【作者】 王云贵；

【导师】 金洁；

【作者基本信息】 浙江大学 ， 内科学， 2010， 博士

【摘要】 第一部分microRNA在急性白血病中的分类及预后中的作用目的：观察23种前体miRNAs在85例初发未治急性白血病中的表达,探讨miRNAs在急性白血病中分类及与预后的关系。方法：采用逆转录实时荧光定量PCR技术检测23种miRNAs在初发未治急性白血病患者骨髓单个核细胞中的表达,并对患者的生存期进行随访。结果：①16个miRNAs在85例AML与ALL中差异表达,9种miRNAs(miR-128a、miR-128b、miR-155、miR-150、miR-17、miR-29a／c、miR-29b、miR-146a、miR-20a)在ALL中表达明显高于AML,而7种miRNAs(miR-223、miR-221、miR-222、miR-27a／b、miR-23a、miR-196b、let-7b)在AML表达明显高于ALL。②32例ALL患者中miR-146a、miR-181a／c和miR-221与ALL患者的总生存期相关：miR-146a、miR-181a／c高表达的患者的生存期短,而miR-221高表达患者有更长的生存期。③5种miRNAs(miR-25、miR-26a、miR-29b、miR-146a、miR-196b)表达水平与53例AML患者生存期有显著相关。miR-25的表达水平与生存期呈正相关,另4种表达水平与生存期呈负相关。对其中的40例非M3亚型的AML病例进行分析,3种miRNAs(miR-26a. miR-29b、miR-146a)的表达水平与生存期均呈负相关。④考虑到miR-146a是我们研究的23个miRNAs中唯一一个与AML和ALL患者总生存期相关的miRNA,追加检测了61例初发未治AML患者骨髓标本miR-146a,结果显示miR-146a低表达患者生存期长。⑤通过检索国际上5个主要的miRNA靶基因数据库,我们发现miR-146a共有约7200个可能的靶基因。我们重点分析了至少3种算法都符合的622个基因。在基因本体论生物过程部分,与被预测的27901个人类可能受miRNA调控的基因相比,622个miR-146a的可能靶基因中负调控生物学过程、负调控细胞增殖、凋亡、细胞周期、细胞周期关卡、细胞周期停滞和DNA损害检查点相关的基因比例明显提高。高表达miR-146a的患者临床预后相对差,可能与miR-146a高表达后抑制了其靶基因表达有关。结论：①9种miRNAs(miR-128a、miR-128b、miR-155、miR-150、miR-17、miR-29a／c、miR-29b、miR-146a、miR-20a)在ALL中表达明显高于AML；而7个miRNAs(miR-223、miR-221、miR-222、miR-27a／b、miR-23a、miR-196b、let-7b)在AML表达明显高于ALL。②miR-146a、miR-181a／c高表达的ALL患者的生存期短,而miR-221高表达的ALL患者生存期长。③miR-25的表达水平与AML患者生存期呈正相关,miR-26a、miR-29b、miR-146a、miR-196b表达水平与AML患者生存期呈负相关。miR-26a、miR-29b、miR-146a的表达水平与非M3亚型的AML患者生存期均呈负相关。④在基因本体论生物过程部分,与被预测的27901个人类可能受miRNA调控的基因相比,622个miR-146a的可能靶基因中与负性调控生物学过程、阴性调控细胞增殖、凋亡、细胞周期、细胞周期关卡、细胞周期停滞和DNA损害检查点相关的基因比例明显提高。miR-146a高表达的患者临床预后相对差,可能与miR-146a高表达后抑制了靶基因表达有关。第二部分microRNA在高三尖杉酯碱诱导急性髓系白血病细胞凋亡中的作用机制目的：探讨microRNA在高三尖杉酯碱诱导急性髓系白血病细胞凋亡中表达变化,并初步探讨其作用机制。方法：采用MTT比色法研究HHT对K562、HL60、U937细胞的体外生长抑制作用；采用流式细胞仪PI染色检测细胞周期和Annexin V/PI双染色检测细胞凋亡；采用miRNA芯片技术检测HHT作用K562细胞后miRNA表达的变化；采用real-timePCR技术检测HHT对K562、HL60、U937细胞成熟miRNA表达的影响；采用real-time PCR技术检测HHT对K562、HL60、U937细胞及原代AML细胞miR-17-92表达的影响；采用real-time PCR技术检测HHT对K562、U937细胞C-Myc和p21mRNA表达水平的影响；采用Western Blot技术检测K562和U937细胞miR-17-92基因簇相关基因蛋白水平表达；采用Western Blot技术检测HHT作用K562细胞后bcr／abl P210蛋白和Bcl-2家族蛋白水平表达变化。结果：①HHT抑制人AML细胞株K562、HL60、U937细胞的生长,均呈浓度和时间依赖性。②HHT对K562细胞无明显的周期抑制作用,HHT能诱导K562、HL60、U937细胞凋亡,并呈浓度依赖性。③共检测了600多个人miRNAs表达变化,发现有13种miRNAs(miR-17,miR-17*,miR-18a,miR-18b,miR-20a,miR-20b,miR-93,miR-106a,miR-126,miR-142-5p,miR-144,miR-233和miR-320)在HHT作用后3个时间点均表达下调,与对照组相比差异大于1.5倍,未发现3个时间点均表达上调的miRNA。进一步通过real-time PCR验证,HHT能下调K562、HL60、U937细胞13种成熟miRNA的表达。④HHT能明显下调K562、HL60、U937和3例原代AML细胞miR-17-92基因簇的初级转录本表达。⑤HHT下调K562、U937细胞C-Myc mRNA表达,并上调p21mRNA表达。⑥HHT能下调K562、U937细胞P-AKT和c-Myc蛋白的表达,上调p21、PTEN、P-PTEN和E2F1蛋白的表达,而BIM和总AKT则无明显变化。⑦HHT作用K562细胞24h后,bcr／abl P210蛋白表达才明显下调,而促凋亡蛋白Bak、Bax表达上调,抗凋亡蛋白Bcl-xl和Mcl-1表达下调。结论：①HHT可以抑制人AML细胞株细胞的增殖并诱导细胞凋亡。②HHT能下调人AML细胞13种miRNA的成熟体表达,其下调miR-17-92基因簇1miRNA表达是先通过下调c-Myc表达,进而下调miR-17-92基因簇的前体转录本表达实现的。HHT作用24h后才明显下调K562细胞bcr/ablP210蛋白表达,说明HHT下调K562细胞的c-Myc表达可能并不通过bcr／abl P210融合蛋白。③HHT上调AML细胞miR-17-92基因簇及其两个旁系同源体的共同靶基因p21、PTEN、和E2F1蛋白表达,并抑制PTEN下游AKT磷酸化,上调p21蛋白表达可能与p21mRNA表达上调有关。这些靶基因表达上调可能与HHT下调miR-17-92基因簇及其两个旁系同源体的相关miRNAs表达有关。④HHT还可以通过上调K562细胞的Bcl-2家族中促凋亡蛋白Bak、Bax表达,下调抗凋亡蛋白Bcl-xl和Mcl-1表达诱导AML细胞凋亡。第三部分miR-17-92基因及miR-17、miR-20a的类似物和抑制剂转染及作用机制目的：通过基因转染进一步研究miR-17-92、miR-17、miR-20a在HHT诱导AML细胞凋亡中的作用机制。方法：通过逆转录病毒载体将miR-17-92基因转染进入K562和U937细胞；采用电转法分别将miR-17和miR-20a的模拟物和抑制剂转染进入K562细胞；采用WesternBlot技术检测相关靶基因表达变化；采用real-time PCR技术检测p21基因表达变化及miRNA表达变化；采用Annexin V/PI双染色方法检测HHT对转染后细胞凋亡影响。结果：①转染miR-17-92基因后,与对照细胞相比,K562和U937细胞的p21、E2F1和PTEN蛋白表达下调,p21mRNA表达下调。50ng／ml HHT处理转染了miR-17-92基因的K562和U937细胞24h后,与对照细胞相比,转染后细胞早期凋亡细胞比例明显下降。②K562细胞转染miR-17和miR-20a的模拟物后,与对照细胞相比,转染后p21、E2F1和PTEN蛋白表达下调,p21mRNA表达下调。K562细胞转染miR-17和miR-20a的抑制剂后,与对照细胞相比,p21、E2F1和PTEN蛋白表达上调,p21mRNA表达上调。50ng／ml HHT处理K562细胞24h后,与对照细胞相比,转染miR-17／miR-20a模拟物后K562细胞早期凋亡比例明显下降,转染miR-17／miR-20a抑制剂后K562细胞早期凋亡比例明显上升。结论：①在K562和U937细胞中,p21、E2F1和PTEN是miR-17-92基因簇的靶基因。②在K562细胞中,p21、E2F1、PTEN表达分别受miR-17和miR-20a调节,是miR-17和miR-20a的靶基因。③结合第二部分结果,说明HHT先下调了AML细胞的miR-17-92基因的表达,进而降低了miR-17和miR-20a的表达,导致靶基因p21、E2F1、PTEN表达上调,从而促进AML细胞凋亡。④提高miR-17-92、miR-17、miR-20a表达均可抑制AML细胞对HHT的敏感性；抑制miR-17或miR-20a功能可促进AML细胞对HHT的敏感性。总结：我们的研究发现在中国病人中一组miRNA—miR-223、miR-128a和miR-128b在AML和ALL中表达不同。在国际上首先发现一些miRNA的表达与急性白血病的总生存期明显相关：miR-146a、miR-181a／c高表达的ALL患者的生存期短,而miR-221高表达的ALL患者生存期长；miR-25的表达水平与AML患者生存期呈正相关,miR-26a、miR-29b、miR-146a、miR-196b表达水平与AML患者生存期呈负相关。分析miR-146a的可能靶基因,其中负性调控细胞生长和促进细胞凋亡的比例明显高于所有可能受miRNA调控的基因中相应基因比例,这可能是急性白血病高表达miR-146a预后相对差的原因之一。HHT能诱导人AML细胞凋亡并下调AML细胞13种miRNAs表达。HHT下调AML细胞miR-17-92基因簇的正向转录因子C-Myc蛋白的表达,导致miR-17-92基因簇表达下调。miR-17-92基因簇表达下调导致miR-17、miR-20a低表达。HHT通过降低miR-17、miR-20a等miRNA表达,上调了其靶基因p21、E2F1、PTEN表达,进而诱导AML细胞凋亡。因而我们认为miR-17-92基因簇及其两个旁系同源体的一些miRNAs,共同通过靶基因p21、E2F1、PTEN介导HHT诱导人AML细胞凋亡,国内外未见相关的报道。更多还原

【Abstract】 Section 1 MicroRNAs expression signatures are associated with lineage and survival in acute leukemiasObjective:The aim of this section was to assess expression of precursors of 23 miRNAs in 85 Chinese adolescent and adult de novo acute leukemia bone marrow specimens and to investigate microRNAs expression signatures in patients with lineage and survival.Methods:Quantitative real-time PCR was used to assess expression of precursors of 23 miRNAs in 85 Chinese adolescent and adult de novo acute leukemia bone marrow specimens.All patients were followed up.Results:①We identified 16 miRNAs differentially expressed between 85 ALL and AML samples. Nine (i.e., miR-128a, miR-128b, miR-155, miR-150, miR-17, miR-29a/c, miR-29b, miR-146a, and miR-20a) were expressed at a significantly higher level in ALL than in AML; In contrast, seven (i.e., miR-223, miR-221, miR-222, miR-27a/b, miR-23a, miR-196b and let-7b) were expressed at a significantly higher level in AML compared to ALL.②We found that three miRNAs including miR-146a, miR-181a/c, and miR-221 were significantly associated with overall survival of the 32 ALL patients. Expression level of the first two precursor miRNAs was associated with a poor outcome whereas that of the latter was associated with a good outcome.③We found that five miRNAs including miR-25, miR-26a, miR-29b, miR-146a, and miR-196b were significantly associated with overall survival of the 53 AML patients. Expression level of the first miRNA was positively whereas that of the latter four was negatively associated with a good outcome. We also found that only three miRNAs including miR-26a, miR-29b, and miR-146a were significantly associated with overall survival of the 40 non-M3 AML patients. Expression level of all three miRNAs was negatively associated with a good outcome.④Because miR-146a is the only miRNA whose expression is significantly associated with overall survival of both AML (including or excluding AML-M3) and ALL patients, we confirmed that the expression signature of miR-146a was significantly inversely associated with overall survival of 61 additional AML patients.⑤Through searching five common human miRNA-target prediction programs/databases, we found that miR-146a has a total of 7,200 predicted targets. We focused on the 622 miR-146a targets that are predicted by at least 3 programs/databases for the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Particularly, in GO BP (biological process) terms, genes related to "negative regulation of biology process", "negative regulation of cellular process", "apoptosis", "cell cycle", "cell cycle checkpoint", "cell cycle arrest", and "DNA damage checkpoint" were significantly more enriched in the 622 miR-146a putative target list than in the 27,901 total putative miRNA target gene list. This might contribute to the association of miR-146a with poor survival.Conclusions:①Nine (i.e., miR-128a, miR-128b, miR-155, miR-150, miR-17, miR-29a/c, miR-29b, miR-146a, and miR-20a) were expressed at a significantly higher level in ALL than in AML, In contrast, seven (i.e., miR-223, miR-221, miR-222, miR-27a/b, miR-23a, miR-196b and let-7b) were expressed at a significantly higher level in AML compared to ALL.②Expression level of miR-146a and miR-181a/c was associated with a poor outcome whereas that of miR-221 was associated with a good outcome in ALL patients.③Expression level of miR-25 was positively whereas that of 4 miRNAs (i.e., miR-26a, miR-29b, miR-146a, miR-196b) was negatively associated with a good outcome in AML patients. Expression level of miR-26a, miR-29b, and miR-146a was negatively associated with a good outcome in non-M3 AML patients.④In GO BP terms, genes related to "negative regulation of biology process" "negative regulation of cellular process", "apoptosis", "cell cycle", "cell cycle checkpoint", "cell cycle arrest", and "DNA damage checkpoint" were significantly more enriched in the 622 miR-146a putative target list than in the 27,901 total putative miRNA target gene list. This might contribute to the association of miR-146a with poor survival.Section 2:The effects and mechanisms of micro RNAs to apoptosis induced by homoharringtonine in acute myeloid leukemia cells Objective: The aim of this section was to compare the miRNA expression profiles of HHT-treated AML cells with those of untreated and to study these mechanisms of microRNAs to apoptosis induced by homoharringtonine in acute myeloid leukemia cell.Methods:We used MTT assay to investigate the effects of HHT on AML cells (i.e., K562, HL60,U937). PI staining was used for cell cycle analysis by flow cytometry. AV-PI staining was used for detecting apoptosis. We used microarray to compare the miRNA expression profiles of HHT-treated K562 cells with those of untreated cells. We then compared miRNA expression in HHT-treated vs untreated AML cells (i.e., K562, HL60, U937) by using real-time PCR. The expression level of miR-17-92 in AML cells (i.e., K562, HL60, U937 and three primary AML patient blasts) was detected by real-time PCR. We compared c-Myc and p21 mRNA expression in HHT-treated vs untreated K562 and U937 cells by using real-time PCR. Western blot was used for detecting expression level of miR-17-92-associated proteins in K562 and U937 cells. We also detected the expression level of P210 bcr/abl and Bcl-2 family proteins in K562 cells by western blot.Results:①HHT could inhibit growth of AML cells (i.e., K562, HL60, U937). The cell growth was inhibited in a time-and dose-dependent manner, corresponding to the reduced cell viability.②HHT could not evidently induce K562 cell cycle arrested. HHT induced apoptosis of K562 and U937 cells in a dose-dependent manner.③From more than 600 miRNAs, we identified that the expression level of 13 miRNAs(i.e.,miR-17, miR-17*, miR-18a, miR-18b, miR-20a, miR-20b, miR-93, miR-106a, miR-126, miR-142-5p, miR-144, miR-233 and miR-320) was downregulated (changed more than 1.5 times vs untreated cells) whereas none was upregulated in 50ng/ml HHT-treated K562 cells in all times(i.e.,3h,12h,24h). The results were confirmed in HHT treated K562, HL60 and U937 cells by using real-time PCR.④The expression level of miR-17-92 pri-miRNA was downregulated in AML cells (i.e., K562, HL60, U937 and three primary AML patient blasts) treated with 50ng/ml HHT.⑤the expression level of C-Myc mRNA and protein was decreased in 50ng/ml HHT-treated K562 and U937 cells. The expression level of P210 bcr/abl was specifically decreased only in 24-hour-HHT-treated K562 cells.⑥The expression level of P-AKT protein was downregulated whereas that of PTEN, P-PTEN, p21 and E2F1 protein was upregulated, and that of BIM and total AKT protein was stable in K562 and U937 cells which were treated with 50ng/ml HHT.⑦The expression level of apopotosis-induced protein Bak and Bax was increased whereas that of Bcl-2 and Mcl-1 proteins was decreased in 50ng/ml HHT treated K562 cells at different time.Conclusions:①HHT inhibited cell growth and induced apoptosis in human AML cells (i.e., K562, HL60, U937).②The expression level of 13 miRNAs was downregulated whereas none that of miRNAs was upregulated in human AML cells (i.e., K562, HL60, U937) treated by HHT. Firsrly, we thought maybe HHT downregulated expression level of miR-17-92 pri-miRNA through downregulated expression level of miR-17-92 activator protein c-Myc. Secondly, some miRNAs expression level of miR-17-92 clusters was downregulated. Expression level of bcr/abl P210 protein was specifically decreased only in 24-hour-HHT-treated K562 cells. So we thought HHT downregulated expression of c-Myc, which bypassed bcr/abl P210 protein.③HHT downregulated expression of p21, PTEN and E2F1, which were target genes of miR-17-92 and its thomologous cluster in AML cells. HHT maybe downregulated expression level of those proteins through miR-17-92 and its two thomologous clusters. The enhanced expression level of p21 protein maybe resulted from enhanced p21 mRNA.HHT maybe inhibited expression of P-AKT through PTEN.④The expression level of apopotosis-induced protein Bak and Bax was increased whereas that of Bcl-2 and Mcl-1 was decreased in HHT-treated K562 cells.Section 3:The study on effcts of cells transfected with miR-17-92 or mimic and inhibitor of miR-17 and miR-20aObjective:To study the effects of miR-17-92, miR-17 and miR-20a to apoptosis induced by homoharringtonine in acute myeloid leukemia cells.Methods:The miR-17-92 gene was transfected into K562 and U937 cells by the retroviral-mediated gene transfer. The mimic and inhibitor of miR-17 and miR-20a was transfected into K562 cells by electroblot. We used real-time PCR to detect p21 expression of mRNA and western blot to detect expression of those targeted proteins. AV-PI staining was used for detecting apoptosis in HHT-treated and HHT-untreated K562 or U937 cells after miR-19-92 gene transfer. We also used AV-PI staining to detect apoptosis in HHT-treated K562 cells after mimic and inhibitor of miR-17 and miR-20a was transfected.Results:①The expression level of some proteins(i.e., E2F1, PTEN and p21) and p21 mRNA was decreased in K562 and U937 cells overexpressing miR-17-92 compared with controls.The percentage of apoptosis cells was deceased in 50ng/ml HHT-treated K562 and U937 cells overexpressing miR-17-92 compared with controls by using AV/PI staining.②The expression level of some proteins(i.e., E2F1, PTEN and p21) and p21 mRNA was decreased in K562 cells overexpressing miR-17 or miR-20a compared with controls.The percentage of apoptosis cells was deceased in 50ng/ml HHT-treated K562 cells overexpressing miR-17 or miR-20a compared with controls. The expression level of some proteins (i.e., E2F1, PTEN and p21) and p21 mRNA was upregulated in K562 cells transfected with miR-17 or miR-20a inhibitor compared with controls.The percentage of apoptosis cells was upregulated in 50ng/ml HHT-treated K562 cells transfected with miR-17 or miR-20a inhibitor compared with controls.Concludions:①In K562 and U937 cells, some genes (i.e., E2F1, PTEN and p21) were real target genes of miR-17-92.②In K562 cells, some gene (i.e., E2F1, PTEN and p21) were real target gene of miR-17 and miR-20a.③Incorporated with the results of second section, at first, HHT downregulated the expression of miR-17/miR-20a through miR-17-92, secondly, HHT downregulated the expression of some proteins (i.e., E2F1, PTEN and p21) through miR-17/miR-20a in AML cells.④Overexpressing miR-17-92 or miR-17/miR-20a decreased sensitivity to HHT induced K562 cells apoptosis, and inhibiting miR-17/miR-20a increased sensitivity to HHT induced K562 cells apoptosis.Summary:Our study shows that a group of microRNAs (e.g., miR-223, miR-128a and miR-128b) are discriminatory microRNAs between AML and ALL in Chinese patients. More importantly, our study also shows that expression of some microRNAs is associated with overall survival of patients with acute leukemias (e.g., miR-146a, miR-181a/c and miR-221 in ALL, and miR-25, miR-26a, miR-29b, miR-146a, and miR-196b in AML), which has not been reported previously. Furthermore, results from the GO and KEGG analysis of potential targets of miR-146a implicate some potential functions of miR-146a (e.g., negatively regulating genes that inhibit cell growth and promote apoptosis), which may be related to the association of miR-146a with poor survival.HHT induced apoptosis and downregulated expression of 13 mature miRNAs in human AML cells. Firstly, HHT downregulated expression of miR-17-92 pri-miRNA through downregulated expression of miR-17-92 activator protein c-Myc. Scondly, the expression of miR-17 and miR-20a were decreased by HHT through miR-17-92. At last some proteins (i.e., p21, PTEN and E2F1) were downregulated through some miRNA of miR-17-92 and its thomologous cluster. So we thought HHT induced human AML cells apotosis through real target protein (i.e., p21, PTEN and E2F1) of some miRNA of miR-17-92 and its thomologous cluster. This maybe one of the mechanisms that HHT induced AML cells apoptosis, which has not been reported previously.更多还原