FLT3靶向RNA干扰的抗白血病效应及NF-κB通路、SMRT在FLT3信号传导中的作用
摘要
急性髓细胞白血病(Acute Myelocytic Leukemia，AML)是造血系统的恶性侵袭性疾病，尽管诱导化疗能使70％以上的患者获得临床缓解，但大部分之后复发，AML患者长期存活率只在40％左右，更有效的治疗策略急待开发。
FMS样酪氨酸激酶3(FMS-like tyrosine kinase 3，FLT3)是Ⅲ型受体酪氨酸激酶(RTK)受体家族的成员，在骨髓中主要表达在CD34~+的造血干／祖细胞及树突状前体细胞上，配体介导的FLT3活化在造血原始细胞的增殖、分化中有重要的作用。然而超过70％的AML细胞表面表达有野生型FLT3、25～35％的表达有突变型FLT3，包括30％左右的FL73近膜结构域短串联重复突变(FLT3-ITD)和7％的酪氨酸激活结构域点突变(FLT3-TDK)。FLT3异常表达和患者的临床白细胞增多症、预后差密切相关。
FLT3作为AML治疗极有前景的分子靶标深受关注。目前已开发了几种FLT3抑制剂，但Ⅰ、Ⅱ期临床实验的结果不令人满意。首先没有一种抑制剂是真正FLT3特异性的，它们也可识别VEGF、c-KIT、FMS及PDGF等受体，临床毒副作用不可避免；另外FLT3-TDK各种点突变引发FLT3结构的细微变化，也导致了某些FLT3抑制剂的耐药问题。更重要的是AML的发病具多基因突变累加的特点，相关基因的研究远未完善；研究证实FLT3信号传导涉及许多中介分子，但大多数在此信号级联中的地位和作用还不清楚，进一步阐明FLT3与其它分子相互作用的机制，FLT3异常激活引发的下游信号途径，将为FLT3靶向治疗研究提供新的视野。
RNA干扰是FLT3靶向抑制的另一策略，它不仅为FLT3功能学研究提供了强有力的工具，也是AML极有前景的基因治疗手段。本研究中首先设计、合成了3条FLT3靶向的短发夹状干扰RNA(FL73-shRNA)，通过转染FLT3过表达的AML细胞株，评价其抑制效应，筛选出效率高的1条；随后体外实验中探讨了FLT3表达的下调对THP-1、HL-60细胞增殖，凋亡的作用。
目前对FLT3信号传导的研究，野生型主要集中在PI3K-AKT、RAS-MAPK、SRC通路上，ITD突变型还包括STAT5通路，对在AML发生、发展中有重要作用的核因子κ-B(Nuclear Factor Kappa B，NF-κB)通路、辅抑制子维甲酸／甲状腺受体沉默因子(Silencing Medtiator for Retinoic Acid and Thyroid Hormone Receptor，SMRT)，研究还少有涉及。NF-κB是一类核内转录因子，它通过调节cyclinD1，c-myc，Bcl-2等基因的转录，可正性调节细胞增殖、凋亡等过程；SMRT是真核生物基因转录抑制调节的基本成分，因此作者通过RNA干扰的方法下调FLT3表达，探讨NF-κB通路、SMRT在FLT3信号传递中的可能作用。并通过建立THP-1白血病细胞Nu／Nu皮下移植瘤模型，体内实验观察FLT3-shRNA干扰单独，或与NF-κB抑制剂Parthenolide(PN)、柔红霉素(Daunorubicin，DNR)联合作用时的抗白血病效应。
第一部分五种髓细胞白血病细胞株FLT3表达及FLT3-ITD突变的研究
方法
1．对5种髓细胞白血病细胞株THP-1、HL-60、K562、Dami及Meg-01，以RT-PCR法检测细胞FLT3 mRNA的表达。
2．FLT3在细胞中有胞浆蛋白和跨膜蛋白两种存在形式，FLT3跨膜蛋白是其活性形式。以Western blotting检测FLT3总蛋白的表达，以流式细胞术(FCM)法检测FLT3跨膜蛋白的表达。
3．鉴于FLT3-ITD突变型与其它类型的FLT3受体激活方式、信号传导途径都有不同，提取各细胞基因组DNA，PCR扩增其易发生I7D突变的近膜区序列，PCR产物连入T-载体并测序，检测是否存在FLT3-I7D突变。
结果
1．5种细胞株中，只有THP-1、HL-60细胞中测及FLT3 mRNA的表达，相对于自身GAPDH mRNA的含量分别是0.83±0.07、0.48±0.05。
2．Western blotting结果显示THP-1、HL-60细胞中有FLT3蛋白的表达，FCM显示FLT3跨膜蛋白在于HP-1、HL-60细胞的表达率分别是53.55±4.44％、27.57±3.42％，前者属强阳性表达、后者属中阳性表达。
3．核酸测序结果显示THP-1、HL-60细胞中扩得的JM区基因片段均为329bp，与野生型FLT3的长度同；经与基因库FLT3序列比对同源性达98％以上，提示均不存在FLT3-ITD突变。
第二部分FT73靶向短发夹状干扰RNA体外转录合成、筛选及作用
方法
1．设计、体外转录合成3条FLT3-shRNA、1条阴性对照shRNA(NC-shRNA)，确定其浓度。
2．用25nM的3种FLT3-shRNA、NC-shRNA转染THP-1细胞，24h、48h及72h分别收获细胞，RT-PCR法检测各组FLT3 mRNA的表达，以GAPDH作内参计算其mRNA的相对含量，初步确定各shRNA的干扰效率。
3．将对mRNA抑制效率较高的FLT3-shRNA1，以5nM、10nM、15nM、20nM、25nM浓度转染THP-1细胞，48h收获细胞测FLT3 mRNA，评价浓度效应关系。
4．将对mRNA抑制效率达50％以上的FLT3-shRNA1、shRNA3转染THP-1细胞，48h、72h收获细胞，以FCM测FLT3跨膜蛋白的表达，72h以Western blotting测FLT3总蛋白的表达。
5．根据上述结果，用15nM的FLT3-shRNA1转染HL-60细胞，48h以RT-PCR测FLT3 mRNA表达，72h以FCM、Western blotting测FLT3蛋白的表达。
结果
1．成功合成3条FLT3-shRNA、1条NC-shRNA。2.25nM的NC-shRNA对FLT3 mRNA表达无影响，所设计的shRNA中，FLT3-shRNA1、shRNA3对其mRNA抑制率超过50％，shRNA1的抑制率强于shRNA3的(p＜0.001)；shRNA1作用最强时间是48h，抑制率是72.95±2.07％；shRNA3是72h，抑制率是54.95±2.07％。
3．在5nM～15nM范围内FLT3-shRNA1对其mRNA抑制率随浓度增加而增大，15nM转染48h抑制率是71.60±1.46％；20nM、25nM的与15nM无差异(P＞0.05)。4.15nM的NC-shRNA对FLT3跨膜蛋白的细胞表达率无影响。FLT3-shRNA1、shRNA3转染48h、72h，表达率均有明显下降，shRNA1作用强于shRNA3的，72h强于48h的(均P＜0.001)；shRNA1作用72h对FLT3跨膜蛋白的抑制率是79.67±0.66％。Western blotting结果也支持shRNA1转染72h对FLT3蛋白的抑制作用更强。
5．15nM的FLT3-shRNA1转染HL-60细胞，48h对FLT3 mRNA的抑制率达(81.66±10.25)％。FCM测72h对FLT3跨膜蛋白的抑制率达(76.76±11.23)％，Western blotting结果也证实72h蛋白表达被明显抑制。
第三部分FLT3靶向RNA干扰抑制THP-1、HL-60细胞增殖、促进凋亡的实验研究
方法
以下试验均设PBS对照组FLT3-shRNAi组，NC-shRNA组及，分别转染以15nM的FLT3-shRNA1、NC-shRNA，或处理以同体积数的PBS。
1．96孔板中THP-1、HL-60细胞以4×10~4／ml密度转染FLT3-shRNAl，培养8天，每天取3孔加入CCK-8，测定细胞增殖活力并绘制增殖曲线。细胞再以4×10~5／ml密度转染，24h、48h、72h测定增殖活力并计算增殖抑制率。
2．如上述处理细胞，48h各组细胞经PI染色，用FCM法测定细胞周期分布。
3．转染48h分别以Annexin V-FITC法、DNA Ladder法检测THP-1、HL-60细胞凋亡状况，另THP-1细胞以TUNEL原位酶标记法检测凋亡。
4．转染48h和72h，用RT-PCR法检测cyclin D1、cyclinA的mRNA表达；提取细胞总蛋白，用Western blotting检测其蛋白表达。
结果
1．与PBS对照相比，NC-shRNA转染THP-1、HL-60后细胞增殖曲线变化不大，但FLT3-shRNA转染后，两细胞增殖曲线均明显低平，缺乏指数增殖特征。转染指数增殖期的细胞后，细胞增殖活力明显下降，在THP-1抑制率是36.66±3.67％、在HL-60是33.10±3.43％。
2．FLT3-shRNA1转染THP-1、HL-60细胞48h，细胞周期都出现G_0／G_1期细胞比例的增加(P＜0.01)，S期细胞比例的下降(P＜0.05)，即出现了G_0／G_1期至S期的阻滞。
3．FLT3-shRNA1转染48h，Annexin V-FITC检测THP-1、HL-60细胞的早期凋亡率都有明显增加(P＜0.05)，两细胞株上都检到了凋亡细胞典型的梯状条带(DNA Ladder)；通过TUNEL细胞原位杂交观察到THP-1晚期凋亡细胞比例的明显增加(P＜0.001)。
4．由FLT3-shRNA1诱导FLT3表达的下调，可导致THP-1、HL-60细胞cyclin D1表达的下降，与NC-shRNA组比有显著差异(P＜0.01)，48h对其mRNA的作用较大，在两细胞抑制率分别是37.09±3.76％、63.69±21.26％；Western blotting结果示48h有cyclin D1蛋白表达的下调，但72h更为明显。另外cyclin A的mRNA、蛋白表达在这一过程中无变化。
第四部分NF-κB通路、核辅抑制子SMRT在FLT3信号传递中的作用
方法
1．THP-1细胞株中用RT-PCR检测NF-κB家族的P65、阻遏物IκB mRNA的表达，用免疫组化、Western blotting法检测其蛋白的表达。
2．96孔板中用0μM～20nM范围内的系列浓度的NF-κB抑制剂Parthenolide(PN)，处理4×10~5／ml密度的THP-1细胞，12h、24h用CCK-8法检测细胞增殖活力，计算细胞增殖半数抑制浓度(IC_(50))。后根据上述结果，用6μM的PN作用于4×10~4／ml密度的THP-1细胞，共培养6天，绘制细胞增殖曲线。
3．试验共分5组，分别处理以PBS、NC-shRNA、FLT3-shRNAi、PN及FLT3-shRNAi＋PN，转染组均转以15nM的shRNA，作用终时间48h；PN组处理以6μM的PN，作用终时间24h。各组细胞收获后，用RT-PCR检测P65、IκB、cyclinD1及SMRTmRNA的表达；用Western blotting检测细胞总蛋白、核蛋白的表达。
4．FLT3-shRNA1转染细胞48h，分别处理以系列浓度的PN，12h、24h用CCK-8法检测细胞增殖活力，计算IC_(50)。
5．THP-1细胞分别处理以PBS、FLT3-shRNAi、PN及FLT3-shRNAi＋PN，细胞收获后以FCM检测细胞周期分布，以Annexin V-FITC法检测细胞凋亡。
结果
1．RT-PCR结果显示THP-1细胞有P65、IκB的mRNA表达，免疫组化示P65、IκB蛋白均主要表达在胞浆内；Western blotting示P65在胞浆、胞核均有表达，而IκB在核内没有表达。
2．PN对THP-1细胞增殖有抑制作用，存在着剂量．效应关系。6μM的PN处理后细胞的增殖曲线明显低平。PN作用后细胞IκB表达明显增加，P65的mRNA、总细胞蛋白表达没有变化，核蛋白明显下降，cyclin D1也有下降；在这一过程中SMRT mRNA、总细胞蛋白表达没有变化，但核蛋白明显上调。
3．FLT3-shRNA1转染THP-1细胞48h，P65、IκB的mRNA、总细胞蛋白表达水平没有变化，P65核蛋白的表达下降，cyclin D1表达也随之下降。SMRT mRNA、总细胞蛋白表达也没有变化，但核蛋白表达有明显的上调。
4．FLT3-shRNA1下调FLT3表达后，THP-1细胞对PN的敏感性增加，12h对PN的IC_(50)在6μM～8μM，24h的IC_(50)在4μM～6μM，与PBS处理对照相比均有下降。再者FLT3-shRNAi、PN联合作用后，比其单独作用时细胞周期中G_0／G_1期细胞比例更高(P＜0.05)，S期也更低(P＜0.05)；Annexin V测细胞早期凋亡率更高(P＜0.05)。分子水平检测显示P65核蛋白在联合组表达量低于PN组，更低于FLT3-shRNAi组；联合组IκB表达的上升与PN组无差异；联合组cyclinD1表达也有更明显的下降。
第五部分Nu／Nu裸鼠移植瘤模型建立及FLT3靶向RNA干扰体内抗白血病的效应
方法
1．指数生长期的THP-1细胞，以1×10~7／只剂量接种到Nu／Nu裸鼠右腋皮下，建立THP-1细胞裸鼠皮下移植瘤模型。
2．当THP-1移植瘤体积长到100～300mm~3，随机分6组，5只／组，各组按既定方案腹腔注射给药，分别处理以PBS(1组)、FLT3-shRNAi(2组)、PN(3组)、shRNAi＋PN(4组)、柔红霉素(DNR，5组)、shRNAi＋DNR(6组)，总计15天。
3．隔日测瘤体积，裸鼠体重；实验结束时处死小鼠称瘤重，计算抑瘤率。
4．对取出的瘤组织，用TUNEL检测瘤细胞凋亡，用免疫组化、RT-PCR、Westernblotting分别检测FLT3、P65、IκB、cyclin D1、SMRT的表达。
结果
1．成功建立了THP-1细胞Nu／Nu裸鼠移植瘤模型，成瘤率为94.44％。
2．腹腔注射FLT3-shRNAi，裸鼠THP-1移植瘤生长受到抑制，其抑制率为28.95％。瘤组织中FLT3的表达明显下降，TUNEL测瘤组织中凋亡细胞比例增加(P＜0.05)；Western blotting显示瘤组织P65核蛋白表达下降，cyclin D1下降，胞核内SMRT表达上升。
3．腹腔注射PN后裸鼠THP-1移植瘤生长受到抑制，其抑制率为32.46％，瘤组织IκB表达上调，胞核内p65表达下降，cyclin D1也下降，胞核内SMRT表达上升。注射FLT3-shRNAi＋PN的裸鼠移植瘤生长被抑制得更为明显，抑瘤率达49.12％(P＜0.05)，瘤组织中凋亡细胞比例上升得更多(P＜0.01)，胞核内p65下降更明显，cyclin D1下降也更明显，但SMRT的上升并不比PN单独治疗组的更高。
4．腹腔注射DNR对裸鼠THP-1移植瘤的抑制率达71.93％，注射FLT3-shRNAi＋DNR的为82.46％。
结论
1．5种髓系白血病细胞株THP-1、HL-60、K562、Dami及Meg-01中，只有THP-1、HL-60有FLT3受体的持续表达，它们都不存有FLT3-ITD突变。
2．所设计、体外转录合成的FLT3-shRNA1，在THP-1、HL-60细胞中可有效地抑制FLT3表达。
3．用FLT3-shRNA1特异性地下调FLT3表达后，可抑制THP-1、HL-60细胞增殖，促进凋亡，体外实验初步证实具有抗白血病的作用。cyclin D1表达下调，细胞周期出现G_0／G_1期到S期的阻滞，是FLT3表达下调后AML细胞增殖抑制的原因之一。
4．THP-1细胞中有NF-κB信号通路的持续激活，NF-κB抑制剂PN可通过增加IκB表达，阻逆P65的核转运，有效地抑制NF-κB信号传递，从而对细胞的增殖起抑制作用。
5．FLT3下调可通过诱导P65核蛋白表达下降，下调NF-κB通路活性，从而抑制细胞增殖。SMRT也是介导FLT3信号传递的重要因子，FLT3-shRNAi可通过增加SMRT的核内表达，增强其转录抑制作用。
6．FLT3-shRNAi可增强THP-1细胞对PN的药物敏感性，FLT3-shRNAi与PN联合作用可通过更强地抑制NF-κB信号通路，体外实验证实有更好的抗白血病效应。
7．裸鼠腹腔注射FLT3-shRNA1可通过下调FLT3表达，PN可通过抑制NF-κB活性，对THP-1细胞移植瘤组织有轻、中度的抑制作用，当二者联用时效果更为显著；FLT3-shRNA1与DNR联用时可增加DNR的抑瘤效果，体内实验证实FLT3靶向RNA干扰具有抗白血病作用。
Acute myelocytic leukemia (AML) is an aggressive hematological malignancy. Presently induction therapies are capable of achieving clinical remission in up to 70% of AML patients; however a large fraction relapse and long-term survival rates still remain 40%, reflecting the need for more effective therapeutic strategies.
FLT3 is a member of the class iii receptor tyrosine kinase family, is preferentially expressed in normal human bone marrow selectively in CD34+ hematopoietic stem/progenitor cells and in dendritic cell progenitors. Ligandmediated activation of the FLT3 receptor is important for normal proliferation of primitive hematopoietic cells. However more than 70% of AML blasts express wild-type FLT3, whereas 25% to 35% carry FLT3 mutations including an internal tandem duplication (FLT3-ITD) within the juxtamembrane domain in nearly 30% of patients and possess and point mutations within the activating loop (FLT3-TDK) in 7% of patients. Aberrant activation of FLT3 is closely associated with leukocytosis and poor prognosis of AML patients.
FLT3 receptor has received considerable attention as an attractive molecular target in AML. To date, several different FLT3 kinase inhibitors have been developed, however, results of phaseⅠ/Ⅱof clinical trials seem unsatisfying. None of these inhibitors are truly FLT3 specific because they often recognize other molecular targets such as VEGF-R_2, c-KIT, FMS and PDGF-R, therefore normal cells may experience significant cytotoxicity, and clinical side effects may be unavoidable. The sensitivity of FLT3 inhibitors to the FLT3 structure confers susceptibility to minor structural changes induced by point mutations within the activating loop, and acquired resistance appears. More important, studies of Flt3 signaling suggest involvement of a variety of intermediates, but for most their place in signaling pathways and identification of their physiologic role is unclear. Thus, an improves understanding of how FLT3 interacts with other molecules, as well as how aberrant activation of FLT3 triggers downstream intracellular signaling pathways, will provide more insights for the development of FLT3 targeting therapy.
Another methodology for molecularly-targeted inhibition to FLT3 is RNA interference (RNAi), which not only provides a powerful system for analysis of FLT3 mechanism but also illustrate the potential benefit of therapeutic approached. Thus, in this study, we designed and synthesized 3 FLT3 targeting small hairpin RNAs (FLT3-shRNA) and screened the most effective one through evaluating their effects on FLT3 suppression, then in vitro studies we explored the efficacy of downregulation of FLT3 induced by RNAi to cell proliferation, apoptosis in THP-1, HL-60 cell lines.
Analyses of signaling passway in the wild-type FLT3 have focused largely on the PI3K-Akt and Ras-MAPK pathways; whereas in the ITD-type FLT3 also involved STAT5 pathways. The aberrant expression of NF-κB, corepressor SMRT(Silencing Medtiator for Retinoic Acid and Thyroid Hormone Receptor) plays an important role in the pathogenesis of AML, However, their roles in FLT3 signaling cascades has not been determined. NF-κB family, taken as transcription factors, participates in cellular processes such as proliferation, apoptosis through regulating transcription of cyclinD1, c-myc, Bcl-2, and so on. SMRT has been identified as fundamental components in the regulation of eukaryotic gene transcription, so we studies whether downregulation of FLT3 influence NF-κB passway, and SMRT in AML cell lines. Under these conditions, we established the subcutaneous xenograft model of THP-1 leukemic tumors in Nu/Nu mice, detected the antitumor activity of FLT3-shRNAi used alone, or combined with NF-κB inhibitor, chemotherapeutic drug in vivo.
PartⅠExpression of FLT3 in Five AML Cell Lines and Detection of FLT3-ITD Mutation
Methods
1. Semi-quantitative RT-PCR was used to examine the FLT3 mRNA in five human AML cell lines that is THP-1, HL-60, K562, Dami and Meg-01.
2. Western blotting was used to detect the FLT3 whole protein, and Flow cytometer (FCM) was used to detect the part of FLT3 transmembrance-protein.
3. In order to determine whether FLT3-ITD Mutation exists in those AML cells, genomic DNA was extracted and was employed for amplifying the sequences of JM domain; their PCR fragments were ligated into T-vector and sequenced.
Results
1. Just THP-1, HL-60 were detected with basal expression of FLT3 mRNA, which related contents to GAPDH were 0.83±0.07%, 0.48±0.05%, respectively.
2. Western blotting showed that just THP-1, HL-60 cells expressed FLT3 protein, FCM showed the percentages of FLT3 positive cells were 53.55±4.44% in THP-1 cells, 27.57±3.42% in HL-60 cell, namely, THP-1 expressed "strong" level as well as HL-60 expressed "intermediate" level.
3. Results from sequencing showed the expected nucleotide size in JM region from THP-1, HL-60 cells were 329bp, the same size as that in wild-type FLT3, the concordance rate with the published data in GenBank (NM-004119) were over 98%, indicated no FLT3-ITD Mutation exists in both THP-1 and HL-60 cells..
PartⅡin vitro Transcription Synthesis, Screening and Efficacy of FLT3 Targeting Short Hairpin RNA
Methods
1. Designed and synthesized three FLT3 targeted shRNAs (FLT3-shRNA), as well as one non-related sequences shRNA (NC-shRNA) by in vitro transcription system, their concentrations were determined.
2. Firstly THP-1 cells were transfected with 25nM of 3 types of FLT3-shRNA as well as NC-shRNA. For 24h, 48h and 72h cells were collected, FLT3 mRNA was detected by RT-PCR, and the related content of FLT3 mRNA to GAPDH was calculated.
3. THP-1 cells were transfected with different contentions FLT3-shRNA1 which was identified with the most effective rate, which is 5nM, 10nM, 15nM, 20nM, 25nM. For 48h cells were collected and FLT3 mRNA was detected.
4. According to the results of RT-PCR, FLT3-shRNA1, shRNA3 were used to transfect THP-1 cells. For 48h, 72h FCM was used to tested expression of FLT3 transmembrance protein, at 72h Western blotting was used to detect the expression of FLT3 whole protein.
5. HL-60 cells were transfected with 15nM FLT3-shRNA1 depending on the results above, For 48h FLT3 mRNA was detected using RT-PCT, 72h FLT3 protein was detected using FCM and Western blotting.
Results
1. Three FLT3-shRNA and NC-shRNA were synthesized successfully.
2. The expression of FLT3 mRNA was not affected by NC-shRNA, whereas it was suppressed by FLT3-shRNA1, shRNA3 over 50%. the inhibiting rates of shRNA1 were higher than those of shRNA3 (P＜0.001); The most effective time for shRNA1 was 48h with inhibiting rate 72.95±2.07%, as well as for shRNA3 was 48h with inhibiting rate 54.95±2.07%.
3. FLT3-shRNA1 suppressed the expression of FLT3 mRNA in a Concentrationdependent manner within 5nM～15nM, the inhibiting rate of 15nM was 71.60±1.46%.
4. Transfected with NC-shRNA didn't affect the expression of FLT3 protein, but FLT3-shRNA1, shRNA3 caused special degradation of the FLT3 transmembrance protein for both 48h and 72h detected by FCM, the more effective one was shRNA1 (P＜0.001), the more effective time was 72h (P＜0.001) with inhibiting rate 79.67±0.66%. And the same result was seen in Western blotting assay.
5. HL-60 cells were transfected with 15nM FLT3-shRNA1, for 48h its inhibiting rate to FLT3 mRNA was 81.66±10.25%, for 72 its inhibiting rate to FLT3 transmembrance protein was 76.76±11.23%, and result form Western blotting confirmed that the expression of FLT3 protein was significantly suppressed too.
PartⅢshRNAi-induced FLT3 Suppression Inhibits Myeloproliferation and Induces Apoptosis of AML Cells
Methods
All tests below were settled for 3 groups, treated with 15nM of FLT3-shRNA, 15nM of NC-shRNA, or the same vol of PBS, respectively.
1. THP-1 and HL-60 cells (4×10~4/ml) in 96-well plates were transfected with FLT3-shRNA and cultured for 8 days, 3 wells were added into CCK-8 and cell viabilities were tested for every day, then growth curves were drawn. Furthermore, cells (4×10~5/ml) were treated as above described, and cell viabilities were tested for 24h, 48h, 72h, and inhibiting rates were calculated.
2. Cells were treated as above; cells were stained with PI and analyzed cell cycle by FCM for 48h
3. For 48h after transfection, cell apoptosis was analyzed by Annexin V-FITC combined with PI staining, DNA ladder in both THP-1 and HL-60 cells, as well as by TUNEL assay in THP-1 cells.
4. For 48h, 72h after transfection, the mRNA level of cyclin D1, cyclin A was determined using RT-PCR, and the protein level was determined in whole cell extracts using Western blotting.
Results
1. Compared with that treated with PBS, little changes in growth curves were found in cells transtected with NC-shRNA in both THP-1 and HL-60 cells, but growth curves transfected with FLT3-shRNAi were low, fiat which lacked the typical character of exponential growth. Furthermore, an obvious loss of cell viability was detected in cells transfected with FLT3-shRNA, the inhibiting rate was 36.66±3.67% in THP-1 cells, 33.10±3.43% in HL-60 cells.
2. FLT3-shRNA transfected cells showed an increase in the percentage of cells in the phase G_0/G_1 (P＜0.01) and a decrease in the percentage of cells in the phase S (P＜ 0.05) in both THP-1 and HL-60 cells.
3. Transfection with FLT3-shRNA for 48h resulted in obvious increase of early apoptosis cell detected by Annexin V-FITC staining (P＜0.05), DNA Ladder which is characteristic of apoptotic cell were seen in both THP-1 and HL-60. And the result of TUNEL in THP-1 cells also showed an increase ofapoptotic cells (P＜0.001).
4. shRNAi-induced suppression of FLT3 resulted in the downregulation of cyclin D1 expression (P＜0.01). The more effective time for cyclin D1 mRNA was 48h after transfection with the inhibiting rate was 37.09±3.76%, 63.69±21.26% in THP-1, HL-60 respectively. Western blotting showed an obvious decrease in cyclin D1 protein level, and 72h was more significant. Moreover the expression level of cyclin A wasn't affected by FLT3-shRNAi.
PartⅣThe Roles of NF-κB Pathway, Corepressor SMRT in the Signaling Transduction of FLT3
Methods
1. RT-PCR was used to examine the P65, IκB mRNA in THP-1 cell lines, immunohistochemistry and Western blotting was used to detect their protein expression.
2. THP-1 (4×10~5/ml) in 96-well plates treated with different concentrations of Pathernolide (PN) within 0μM～20nM, cell viabilities were tested by CCK-8 and inhibitory concentration 50% (IC_(50)) were calculated for 12h, 24h. According the results above, cells (4×10~4/ml)were treated with 6μM of PN and cultured for 6 days, 3 wells were tested for cell viabilities for every day, and growth curves were drawn.
3. All tests below were settled for 5 groups, treated with PBS (group 1), 15nM of NC-shRNA(group 2), 15nM of FLT3-shRNA(group 3), 6μM of PN(group 4), 15nM of FLT3-shRNA + 6μM of PN(group 5), respectively. All the final time of shRNA was 48h, as well as the final time of PN was 24h. For P65、IκB, cyclinD1 and SMRT, RT-PCR was used to detected their mRNA expression, Western blotting was used to detected their protein expression in whole cell extracts or nuclear extracts.
4. After transfected with 15nM of FLT3-shRNA1 for 48h, THP-1 cell were treated by designed concentrations of PN as described above, the IC_(50) were determined for another 12h, 24h,
5. THP-1 cells were treated with PBS, FLT3-shRNAi, PN, FLT-shRNAi+PN, respectively. After cell collection FCM was used to determine the distribution of cell cycle, Annexin V-FITC staining was employed to analyze cell apoptosis.
Results
1. RT-PCR showed that P65, IκB mRNA expressed in THP-1 cells, Immunocytochemistry also showed P65, IκB protein, both mainly in cytoplasms, whereas Western blotting indicated P65 expressed not only in cytoplasm but also in nuclear, as well as IκB just in cytoplasm.
2. Growth inhibition was seen in cells treated with PN in a concentration-dependant manner, the growth curve treated with 6μM of PN was lower. After 6μM of PN treatment, the IκB mRNA, protein increased obviously, P65 protein in nuclear extracts decreased, cyclin D1 decreased, as well as SMRT in nuclear extracts increased.
3. The P65, IκB mRNA, protein in the whole cell extracts weren't affected by transfected with FLT3-shRNA for 48h, but P65 protein in the nuclear extracts decreased, cyclin D1 also downregulated as well. The expression of SMRT mRNA, protein in the whole cell extracts weren't affected but SMRT protein in the nuclear extracts decreased.
4. The sensitivity of PN of THP-1 cells was increased due to the downregulation of FLT3, the IC50 for 12h was 6μM～8μM, for 24h was 4μM～6μM, both lower than those in controls. Cells treated with FLT3-shRNAi or PN respectively showed an increase in the percentage of cells in phase G_0/G_1 (P＜0.05), and a decrease in the percentage of cells in phase S (P＜0.05), the results were more prominence while cells treated with FLT3-shRNAi+PN (P＜0.05), Likely, the increased levels of early apoptosis were seen when cells treated with FLT3-shRNAi or PN, the results were more prominence when treated the both (P＜0.05), Results of Western blotting indicated that P65 nuclear protein, cyclin D1 protein decreased in cells treated with FLT3-shRNAi, PN alone, and more decreased when treated FLT3-shRNAi+PN.
PartⅤEstablishment of THP-1 Cell Xenograft Tumor Model in Nu/Nu Mice and Antileukemic Efficacy of FLT3 Targeting RNA Interference in vivo
Methods
1. THP-1 cells at exponential phase (1×10~7 cells per mouse) were implanted S.C. into the right flank of Nu/Nu mice, established xenograft tumor model of THP-1 cell line.
2. Treatments were initiated when tumors were 100～300mm~3, mice were randomly assigned into six groups (5 mice per group). FLT3-shRNAi, PN, daunorubicin (DNR) were administered i.p. alone or combinations as protocol, all for 15days.
3. Tumor volumes and body. weights were assessed every 2 day following the treatment. The mice were sacrificed 2 days after the last treatment, tumor massed was weighted, percentage of tumor growth inhibition was calculated...
4. For tumor tissues in each group, TUNEL methods were adopted to assay cell apoptosis, immunohistochemistry, RT-PCR, and Western blotting were employed to determine the mRNA, protein expression of FLT3, P65, IκB, cyclin D1, SMRT, respectively.
Results
1. Treatment with FLT3-shRNAi led to tumor growth inhibition in Nu/Nu mice bearing THP-1 xenograft tumor, the percentage of growth inhibition was 28.95%, the percentage of apoptotic cell increased (P＜0.05). Western blotting showed FLT3 expression decreased in tumor tissues, P65 protein in nuclear as well as cyclin D1 decreased, SMRT protein in nuclear increased.
2. Treatment with PN led to tumor growth inhibition in Nu/Nu mice, the percentage of growth inhibition was 32.46%, IκB expression in tumor tissues upregulated as well as p65 protein in nuclear, cyclin D1 protein downregulated, and SMRT protein in nuclear upregulated at the same time. More important, Treatment with FLT3-shRNAi+PN led to more efficiency on tumor inhibition which the percentage of growth inhibition was up to 49.12% (P＜0.05), more apoptotic cells in tumor tissues,(P＜0.01). and the decrease of p65 protein in nuclear, cyclin D1 was more prominence, however the increase of SMRT in nuclear wasn't more than that treated with PN alone.
3. DNR had strong effects on tumor inhibition (72.05%), and when it combined with FLT3-shRNAi, the percentage of inhibition increased to 82.52%.
Conclusions
1. Just THP-1, HL-60 cells constitutively express FLT3 in the five AML cell lines, and no FLT3-ITD mutation exists in them.
2. FLT3-shRNA1 can effectively down-regulate FLT3 expression.
3. shRNAi-mediated FLT3 suppression inhibits myeloproliferation, induces cell apoptosis in both THP-1, HL-60 cells, thus tentatively confirms the anti-leukemic efficacy of the FLT3 targeting RNA interference in vitro studies. FLT3 suppression also down-regulates the expression of cyclin D1 in mRNA and protein level, and obviouly inhibits cell cycle from phase G_0/G_1 to phase S, which illustrates one of the important reasons for the myeloproliferation inhibition.
4. Constitutively activated NF-κB signaling pathway exists in the THP-1 cells, Parthernolide(PN), a NF-κB inhibitor, effectively inhibits NF-κB signaling by upregulate the IκB expression and reverse P65 nuclear transportation, thus, suppress the cell growth.
5. shRNAi-mediated FLT3 suppression can inhibit NF-κB activation through downregulates P65 protein in nuclear, thus, cell growth inhibition appears. Besides, SMRT protein in nuclear upregulates, and enhancement of transcriptional repression may be another reason of growth inhibition.
6. shRNAi-mediated FLT3 suppression increases the sensitivity of THP-1 to treatment with PN, the combination of FLT3-shRNAi with PN has a cooperation-suppression of NF-κB passway, and illustrates the cooperation effect of anti-leukemic efficacy in vitro studies.
FMS样酪氨酸激酶3(FMS-like tyrosine kinase 3，FLT3)是Ⅲ型受体酪氨酸激酶(RTK)受体家族的成员，在骨髓中主要表达在CD34~+的造血干／祖细胞及树突状前体细胞上，配体介导的FLT3活化在造血原始细胞的增殖、分化中有重要的作用。然而超过70％的AML细胞表面表达有野生型FLT3、25～35％的表达有突变型FLT3，包括30％左右的FL73近膜结构域短串联重复突变(FLT3-ITD)和7％的酪氨酸激活结构域点突变(FLT3-TDK)。FLT3异常表达和患者的临床白细胞增多症、预后差密切相关。
FLT3作为AML治疗极有前景的分子靶标深受关注。目前已开发了几种FLT3抑制剂，但Ⅰ、Ⅱ期临床实验的结果不令人满意。首先没有一种抑制剂是真正FLT3特异性的，它们也可识别VEGF、c-KIT、FMS及PDGF等受体，临床毒副作用不可避免；另外FLT3-TDK各种点突变引发FLT3结构的细微变化，也导致了某些FLT3抑制剂的耐药问题。更重要的是AML的发病具多基因突变累加的特点，相关基因的研究远未完善；研究证实FLT3信号传导涉及许多中介分子，但大多数在此信号级联中的地位和作用还不清楚，进一步阐明FLT3与其它分子相互作用的机制，FLT3异常激活引发的下游信号途径，将为FLT3靶向治疗研究提供新的视野。
RNA干扰是FLT3靶向抑制的另一策略，它不仅为FLT3功能学研究提供了强有力的工具，也是AML极有前景的基因治疗手段。本研究中首先设计、合成了3条FLT3靶向的短发夹状干扰RNA(FL73-shRNA)，通过转染FLT3过表达的AML细胞株，评价其抑制效应，筛选出效率高的1条；随后体外实验中探讨了FLT3表达的下调对THP-1、HL-60细胞增殖，凋亡的作用。
目前对FLT3信号传导的研究，野生型主要集中在PI3K-AKT、RAS-MAPK、SRC通路上，ITD突变型还包括STAT5通路，对在AML发生、发展中有重要作用的核因子κ-B(Nuclear Factor Kappa B，NF-κB)通路、辅抑制子维甲酸／甲状腺受体沉默因子(Silencing Medtiator for Retinoic Acid and Thyroid Hormone Receptor，SMRT)，研究还少有涉及。NF-κB是一类核内转录因子，它通过调节cyclinD1，c-myc，Bcl-2等基因的转录，可正性调节细胞增殖、凋亡等过程；SMRT是真核生物基因转录抑制调节的基本成分，因此作者通过RNA干扰的方法下调FLT3表达，探讨NF-κB通路、SMRT在FLT3信号传递中的可能作用。并通过建立THP-1白血病细胞Nu／Nu皮下移植瘤模型，体内实验观察FLT3-shRNA干扰单独，或与NF-κB抑制剂Parthenolide(PN)、柔红霉素(Daunorubicin，DNR)联合作用时的抗白血病效应。
第一部分五种髓细胞白血病细胞株FLT3表达及FLT3-ITD突变的研究
方法
1．对5种髓细胞白血病细胞株THP-1、HL-60、K562、Dami及Meg-01，以RT-PCR法检测细胞FLT3 mRNA的表达。
2．FLT3在细胞中有胞浆蛋白和跨膜蛋白两种存在形式，FLT3跨膜蛋白是其活性形式。以Western blotting检测FLT3总蛋白的表达，以流式细胞术(FCM)法检测FLT3跨膜蛋白的表达。
3．鉴于FLT3-ITD突变型与其它类型的FLT3受体激活方式、信号传导途径都有不同，提取各细胞基因组DNA，PCR扩增其易发生I7D突变的近膜区序列，PCR产物连入T-载体并测序，检测是否存在FLT3-I7D突变。
结果
1．5种细胞株中，只有THP-1、HL-60细胞中测及FLT3 mRNA的表达，相对于自身GAPDH mRNA的含量分别是0.83±0.07、0.48±0.05。
2．Western blotting结果显示THP-1、HL-60细胞中有FLT3蛋白的表达，FCM显示FLT3跨膜蛋白在于HP-1、HL-60细胞的表达率分别是53.55±4.44％、27.57±3.42％，前者属强阳性表达、后者属中阳性表达。
3．核酸测序结果显示THP-1、HL-60细胞中扩得的JM区基因片段均为329bp，与野生型FLT3的长度同；经与基因库FLT3序列比对同源性达98％以上，提示均不存在FLT3-ITD突变。
第二部分FT73靶向短发夹状干扰RNA体外转录合成、筛选及作用
方法
1．设计、体外转录合成3条FLT3-shRNA、1条阴性对照shRNA(NC-shRNA)，确定其浓度。
2．用25nM的3种FLT3-shRNA、NC-shRNA转染THP-1细胞，24h、48h及72h分别收获细胞，RT-PCR法检测各组FLT3 mRNA的表达，以GAPDH作内参计算其mRNA的相对含量，初步确定各shRNA的干扰效率。
3．将对mRNA抑制效率较高的FLT3-shRNA1，以5nM、10nM、15nM、20nM、25nM浓度转染THP-1细胞，48h收获细胞测FLT3 mRNA，评价浓度效应关系。
4．将对mRNA抑制效率达50％以上的FLT3-shRNA1、shRNA3转染THP-1细胞，48h、72h收获细胞，以FCM测FLT3跨膜蛋白的表达，72h以Western blotting测FLT3总蛋白的表达。
5．根据上述结果，用15nM的FLT3-shRNA1转染HL-60细胞，48h以RT-PCR测FLT3 mRNA表达，72h以FCM、Western blotting测FLT3蛋白的表达。
结果
1．成功合成3条FLT3-shRNA、1条NC-shRNA。2.25nM的NC-shRNA对FLT3 mRNA表达无影响，所设计的shRNA中，FLT3-shRNA1、shRNA3对其mRNA抑制率超过50％，shRNA1的抑制率强于shRNA3的(p＜0.001)；shRNA1作用最强时间是48h，抑制率是72.95±2.07％；shRNA3是72h，抑制率是54.95±2.07％。
3．在5nM～15nM范围内FLT3-shRNA1对其mRNA抑制率随浓度增加而增大，15nM转染48h抑制率是71.60±1.46％；20nM、25nM的与15nM无差异(P＞0.05)。4.15nM的NC-shRNA对FLT3跨膜蛋白的细胞表达率无影响。FLT3-shRNA1、shRNA3转染48h、72h，表达率均有明显下降，shRNA1作用强于shRNA3的，72h强于48h的(均P＜0.001)；shRNA1作用72h对FLT3跨膜蛋白的抑制率是79.67±0.66％。Western blotting结果也支持shRNA1转染72h对FLT3蛋白的抑制作用更强。
5．15nM的FLT3-shRNA1转染HL-60细胞，48h对FLT3 mRNA的抑制率达(81.66±10.25)％。FCM测72h对FLT3跨膜蛋白的抑制率达(76.76±11.23)％，Western blotting结果也证实72h蛋白表达被明显抑制。
第三部分FLT3靶向RNA干扰抑制THP-1、HL-60细胞增殖、促进凋亡的实验研究
方法
以下试验均设PBS对照组FLT3-shRNAi组，NC-shRNA组及，分别转染以15nM的FLT3-shRNA1、NC-shRNA，或处理以同体积数的PBS。
1．96孔板中THP-1、HL-60细胞以4×10~4／ml密度转染FLT3-shRNAl，培养8天，每天取3孔加入CCK-8，测定细胞增殖活力并绘制增殖曲线。细胞再以4×10~5／ml密度转染，24h、48h、72h测定增殖活力并计算增殖抑制率。
2．如上述处理细胞，48h各组细胞经PI染色，用FCM法测定细胞周期分布。
3．转染48h分别以Annexin V-FITC法、DNA Ladder法检测THP-1、HL-60细胞凋亡状况，另THP-1细胞以TUNEL原位酶标记法检测凋亡。
4．转染48h和72h，用RT-PCR法检测cyclin D1、cyclinA的mRNA表达；提取细胞总蛋白，用Western blotting检测其蛋白表达。
结果
1．与PBS对照相比，NC-shRNA转染THP-1、HL-60后细胞增殖曲线变化不大，但FLT3-shRNA转染后，两细胞增殖曲线均明显低平，缺乏指数增殖特征。转染指数增殖期的细胞后，细胞增殖活力明显下降，在THP-1抑制率是36.66±3.67％、在HL-60是33.10±3.43％。
2．FLT3-shRNA1转染THP-1、HL-60细胞48h，细胞周期都出现G_0／G_1期细胞比例的增加(P＜0.01)，S期细胞比例的下降(P＜0.05)，即出现了G_0／G_1期至S期的阻滞。
3．FLT3-shRNA1转染48h，Annexin V-FITC检测THP-1、HL-60细胞的早期凋亡率都有明显增加(P＜0.05)，两细胞株上都检到了凋亡细胞典型的梯状条带(DNA Ladder)；通过TUNEL细胞原位杂交观察到THP-1晚期凋亡细胞比例的明显增加(P＜0.001)。
4．由FLT3-shRNA1诱导FLT3表达的下调，可导致THP-1、HL-60细胞cyclin D1表达的下降，与NC-shRNA组比有显著差异(P＜0.01)，48h对其mRNA的作用较大，在两细胞抑制率分别是37.09±3.76％、63.69±21.26％；Western blotting结果示48h有cyclin D1蛋白表达的下调，但72h更为明显。另外cyclin A的mRNA、蛋白表达在这一过程中无变化。
第四部分NF-κB通路、核辅抑制子SMRT在FLT3信号传递中的作用
方法
1．THP-1细胞株中用RT-PCR检测NF-κB家族的P65、阻遏物IκB mRNA的表达，用免疫组化、Western blotting法检测其蛋白的表达。
2．96孔板中用0μM～20nM范围内的系列浓度的NF-κB抑制剂Parthenolide(PN)，处理4×10~5／ml密度的THP-1细胞，12h、24h用CCK-8法检测细胞增殖活力，计算细胞增殖半数抑制浓度(IC_(50))。后根据上述结果，用6μM的PN作用于4×10~4／ml密度的THP-1细胞，共培养6天，绘制细胞增殖曲线。
3．试验共分5组，分别处理以PBS、NC-shRNA、FLT3-shRNAi、PN及FLT3-shRNAi＋PN，转染组均转以15nM的shRNA，作用终时间48h；PN组处理以6μM的PN，作用终时间24h。各组细胞收获后，用RT-PCR检测P65、IκB、cyclinD1及SMRTmRNA的表达；用Western blotting检测细胞总蛋白、核蛋白的表达。
4．FLT3-shRNA1转染细胞48h，分别处理以系列浓度的PN，12h、24h用CCK-8法检测细胞增殖活力，计算IC_(50)。
5．THP-1细胞分别处理以PBS、FLT3-shRNAi、PN及FLT3-shRNAi＋PN，细胞收获后以FCM检测细胞周期分布，以Annexin V-FITC法检测细胞凋亡。
结果
1．RT-PCR结果显示THP-1细胞有P65、IκB的mRNA表达，免疫组化示P65、IκB蛋白均主要表达在胞浆内；Western blotting示P65在胞浆、胞核均有表达，而IκB在核内没有表达。
2．PN对THP-1细胞增殖有抑制作用，存在着剂量．效应关系。6μM的PN处理后细胞的增殖曲线明显低平。PN作用后细胞IκB表达明显增加，P65的mRNA、总细胞蛋白表达没有变化，核蛋白明显下降，cyclin D1也有下降；在这一过程中SMRT mRNA、总细胞蛋白表达没有变化，但核蛋白明显上调。
3．FLT3-shRNA1转染THP-1细胞48h，P65、IκB的mRNA、总细胞蛋白表达水平没有变化，P65核蛋白的表达下降，cyclin D1表达也随之下降。SMRT mRNA、总细胞蛋白表达也没有变化，但核蛋白表达有明显的上调。
4．FLT3-shRNA1下调FLT3表达后，THP-1细胞对PN的敏感性增加，12h对PN的IC_(50)在6μM～8μM，24h的IC_(50)在4μM～6μM，与PBS处理对照相比均有下降。再者FLT3-shRNAi、PN联合作用后，比其单独作用时细胞周期中G_0／G_1期细胞比例更高(P＜0.05)，S期也更低(P＜0.05)；Annexin V测细胞早期凋亡率更高(P＜0.05)。分子水平检测显示P65核蛋白在联合组表达量低于PN组，更低于FLT3-shRNAi组；联合组IκB表达的上升与PN组无差异；联合组cyclinD1表达也有更明显的下降。
第五部分Nu／Nu裸鼠移植瘤模型建立及FLT3靶向RNA干扰体内抗白血病的效应
方法
1．指数生长期的THP-1细胞，以1×10~7／只剂量接种到Nu／Nu裸鼠右腋皮下，建立THP-1细胞裸鼠皮下移植瘤模型。
2．当THP-1移植瘤体积长到100～300mm~3，随机分6组，5只／组，各组按既定方案腹腔注射给药，分别处理以PBS(1组)、FLT3-shRNAi(2组)、PN(3组)、shRNAi＋PN(4组)、柔红霉素(DNR，5组)、shRNAi＋DNR(6组)，总计15天。
3．隔日测瘤体积，裸鼠体重；实验结束时处死小鼠称瘤重，计算抑瘤率。
4．对取出的瘤组织，用TUNEL检测瘤细胞凋亡，用免疫组化、RT-PCR、Westernblotting分别检测FLT3、P65、IκB、cyclin D1、SMRT的表达。
结果
1．成功建立了THP-1细胞Nu／Nu裸鼠移植瘤模型，成瘤率为94.44％。
2．腹腔注射FLT3-shRNAi，裸鼠THP-1移植瘤生长受到抑制，其抑制率为28.95％。瘤组织中FLT3的表达明显下降，TUNEL测瘤组织中凋亡细胞比例增加(P＜0.05)；Western blotting显示瘤组织P65核蛋白表达下降，cyclin D1下降，胞核内SMRT表达上升。
3．腹腔注射PN后裸鼠THP-1移植瘤生长受到抑制，其抑制率为32.46％，瘤组织IκB表达上调，胞核内p65表达下降，cyclin D1也下降，胞核内SMRT表达上升。注射FLT3-shRNAi＋PN的裸鼠移植瘤生长被抑制得更为明显，抑瘤率达49.12％(P＜0.05)，瘤组织中凋亡细胞比例上升得更多(P＜0.01)，胞核内p65下降更明显，cyclin D1下降也更明显，但SMRT的上升并不比PN单独治疗组的更高。
4．腹腔注射DNR对裸鼠THP-1移植瘤的抑制率达71.93％，注射FLT3-shRNAi＋DNR的为82.46％。
结论
1．5种髓系白血病细胞株THP-1、HL-60、K562、Dami及Meg-01中，只有THP-1、HL-60有FLT3受体的持续表达，它们都不存有FLT3-ITD突变。
2．所设计、体外转录合成的FLT3-shRNA1，在THP-1、HL-60细胞中可有效地抑制FLT3表达。
3．用FLT3-shRNA1特异性地下调FLT3表达后，可抑制THP-1、HL-60细胞增殖，促进凋亡，体外实验初步证实具有抗白血病的作用。cyclin D1表达下调，细胞周期出现G_0／G_1期到S期的阻滞，是FLT3表达下调后AML细胞增殖抑制的原因之一。
4．THP-1细胞中有NF-κB信号通路的持续激活，NF-κB抑制剂PN可通过增加IκB表达，阻逆P65的核转运，有效地抑制NF-κB信号传递，从而对细胞的增殖起抑制作用。
5．FLT3下调可通过诱导P65核蛋白表达下降，下调NF-κB通路活性，从而抑制细胞增殖。SMRT也是介导FLT3信号传递的重要因子，FLT3-shRNAi可通过增加SMRT的核内表达，增强其转录抑制作用。
6．FLT3-shRNAi可增强THP-1细胞对PN的药物敏感性，FLT3-shRNAi与PN联合作用可通过更强地抑制NF-κB信号通路，体外实验证实有更好的抗白血病效应。
7．裸鼠腹腔注射FLT3-shRNA1可通过下调FLT3表达，PN可通过抑制NF-κB活性，对THP-1细胞移植瘤组织有轻、中度的抑制作用，当二者联用时效果更为显著；FLT3-shRNA1与DNR联用时可增加DNR的抑瘤效果，体内实验证实FLT3靶向RNA干扰具有抗白血病作用。
Acute myelocytic leukemia (AML) is an aggressive hematological malignancy. Presently induction therapies are capable of achieving clinical remission in up to 70% of AML patients; however a large fraction relapse and long-term survival rates still remain 40%, reflecting the need for more effective therapeutic strategies.
FLT3 is a member of the class iii receptor tyrosine kinase family, is preferentially expressed in normal human bone marrow selectively in CD34+ hematopoietic stem/progenitor cells and in dendritic cell progenitors. Ligandmediated activation of the FLT3 receptor is important for normal proliferation of primitive hematopoietic cells. However more than 70% of AML blasts express wild-type FLT3, whereas 25% to 35% carry FLT3 mutations including an internal tandem duplication (FLT3-ITD) within the juxtamembrane domain in nearly 30% of patients and possess and point mutations within the activating loop (FLT3-TDK) in 7% of patients. Aberrant activation of FLT3 is closely associated with leukocytosis and poor prognosis of AML patients.
FLT3 receptor has received considerable attention as an attractive molecular target in AML. To date, several different FLT3 kinase inhibitors have been developed, however, results of phaseⅠ/Ⅱof clinical trials seem unsatisfying. None of these inhibitors are truly FLT3 specific because they often recognize other molecular targets such as VEGF-R_2, c-KIT, FMS and PDGF-R, therefore normal cells may experience significant cytotoxicity, and clinical side effects may be unavoidable. The sensitivity of FLT3 inhibitors to the FLT3 structure confers susceptibility to minor structural changes induced by point mutations within the activating loop, and acquired resistance appears. More important, studies of Flt3 signaling suggest involvement of a variety of intermediates, but for most their place in signaling pathways and identification of their physiologic role is unclear. Thus, an improves understanding of how FLT3 interacts with other molecules, as well as how aberrant activation of FLT3 triggers downstream intracellular signaling pathways, will provide more insights for the development of FLT3 targeting therapy.
Another methodology for molecularly-targeted inhibition to FLT3 is RNA interference (RNAi), which not only provides a powerful system for analysis of FLT3 mechanism but also illustrate the potential benefit of therapeutic approached. Thus, in this study, we designed and synthesized 3 FLT3 targeting small hairpin RNAs (FLT3-shRNA) and screened the most effective one through evaluating their effects on FLT3 suppression, then in vitro studies we explored the efficacy of downregulation of FLT3 induced by RNAi to cell proliferation, apoptosis in THP-1, HL-60 cell lines.
Analyses of signaling passway in the wild-type FLT3 have focused largely on the PI3K-Akt and Ras-MAPK pathways; whereas in the ITD-type FLT3 also involved STAT5 pathways. The aberrant expression of NF-κB, corepressor SMRT(Silencing Medtiator for Retinoic Acid and Thyroid Hormone Receptor) plays an important role in the pathogenesis of AML, However, their roles in FLT3 signaling cascades has not been determined. NF-κB family, taken as transcription factors, participates in cellular processes such as proliferation, apoptosis through regulating transcription of cyclinD1, c-myc, Bcl-2, and so on. SMRT has been identified as fundamental components in the regulation of eukaryotic gene transcription, so we studies whether downregulation of FLT3 influence NF-κB passway, and SMRT in AML cell lines. Under these conditions, we established the subcutaneous xenograft model of THP-1 leukemic tumors in Nu/Nu mice, detected the antitumor activity of FLT3-shRNAi used alone, or combined with NF-κB inhibitor, chemotherapeutic drug in vivo.
PartⅠExpression of FLT3 in Five AML Cell Lines and Detection of FLT3-ITD Mutation
Methods
1. Semi-quantitative RT-PCR was used to examine the FLT3 mRNA in five human AML cell lines that is THP-1, HL-60, K562, Dami and Meg-01.
2. Western blotting was used to detect the FLT3 whole protein, and Flow cytometer (FCM) was used to detect the part of FLT3 transmembrance-protein.
3. In order to determine whether FLT3-ITD Mutation exists in those AML cells, genomic DNA was extracted and was employed for amplifying the sequences of JM domain; their PCR fragments were ligated into T-vector and sequenced.
Results
1. Just THP-1, HL-60 were detected with basal expression of FLT3 mRNA, which related contents to GAPDH were 0.83±0.07%, 0.48±0.05%, respectively.
2. Western blotting showed that just THP-1, HL-60 cells expressed FLT3 protein, FCM showed the percentages of FLT3 positive cells were 53.55±4.44% in THP-1 cells, 27.57±3.42% in HL-60 cell, namely, THP-1 expressed "strong" level as well as HL-60 expressed "intermediate" level.
3. Results from sequencing showed the expected nucleotide size in JM region from THP-1, HL-60 cells were 329bp, the same size as that in wild-type FLT3, the concordance rate with the published data in GenBank (NM-004119) were over 98%, indicated no FLT3-ITD Mutation exists in both THP-1 and HL-60 cells..
PartⅡin vitro Transcription Synthesis, Screening and Efficacy of FLT3 Targeting Short Hairpin RNA
Methods
1. Designed and synthesized three FLT3 targeted shRNAs (FLT3-shRNA), as well as one non-related sequences shRNA (NC-shRNA) by in vitro transcription system, their concentrations were determined.
2. Firstly THP-1 cells were transfected with 25nM of 3 types of FLT3-shRNA as well as NC-shRNA. For 24h, 48h and 72h cells were collected, FLT3 mRNA was detected by RT-PCR, and the related content of FLT3 mRNA to GAPDH was calculated.
3. THP-1 cells were transfected with different contentions FLT3-shRNA1 which was identified with the most effective rate, which is 5nM, 10nM, 15nM, 20nM, 25nM. For 48h cells were collected and FLT3 mRNA was detected.
4. According to the results of RT-PCR, FLT3-shRNA1, shRNA3 were used to transfect THP-1 cells. For 48h, 72h FCM was used to tested expression of FLT3 transmembrance protein, at 72h Western blotting was used to detect the expression of FLT3 whole protein.
5. HL-60 cells were transfected with 15nM FLT3-shRNA1 depending on the results above, For 48h FLT3 mRNA was detected using RT-PCT, 72h FLT3 protein was detected using FCM and Western blotting.
Results
1. Three FLT3-shRNA and NC-shRNA were synthesized successfully.
2. The expression of FLT3 mRNA was not affected by NC-shRNA, whereas it was suppressed by FLT3-shRNA1, shRNA3 over 50%. the inhibiting rates of shRNA1 were higher than those of shRNA3 (P＜0.001); The most effective time for shRNA1 was 48h with inhibiting rate 72.95±2.07%, as well as for shRNA3 was 48h with inhibiting rate 54.95±2.07%.
3. FLT3-shRNA1 suppressed the expression of FLT3 mRNA in a Concentrationdependent manner within 5nM～15nM, the inhibiting rate of 15nM was 71.60±1.46%.
4. Transfected with NC-shRNA didn't affect the expression of FLT3 protein, but FLT3-shRNA1, shRNA3 caused special degradation of the FLT3 transmembrance protein for both 48h and 72h detected by FCM, the more effective one was shRNA1 (P＜0.001), the more effective time was 72h (P＜0.001) with inhibiting rate 79.67±0.66%. And the same result was seen in Western blotting assay.
5. HL-60 cells were transfected with 15nM FLT3-shRNA1, for 48h its inhibiting rate to FLT3 mRNA was 81.66±10.25%, for 72 its inhibiting rate to FLT3 transmembrance protein was 76.76±11.23%, and result form Western blotting confirmed that the expression of FLT3 protein was significantly suppressed too.
PartⅢshRNAi-induced FLT3 Suppression Inhibits Myeloproliferation and Induces Apoptosis of AML Cells
Methods
All tests below were settled for 3 groups, treated with 15nM of FLT3-shRNA, 15nM of NC-shRNA, or the same vol of PBS, respectively.
1. THP-1 and HL-60 cells (4×10~4/ml) in 96-well plates were transfected with FLT3-shRNA and cultured for 8 days, 3 wells were added into CCK-8 and cell viabilities were tested for every day, then growth curves were drawn. Furthermore, cells (4×10~5/ml) were treated as above described, and cell viabilities were tested for 24h, 48h, 72h, and inhibiting rates were calculated.
2. Cells were treated as above; cells were stained with PI and analyzed cell cycle by FCM for 48h
3. For 48h after transfection, cell apoptosis was analyzed by Annexin V-FITC combined with PI staining, DNA ladder in both THP-1 and HL-60 cells, as well as by TUNEL assay in THP-1 cells.
4. For 48h, 72h after transfection, the mRNA level of cyclin D1, cyclin A was determined using RT-PCR, and the protein level was determined in whole cell extracts using Western blotting.
Results
1. Compared with that treated with PBS, little changes in growth curves were found in cells transtected with NC-shRNA in both THP-1 and HL-60 cells, but growth curves transfected with FLT3-shRNAi were low, fiat which lacked the typical character of exponential growth. Furthermore, an obvious loss of cell viability was detected in cells transfected with FLT3-shRNA, the inhibiting rate was 36.66±3.67% in THP-1 cells, 33.10±3.43% in HL-60 cells.
2. FLT3-shRNA transfected cells showed an increase in the percentage of cells in the phase G_0/G_1 (P＜0.01) and a decrease in the percentage of cells in the phase S (P＜ 0.05) in both THP-1 and HL-60 cells.
3. Transfection with FLT3-shRNA for 48h resulted in obvious increase of early apoptosis cell detected by Annexin V-FITC staining (P＜0.05), DNA Ladder which is characteristic of apoptotic cell were seen in both THP-1 and HL-60. And the result of TUNEL in THP-1 cells also showed an increase ofapoptotic cells (P＜0.001).
4. shRNAi-induced suppression of FLT3 resulted in the downregulation of cyclin D1 expression (P＜0.01). The more effective time for cyclin D1 mRNA was 48h after transfection with the inhibiting rate was 37.09±3.76%, 63.69±21.26% in THP-1, HL-60 respectively. Western blotting showed an obvious decrease in cyclin D1 protein level, and 72h was more significant. Moreover the expression level of cyclin A wasn't affected by FLT3-shRNAi.
PartⅣThe Roles of NF-κB Pathway, Corepressor SMRT in the Signaling Transduction of FLT3
Methods
1. RT-PCR was used to examine the P65, IκB mRNA in THP-1 cell lines, immunohistochemistry and Western blotting was used to detect their protein expression.
2. THP-1 (4×10~5/ml) in 96-well plates treated with different concentrations of Pathernolide (PN) within 0μM～20nM, cell viabilities were tested by CCK-8 and inhibitory concentration 50% (IC_(50)) were calculated for 12h, 24h. According the results above, cells (4×10~4/ml)were treated with 6μM of PN and cultured for 6 days, 3 wells were tested for cell viabilities for every day, and growth curves were drawn.
3. All tests below were settled for 5 groups, treated with PBS (group 1), 15nM of NC-shRNA(group 2), 15nM of FLT3-shRNA(group 3), 6μM of PN(group 4), 15nM of FLT3-shRNA + 6μM of PN(group 5), respectively. All the final time of shRNA was 48h, as well as the final time of PN was 24h. For P65、IκB, cyclinD1 and SMRT, RT-PCR was used to detected their mRNA expression, Western blotting was used to detected their protein expression in whole cell extracts or nuclear extracts.
4. After transfected with 15nM of FLT3-shRNA1 for 48h, THP-1 cell were treated by designed concentrations of PN as described above, the IC_(50) were determined for another 12h, 24h,
5. THP-1 cells were treated with PBS, FLT3-shRNAi, PN, FLT-shRNAi+PN, respectively. After cell collection FCM was used to determine the distribution of cell cycle, Annexin V-FITC staining was employed to analyze cell apoptosis.
Results
1. RT-PCR showed that P65, IκB mRNA expressed in THP-1 cells, Immunocytochemistry also showed P65, IκB protein, both mainly in cytoplasms, whereas Western blotting indicated P65 expressed not only in cytoplasm but also in nuclear, as well as IκB just in cytoplasm.
2. Growth inhibition was seen in cells treated with PN in a concentration-dependant manner, the growth curve treated with 6μM of PN was lower. After 6μM of PN treatment, the IκB mRNA, protein increased obviously, P65 protein in nuclear extracts decreased, cyclin D1 decreased, as well as SMRT in nuclear extracts increased.
3. The P65, IκB mRNA, protein in the whole cell extracts weren't affected by transfected with FLT3-shRNA for 48h, but P65 protein in the nuclear extracts decreased, cyclin D1 also downregulated as well. The expression of SMRT mRNA, protein in the whole cell extracts weren't affected but SMRT protein in the nuclear extracts decreased.
4. The sensitivity of PN of THP-1 cells was increased due to the downregulation of FLT3, the IC50 for 12h was 6μM～8μM, for 24h was 4μM～6μM, both lower than those in controls. Cells treated with FLT3-shRNAi or PN respectively showed an increase in the percentage of cells in phase G_0/G_1 (P＜0.05), and a decrease in the percentage of cells in phase S (P＜0.05), the results were more prominence while cells treated with FLT3-shRNAi+PN (P＜0.05), Likely, the increased levels of early apoptosis were seen when cells treated with FLT3-shRNAi or PN, the results were more prominence when treated the both (P＜0.05), Results of Western blotting indicated that P65 nuclear protein, cyclin D1 protein decreased in cells treated with FLT3-shRNAi, PN alone, and more decreased when treated FLT3-shRNAi+PN.
PartⅤEstablishment of THP-1 Cell Xenograft Tumor Model in Nu/Nu Mice and Antileukemic Efficacy of FLT3 Targeting RNA Interference in vivo
Methods
1. THP-1 cells at exponential phase (1×10~7 cells per mouse) were implanted S.C. into the right flank of Nu/Nu mice, established xenograft tumor model of THP-1 cell line.
2. Treatments were initiated when tumors were 100～300mm~3, mice were randomly assigned into six groups (5 mice per group). FLT3-shRNAi, PN, daunorubicin (DNR) were administered i.p. alone or combinations as protocol, all for 15days.
3. Tumor volumes and body. weights were assessed every 2 day following the treatment. The mice were sacrificed 2 days after the last treatment, tumor massed was weighted, percentage of tumor growth inhibition was calculated...
4. For tumor tissues in each group, TUNEL methods were adopted to assay cell apoptosis, immunohistochemistry, RT-PCR, and Western blotting were employed to determine the mRNA, protein expression of FLT3, P65, IκB, cyclin D1, SMRT, respectively.
Results
1. Treatment with FLT3-shRNAi led to tumor growth inhibition in Nu/Nu mice bearing THP-1 xenograft tumor, the percentage of growth inhibition was 28.95%, the percentage of apoptotic cell increased (P＜0.05). Western blotting showed FLT3 expression decreased in tumor tissues, P65 protein in nuclear as well as cyclin D1 decreased, SMRT protein in nuclear increased.
2. Treatment with PN led to tumor growth inhibition in Nu/Nu mice, the percentage of growth inhibition was 32.46%, IκB expression in tumor tissues upregulated as well as p65 protein in nuclear, cyclin D1 protein downregulated, and SMRT protein in nuclear upregulated at the same time. More important, Treatment with FLT3-shRNAi+PN led to more efficiency on tumor inhibition which the percentage of growth inhibition was up to 49.12% (P＜0.05), more apoptotic cells in tumor tissues,(P＜0.01). and the decrease of p65 protein in nuclear, cyclin D1 was more prominence, however the increase of SMRT in nuclear wasn't more than that treated with PN alone.
3. DNR had strong effects on tumor inhibition (72.05%), and when it combined with FLT3-shRNAi, the percentage of inhibition increased to 82.52%.
Conclusions
1. Just THP-1, HL-60 cells constitutively express FLT3 in the five AML cell lines, and no FLT3-ITD mutation exists in them.
2. FLT3-shRNA1 can effectively down-regulate FLT3 expression.
3. shRNAi-mediated FLT3 suppression inhibits myeloproliferation, induces cell apoptosis in both THP-1, HL-60 cells, thus tentatively confirms the anti-leukemic efficacy of the FLT3 targeting RNA interference in vitro studies. FLT3 suppression also down-regulates the expression of cyclin D1 in mRNA and protein level, and obviouly inhibits cell cycle from phase G_0/G_1 to phase S, which illustrates one of the important reasons for the myeloproliferation inhibition.
4. Constitutively activated NF-κB signaling pathway exists in the THP-1 cells, Parthernolide(PN), a NF-κB inhibitor, effectively inhibits NF-κB signaling by upregulate the IκB expression and reverse P65 nuclear transportation, thus, suppress the cell growth.
5. shRNAi-mediated FLT3 suppression can inhibit NF-κB activation through downregulates P65 protein in nuclear, thus, cell growth inhibition appears. Besides, SMRT protein in nuclear upregulates, and enhancement of transcriptional repression may be another reason of growth inhibition.
6. shRNAi-mediated FLT3 suppression increases the sensitivity of THP-1 to treatment with PN, the combination of FLT3-shRNAi with PN has a cooperation-suppression of NF-κB passway, and illustrates the cooperation effect of anti-leukemic efficacy in vitro studies.
引文
24. Meierhoff G, Dehmel U, Gruss HJ, et al. Expression of FLT3 receptor and FLT3-1igand in human leukemia-lymphoma cell lines. Leukemia, 1995, 9(8): 1368-1372.
25．J．萨姆布鲁克和 DW．拉赛尔著．分子克隆实验指南，黄培堂等译．科学出版社，2002年8月出版；第三版．
26．卢圣栋．现代分子生物学实验技术．中国协和医科大学出版社，1999年12月出版；第二版．
27. Levis M, Murphy KM, Pham R, et al. Intemal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood, 2005; 106(2): 673-680.
28．王莉红，周春林，张新伟，等．FLT3基因内部串联重复突变与急性白血病的关系及临床意义．中华血液学杂志，2004；25(7)：393-396．
29. Wodnar-Filipowicz A. Flt3 ligand: role in control of hematopoietic and immune functions of the bone marrow. News Physiol Sci, 2003; 18: 247-251.
30. Tumer AM, Lin NL, Issarachai S, et al. FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells. Blood, 88(9): 3383-3390.
31. Schnittger S, Schoch C, Dugas M, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood, 2002; 100(1): 59-66.
32. Ishiko J, Mizuki M, Matsumura I, et al. Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant. Oncogene, 2005; 24(55): 8144-8153.
33. Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000; 404(6775): 293-296.
34. Zhang J, Hua ZC. Targeted gene silencing by small interfering RNA-based knock-down technology. Curr Pharm Biotechnol, 2004, 5(1): 1-7.
35. Chakraborty C. Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Current drug targets, 2007; 8(3): 469-482.
36. Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in 275(17): 12470-12474.
78. Jonas BA, Privalsky ML. SMRT and N-CoR corepressors are regulated by distinct kinase signaling pathways. J Biol Chem, 2004; 279(52): 54676-54686.
79. Ko HM, Jung HH, Seo KH, et al. Platelet-activating factor-induced NF-kappa B activation enhances VEGF expression through a decrease in p53 activity. FEBS Lett, 2006; 580(13): 3006-3012.
80. Putral LN, Gu W, et al. RNA interference for the treatment of cancer. Drug News Perspect, 2006; 19(6): 317-324.
81. Stephanie Filleur, Aurelie Courtin, Slimane Ait-Si-Ali, et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res, 2003, 63(7): 3919-3922.
82. Sung-Suk Chae, Ji-Hye Paik, Henry Furneaux. Requirement for sphingosine 1-phosphat.e receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference. J Clin Invest, 2004, 114(8): 1082-1089.
83. Oka D, Nishimura K, Shiba M, et al. Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation ofNF-kappaB. Int J Cancer, 2007; 120(12): 2576-2525.
84. Taguchi T, Takao T, Iwasaki Y, et al. Suppressive effects of dehydroepiandrosterone and the nuclear factor-kappaB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo. J Endocrinol, 2006; 188(2): 321-331.
85．孙以方．医学实验动物学．兰州大学出版社，2005年8月出版；第一版．
86. Sausville EA, Burger AM. Contributions of human tumor xenografts to anticancer drug development. Cancer Res, 2006; 66(7): 3351-3354.
87. Zhang SY, Zhu J, Chen GQ, et al. Establishment of a human acute promyelocytic leukemia-ascites model in SCID mice. Blood, 1996; 87(8): 3404-3409.
88. Gao L, Chen L, Fei XH, et al. STI571 combined with vincristine greatly suppressed the tumor formation of multidrug-resistant K562 cells in a human-nude mice xenograft model. Chin Med J (Engl), 2006; 119(11): 911-918.
1. Stone RM, O'Donnell MR, Sekeres MA. Acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2004;: 98-117.
2. Cucuianu A. Pathogenic pathways in acute myeloid leukemias. Rom J Physiol. 2004; 41(1-2): 109-118.
3. Clark JJ, Smith FO, Arceci RJ, et al. Update in childhood acute myeloid leukemia: recent developments in the molecular basis of disease and novel therapies. Curr Opin Hematol. 2003; 10(1): 31-39.
4. Larson RA, Le Beau MM. Therapy-related myeloid leukaemia: a model for leukemogenesis in humans. Chem Biol Interact, 2005; 153-154: 187-95.
5. Smith BD, Bao T, Karp JE. New concepts in the treatment of acute myeloid malignancies: selected pathways for targeted therapy. J Biol Regul Homeost Agents, 2005; 19(1-2): 23-32.
6. John AM, Thomas NS, Mufti GJ, et al. Targeted therapies in myeloid leukemia. Semin Cancer Biol, 2004; 14(1): 41-62.
7．邱慧颖，王健民，薛永权．急性髓系白血病细胞及分子遗传学研究进展．中华血液学杂志，2004；25(12)：761-764．
8．陈赛娟，陈丽娟，周光飚．白血病基因产物靶向治疗的基础和临床研究．中国实验血液学杂志，2005；13(1)：1-8．
9. Cilloni D, Messa E, Messa F, et al. Genetic abnormalities as targets for molecular therapies in myelodysplastic syndromes. Ann N Y Acad Sci, 2006; 1089: 411-423.
10. Inokuchi K. Chronic myelogenous leukemia: from molecular biology to clinical aspects and novel targeted therapies. J Nippon Med Sch, 2006; 73(4): 178-192.
11. Kovitz C, Kantarjian H, Garcia-Manero G, et al. Myelodysplastic syndromes and acute leukemia developing after imatinib mesylate therapy for chronic myeloid leukemia. Blood, 2006; 108(8): 2811-2813.
12. Cortes JE, Talpaz M, O'Brien S, et al. Staging of chronic myeloid leukemia in the imatinib era: an evaluation of the World Health Organization proposal. Cancer,
25．J．萨姆布鲁克和 DW．拉赛尔著．分子克隆实验指南，黄培堂等译．科学出版社，2002年8月出版；第三版．
26．卢圣栋．现代分子生物学实验技术．中国协和医科大学出版社，1999年12月出版；第二版．
27. Levis M, Murphy KM, Pham R, et al. Intemal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood, 2005; 106(2): 673-680.
28．王莉红，周春林，张新伟，等．FLT3基因内部串联重复突变与急性白血病的关系及临床意义．中华血液学杂志，2004；25(7)：393-396．
29. Wodnar-Filipowicz A. Flt3 ligand: role in control of hematopoietic and immune functions of the bone marrow. News Physiol Sci, 2003; 18: 247-251.
30. Tumer AM, Lin NL, Issarachai S, et al. FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells. Blood, 88(9): 3383-3390.
31. Schnittger S, Schoch C, Dugas M, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood, 2002; 100(1): 59-66.
32. Ishiko J, Mizuki M, Matsumura I, et al. Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant. Oncogene, 2005; 24(55): 8144-8153.
33. Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000; 404(6775): 293-296.
34. Zhang J, Hua ZC. Targeted gene silencing by small interfering RNA-based knock-down technology. Curr Pharm Biotechnol, 2004, 5(1): 1-7.
35. Chakraborty C. Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Current drug targets, 2007; 8(3): 469-482.
36. Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in 275(17): 12470-12474.
78. Jonas BA, Privalsky ML. SMRT and N-CoR corepressors are regulated by distinct kinase signaling pathways. J Biol Chem, 2004; 279(52): 54676-54686.
79. Ko HM, Jung HH, Seo KH, et al. Platelet-activating factor-induced NF-kappa B activation enhances VEGF expression through a decrease in p53 activity. FEBS Lett, 2006; 580(13): 3006-3012.
80. Putral LN, Gu W, et al. RNA interference for the treatment of cancer. Drug News Perspect, 2006; 19(6): 317-324.
81. Stephanie Filleur, Aurelie Courtin, Slimane Ait-Si-Ali, et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res, 2003, 63(7): 3919-3922.
82. Sung-Suk Chae, Ji-Hye Paik, Henry Furneaux. Requirement for sphingosine 1-phosphat.e receptor-1 in tumor angiogenesis demonstrated by in vivo RNA interference. J Clin Invest, 2004, 114(8): 1082-1089.
83. Oka D, Nishimura K, Shiba M, et al. Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation ofNF-kappaB. Int J Cancer, 2007; 120(12): 2576-2525.
84. Taguchi T, Takao T, Iwasaki Y, et al. Suppressive effects of dehydroepiandrosterone and the nuclear factor-kappaB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo. J Endocrinol, 2006; 188(2): 321-331.
85．孙以方．医学实验动物学．兰州大学出版社，2005年8月出版；第一版．
86. Sausville EA, Burger AM. Contributions of human tumor xenografts to anticancer drug development. Cancer Res, 2006; 66(7): 3351-3354.
87. Zhang SY, Zhu J, Chen GQ, et al. Establishment of a human acute promyelocytic leukemia-ascites model in SCID mice. Blood, 1996; 87(8): 3404-3409.
88. Gao L, Chen L, Fei XH, et al. STI571 combined with vincristine greatly suppressed the tumor formation of multidrug-resistant K562 cells in a human-nude mice xenograft model. Chin Med J (Engl), 2006; 119(11): 911-918.
1. Stone RM, O'Donnell MR, Sekeres MA. Acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2004;: 98-117.
2. Cucuianu A. Pathogenic pathways in acute myeloid leukemias. Rom J Physiol. 2004; 41(1-2): 109-118.
3. Clark JJ, Smith FO, Arceci RJ, et al. Update in childhood acute myeloid leukemia: recent developments in the molecular basis of disease and novel therapies. Curr Opin Hematol. 2003; 10(1): 31-39.
4. Larson RA, Le Beau MM. Therapy-related myeloid leukaemia: a model for leukemogenesis in humans. Chem Biol Interact, 2005; 153-154: 187-95.
5. Smith BD, Bao T, Karp JE. New concepts in the treatment of acute myeloid malignancies: selected pathways for targeted therapy. J Biol Regul Homeost Agents, 2005; 19(1-2): 23-32.
6. John AM, Thomas NS, Mufti GJ, et al. Targeted therapies in myeloid leukemia. Semin Cancer Biol, 2004; 14(1): 41-62.
7．邱慧颖，王健民，薛永权．急性髓系白血病细胞及分子遗传学研究进展．中华血液学杂志，2004；25(12)：761-764．
8．陈赛娟，陈丽娟，周光飚．白血病基因产物靶向治疗的基础和临床研究．中国实验血液学杂志，2005；13(1)：1-8．
9. Cilloni D, Messa E, Messa F, et al. Genetic abnormalities as targets for molecular therapies in myelodysplastic syndromes. Ann N Y Acad Sci, 2006; 1089: 411-423.
10. Inokuchi K. Chronic myelogenous leukemia: from molecular biology to clinical aspects and novel targeted therapies. J Nippon Med Sch, 2006; 73(4): 178-192.
11. Kovitz C, Kantarjian H, Garcia-Manero G, et al. Myelodysplastic syndromes and acute leukemia developing after imatinib mesylate therapy for chronic myeloid leukemia. Blood, 2006; 108(8): 2811-2813.
12. Cortes JE, Talpaz M, O'Brien S, et al. Staging of chronic myeloid leukemia in the imatinib era: an evaluation of the World Health Organization proposal. Cancer,