pack-Eco,or the packaging plasmid pV Pack-GP using a method of calcium phosphate precipitation to product retrovirus.The title of virus was measured using a bioassay with the 3T3 cell line.The expression of the fusion proteins in the tranduced cells was confirmed.The title of retrovirus can satisfy the continually experimental demand.
2.The role of AML1a in abnormal hematopoiesis and leukemogenesis.
1) Bone marrow mononuclear cells(BMMNCs) were infected with the retroviral vector MSCV expressing a FLAG-AML1a fusion protein and a yellow fluorescent protein(YFP).The cells were cultured in Iscove modified Dulbecco medium(IMDM) supplemented with murine IL-3(mIL-3),murine IL-6(mIL-6) and marine SCF (mSCF).The change of surface marker of each hematopoietic linage was analyzed by FACS in 11d,23d and 35d after transduction.The result showed that the expression of Sca-1 and Thy1.2 in AML1a transduced BMMNC was higher than that of the control,which indicating that AML1a may prone to block the differentiation of hematopoietic stem cell in the relatively early lymphocytic stage 2) BMMNCs from mice were transduced with AML1a and transplanted into lethally irradiated mice, which develop lymphoblastic leukemia after transplantation.Lethally irradiated female C57 mice received BMCs infected with the retroviral vector MSCV expressing a FLAG-AML1a fusion protein and a yellow fluorescent protein(YFP). BMCs infected with the vector expressing YFP only was used as a control,Number of YFP-positive cells in the peripheral blood as well as general health condition of the mice was monitored.9 out a total of 12 mice in the AML1a group developed leukemia at 3-11 months.Autopsy of these mice revealed spleenon(?)agly and hepatomegaly with thymoma and lymph node enlargement.Bone marrow,spleen, lung,liver,kidney and thymus were infiltrated with leukemia cells.A further analysis revealed that the BMCs in mice with leukemia mainly exhibited 2 phenotypes.One was Sca-1~+ cytoplasmic CD3~+(cCD3~+),a common type of T-lymphoblastic leukemia. The other was CD3~+CD4~+CD8~+,a type of T lymphocytic leukemia.All mice received secondary transplantation rapidly developed lymphocytic leukemia.Taken together, these results indicate that overexpression of AML1a may be an important contributing factor to leukemogenesis.
3) Cell cycle of BMCs from the secondary recipient of leukemia cells was examined.Representative cycle distribution of the BMCs was shown that BMCs in the AML1a group obviously were of higher percentage in G0/G1 phase and lower percentage in S phase in comparison to the control group,which indicated that cell cycle of transduced AML1a hematopoietic cells was arrested in the G0/G1 phase AML1a may alter the process of normal cell proliferation and division and lead to the malignant transformation of hemotopoietic cells.
In conclusion,we transduced AML1a into BMMNC of mice by retrovirus and transplanted these cells into lethally-irretiated mice.As a result,this study has revealed,for the first time,a new effect of AML1a in leukemogenesis.It indicates that AML1a may play a critical role in leukemogenesis,especially in the development of lymphoid leukemia.In addition,lymphoblastic leukemia model established in this study may serve as a valuable tool for future studies.These results may also have implications in the treatment of leukemia.
PartⅡAnalysis of microRNAs expression profiles of acute promyelocytic leukemia cell line NB4 treated with all-trans retinoic acid
MicroRNAs(miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators to regulate diverse biological processes. Each miRNA can control hundreds of gene targets.Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes.Thus, miRNAs might prove useful in the diagnosis and treatment of cancer.To explore the profile of microRNAs in acute promyelocytic leukemia(APL) cell line NB4 treated with all-trans retinoic acid(ATRA) and to look for the microRNAs which affect on cell differentiation.The differentiation of NB4 cell treated with ATRA was verified by cell morphology and flow cytometry.The difference of microRNAs expression profiles of NB4 cells treated with ATRA at different time was analyzed by means of microRNAs array.Real-time RT-PCR was used to testify the change of microRNAs expression,and the target genes of microRNAs were predicted.NB4 cells can be induced to terminal differentiation after 3 days exposure to ATRA and the differentiation antigen CD11b increased significantly from 3%to 96%.Using microRNAs array,24 differentially expressed microRNAs were screened from 576 human microRNAs candidates in which,21 microRNAs were up-regulated and 3 down-regulated.Further more,by microRNA-target gene prediction assay,the predicted target genes of the 16 up-regulated microRNAs were in volved in leukemogenesis related HOX genes.The expression of the two most significantly up-regulated microRNAs(miR-210 and miR-342) was confirmed by real time RT-PCR.The ratio of the two microRNAs after normalization was 2.16 and 4.61 respectively.The differentially expressed microRNAs in ATRA treated NB4 cells may be associated with the differentiation.Furthermore,the change of microRNAs expression may provide basis for the control mode of repressing gene expression at post-transcription level.
引文
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