白血病Hedgehog信号通路激活与HHIP基因甲基化的关系及As_2O_3去甲基化机制研究
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摘要
白血病的病因及发病机制非常复杂,目前还不完全清楚。表观遗传学调控是基于非基因序列改变所致可遗传的基因表达水平变化的一种调控方式,包括DNA甲基化、组蛋白去乙酰化、小RNA(miRNA)沉默等,表观遗传调控导致的抑癌基因表达沉默是诱发白血病的重要因素之一,近年研究发现,DNA甲基化是人类白血病中普遍存在的现象,与白血病的发生密切相关。
抑癌基因启动子甲基化导致转录失活是一个可逆的表观遗传学修饰过程,恢复或改变基因的高甲基化状态,从而改变基因组表达,对细胞的功能状态进行调节,是去甲基化治疗的理论基础。去甲基化治疗作为一种新的抗肿瘤方法已在白血病及骨髓增生异常综合征(MDS)的治疗中展示了良好的应用前景,特别是为难治复发白血病的治疗开辟了一条新的途径。目前临床上常用的去甲基化药物是5-氮杂-2’-脱氧胞苷(5-aza-2'deoxyeytidine,地西他滨)等。三氧化二砷(As203)是一种传统中药,最初用于治疗急性早幼粒细胞白血病(APL)并取得良好效果,As2O3治疗APL的主要机制与诱导白血病细胞的凋亡有关,随着研究的深入,发现砷剂对其他类型白血病细胞株及实体肿瘤细胞亦具有广泛的抑制增殖,诱导凋亡的作用。最近又有不可思议的新发现,As2O3可逆转骨髓瘤细胞及淋巴瘤细胞中某些抑癌基因的高甲基化状态,也就是说As2O3还具有去甲基化作用。As2O3对白血病细胞除诱导凋亡外,是否还有其他作用机制?As2O3对白血病发病过程中起重要作用的抑癌基因是否也有去甲基化作用?目前尚未见报道。
Hedgehog (Hh)信号通路是体内重要信号传导通路之一,它在人类和动物的正常胚胎发育和器官形成过程中发挥重要作用,但相对于WNT、NOTCH等其他信号通路,该通路的研究较少,也不够深入。通常情况下,该信号通路在机体发育成熟后进入失活状态,但其通路成员的突变或错误表达会激活该通路,导致肿瘤的发生和发展。目前已在多种肿瘤中发现Hh通路的异常激活,但其激活的具体机制尚不明确。在Hh通路被激活的肿瘤中,HHh激活的机制也是不同的,部分是由于Hh信号通路某个成员的突变,但约有一半并没有发现通路成员的突变,因而推测Hh通路的激活还有其他调控机制。研究发现肝癌、脑膜瘤等实体瘤中存在Hh信号通路成员HHIP启动子甲基化现象,提示表观遗传学可能参与了Hh通路的调控。
为进一步探讨Hh信号通路在白血病中的作用及激活的机制,Hh通路抑制因子HHIP的甲基化是否也参与了Hh通路的激活及白血病的发生,我们从RNA水平及蛋白水平上对As2O3处理前后该通路成员进行了检测,用甲基化特异性PCR检测Hh通路抑癌基因HHIP的甲基化状态,对进一步阐明白血病发病机制的未知领域,发现新的治疗靶点有重要意义。
目的
一、分别从mRNA水平及蛋白水平检测Hh信号通路成员及靶基因G1i、PTCH表达,了解白血病细胞系Hh通路的活性水平。
二、用甲基化特异性PCR检测白血病细胞系Hh通路抑制因子HHIP的甲基化状态,并探讨HHIP的高甲基化是否与Hh通路的异常激活有关。
三、观察三氧化二砷(As2O3)能否通过逆转HHIP基因甲基化而阻断Hh异常激活,从而明确Hh通路是否存在去甲基化药物的作用靶点。
四、探讨As2O3去甲基化作用的最佳浓度及具体机制。
方法
选用急性髓系白血病细胞系HL60、慢性粒细胞白血病细胞系K562、急性B淋巴细胞白血病细胞系BALL-1、急性T淋巴细胞白血病细胞系Jurkat作为研究对象,对照组为正常人外周血淋巴细胞。细胞株用含10%小牛血清的RPM I1640培养液、在37℃和5%C02培养箱中培养传代。
一、白血病细胞系Hh通路的活性水平
1、 RT-PCR检测SHH、PTCH、Gli、HHIP基因mRNA表达
用Total RNA提取试剂RNAiso提取细胞系总RNA,用逆转录聚合酶链反应(RT-PCR)检测各白血病细胞系及正常人外周血淋巴细胞Hedgehog通路成员SHH.PTCH、Gli、HHIP基因mRNA表达。
2、Western-blot检测Hh通路成员蛋白表达水平
采用蛋白裂解液提取蛋白,SDS-PAGE聚丙烯酰胺凝胶电泳,电泳后将蛋白转移到硝酸纤维素膜上,加一抗和酶标的山羊抗兔的IgG,最后加底物显色5min,暗室发光并显影定影。使用凝胶分析系统测定条带的面积和灰度值,以积分灰度值与内参的比值代表蛋白的相对表达量。
二、白血病细胞HHIP基因甲基化状态检测及对G1i表达的影响
1、采用甲基化特异性PCR (MS-PCR)检测4种白血病细胞系及正常人外周血淋巴细胞Hedgehog信号通路HHIP基因启动子区域甲基化情况。
2、用siRNA技术封闭HHIP基因表达,然后用RT-PCR检测Gli表达变化,进一步验证HHIP基因对Gli的调控作用。
三、As2O3去甲基化作用机制研究
用不同浓度的As2O3处理BALL-1细胞系,按照As2O3浓度分为0、2.5、5.0、10.0umol/L组,药后常规培养,培养过程不换液。
1、CCK-8检测细胞增殖
于药物处理后24、48、72、96小时加入1Oul CCK-8溶液,继续孵育2-3小时,置酶标仪上450nm单波长测吸光度OD值,计算抑制率,并以时间为横轴,OD值为纵轴绘制生长曲线。
2、流式细胞术(FCM)检测细胞周期变化
As2O3处理48小时后,收集各组细胞,FCM检测As2O3处理后细胞周期变化。
3、As2O3对BALL-1细胞HHIP基因甲基化模式的影响
As203对BALL-1细胞作用24小时、48小时、72小时后,提取细胞基因组DNA,采用CpGenome DNA Methylation kit试剂盒对DNA进行修饰,然后用MS-PCR检测BALL-1细胞系HHIP基因启动子区域甲基化情况。
4、As2O3对BALL-1细胞HHIP基因及Hh通路靶基因Gli、下游基因cyclinD2表达的影响
As2O3作用于BALL-1细胞48小时后,用Total RNA提取试剂RNAiso提取BALL-1细胞系总RNA,用RT-PCR检测HHIP、Gli、cyclinD2基因mRNA表达。用Western-blot检测药物处理后HHIP蛋白表达。
5、As2O3对DNA甲基转移酶dnmtl.dnmt3b mRNA及去甲基化酶mbd2mRNA表达的影响
As2O3作用于BALL-1细胞48小时后,用Total RNA提取试剂RNAiso提取BALL-1细胞系总RNA,用RT-PCR检测dnmtl.dnmt3b.mbd2mRNA表达。
结果
一、白血病细胞Hh通路的活性水平
PTCH.Gli既是Hh信号通路的成员,也是该通路的靶基因,通常用PTCH. G1i表达来代表Hh通路的活性。
4种白血病细胞系中,BALL-1.JUrkat细胞系Gli mRNA表达均明显高于对照组(1.163±0.066vs0.828±0.067,1.123±0.024VS0.828±0.067,P<0.05):HL60细胞PTCH表达高于对照组(0.783±0.045vs0.553±0.064,P<0.05),所有白血病细胞系中SHH配体表达与正常人淋巴细胞相比,无明显差异(P>0.05)；除K562细胞系外,HL60(0.638±0.130vs0.744±0.061).BALL-1(0.416±0.064vs0.744±0.061).Jurkat(0.544±0.098vs0.744±0.061中HHIP表达都明显减低(P<0.05).BALL-l细胞Gli蛋白表达高于对照组(1.223±0.102vs0.830±0.046,P<0.05),以上结果提示在BALL-1、Jurkat.HL60细胞中,Hh通路是激活的。
二、HHIP基因的MS-PCR检测结果及HH工P甲基化对Gli表达的影响
1、在4种白血病细胞系中检测到HHIP发生不同程度甲基化,其中BALL-1、 Jurkat中HH工P基因启动子区域CpG岛高甲基化,HL6O、K562细胞HHIP基因检测到部分甲基化,而正常人淋巴细胞HHIP基因未发生甲基化。
2.HHIPsiRNA孵育BALL细胞48h后,HHIP的mRNA转录受抑制,电泳条带变暗,HHIP/GAPDH从0.983降至0.409;GlilmRNA转录水平升高,条带变亮,Gli/GAPDH从1.045增至1.383,提示在BALL细胞中HHIP可能Gli的上游负性调控因子,可抑制Gli的转录。
三、As2O3去甲基化机制研究
1、As2O3对BALL-1细胞生长的抑制作用
各药物处理组的细胞增殖抑制率随时间和浓度增加而显著升高,与阴性对照组相比差异有统计学意义(P<0.05),其抑制率呈时间和浓度依赖性。
2、As2O3对细胞周期的影响
不同浓度As2O3处理后,BALL-1细胞不同程度阻滞于G0、G1期,G0/G1期细胞百分比升高,S、G2/M期百分比下降(P<0.05)。提示As2O3能引起BALL-1细胞的G0/G1期停滞,从而抑制细胞增殖。
3、As2O3对HHIP基因甲基化模式的影响
未加药组细胞HHIP-M扩增结果阳性,HHIP-U扩增结果为阴性,提示BALL-1细胞常态下HHIP基因呈高度甲基化；随着As2O3浓度的升高,HHIP-M条带逐渐减弱,而HHIP-U条带逐渐增强,提示BALL-1细胞HHIP基因甲基化程度逐渐降低,而非甲基化程度逐渐升高。以上结果表明：As2O3晚可逆转HHIP基因启动子区的高甲基化状态,且呈浓度依赖性。
4、As2O3对HHIP基因及Hh通路靶基因Gli、下游基因cyclinD表达的影响
5.Oumol/L As2O3处理BALL-1细胞72小时后,HHIP mRNA及蛋白表达明显上调(0.826±0.045vs0.253±0.012,0.328±0.021vs0.024±0.005, P<0.05)。说明5.Oumol/L以上浓度As2O3对BALL-1细胞有去甲基化作用。Hh通路靶基因Gli mRNA表达较药物处理前减低(1.030±0.088vs1.328±0.071P<0.05)。而下游基因cyclinDmRNA表达在药物处理自矿后无明显差异(P>0.05)。
5、As2O3对DNA甲基转移酶及去甲基化酶表达的影响
未加药组BALL-1细胞dnmt1、 dnmt3b基因呈强表达；加药组随As2O3浓度的增加,dnmt1、 dnmt3b基因的表达逐渐减弱,与未加药组差异有显著性(0.548±0.044vs0.886±0.028,0.463±0.043vs0.877±0.039,P<0.05). As2O3处理前后mbd2mRNA的表达无明显变化(P>0.05)。提示As2O3可下调DNA甲基转移酶dnmt1、 dnmt3bmRNA的表达,从而发挥去甲基化作用。
结论
一、BALL-1、Jurkat、HL60细胞系Gli或PTCH表达增加,Hedgehog信号通路异常激活,提示白血病发生可能与Hedgehog信号通路的异常激活有关。
二、HHIP基因启动子甲基化使HHIP基因沉默,失去对Hh通路的调控作用,可能是Hedgehog信号通路的异常激活的机制之一。
三、As2O3对BALL-1细胞HHIP基因有去甲基化作用,且呈浓度依赖性,5.0umol/L以上浓度的As2O3对BALL-1细胞HHIP基因有明显的去甲基化作用。
四、As2O3能抑制DNA甲基化转移酶的活性,从而逆转抑癌基因的甲基化水平,使其重新激活,发挥肿瘤抑制作用。
Background
The etiology and pathogensis of leukemia is very complex, and it is not entirely clear. Epigenetic regulation is a kind of control mode which based on non sequential genetic alterations induced heritable gene expression level changes, including DNA methylation, histone acetylation, small RNA (miRNA) silence. In leukemia development process, it plays a very important role. Epigenetic regulation has led to the tumor suppressor gene expression of silence is one of the important factors inducing leukemia. In recent years, some studys have proved that DNA methylation was a common phenomenon in human leukemia, and closely related to the onset of leukemia.
Demethylation therapy as a new antitumor method has been shows a good application prospect in leukemia treatment, especially opened up a new way in the treatment of refractory and relapsed leukemia.The clinical application of demethylation drugs have begun to hematological malignancies such as acute leukemia and myelodysplastic syndrome(MDS) treatment, such as5-aza-2'-deoxycytidine (deoxyeytidine) etc. As2O3is a kind of traditional Chinese medicine, initially for the treatment of acute promyelocytic leukemia and achieved good results. The main mechanism of As2O3in treatment of acute leukemia is inducing leukemia cell apoptosis. Recent studies found that, As2O3can reverse the methylation of certain tumor suppressor gene in myeloma cells and lymphoma cells, so in induction to apoptosis, As2O3may be has other mechanisms of action.
The Hedgehog signaling pathway plays an important role in human and animal normal embryonic development and organ formation.Its channel members including hedgehog, PTCH, SMO, and Gli.The channel member mutations or error expression will activate the pathways, ultimately leading to cancer occurrence and development. Recent research demonstrated that the pathways relevant to tumors including basal cell carcinoma, pancreatic cancer, colon cancer, stomach cancer, small cell lung cancer, melanoma, breast cancer, ovarian cancer and other. In the tumors that Hh pathway is activated, approximately half did not find the channel member mutation. so we hypothesized that Hh pathway activation may have other regulation mechanism. Although the existing research has proved that Hh pathway has close relationship with tumor, but Hh pathway through what mechanisms contribute to tumorigenesis remain unclear. Recent studies found that some tumor in the presence of Hh signal pathway members promoter methylation. There are also reports indicate that Hh pathway is activate in some leukemia.
For the further study of Hh signaling role in leukemia and activation mechanism, Whether aberrant methylation of tumor suppressor genes are involved in the Hh pathway activation and the occurrence of leukemia, we detect the channel member from the RNA levels and protein level before and after As2O3treatment, detect Hh pathway tumor suppressor gene HHIP methylation status by methylation specific PCR. The study will be have important significance on the further elucidation of the pathogenesis and the discovery of new therapeutic targets in leukemia.
Objective
The study was designed to investigate whether Hh pathway is activate in acute leukemia, and whether the activation of Hedgehog pathway is related to HHIP methylation. To explore the effect of DNA methylation on the pathogenesis of leukemia. Through the research of arsenic trioxide's demethylation effect mechanism on HHIP gene, to explore whether Hh pathway exists target of demethylation drugs.
Methods
Selection of acute myeloid leukemia cell line HL60, chronic myeloid leukemia cell line K562, acute T lymphocytic leukemia cell lines Jurkat, B cell acute lymphoblastic leukemia cell line BALL-1, cell lines containing10%calf serum RPMI1640culture medium, at37degrees and5%C02culture box culture.
1、 The activity level of Hh pathway in leukemia cell lines
1.1using reverse transcription polymerase chain reaction (RT-PCR) to detecte Hedgehog pathway members SHH, PTCH, Gli, HHIP gene expression of the leukemia cell lines and leukemia patients.
1.2Using Western-blot to detect the corresponding protein expression.
2、 Methylation status of HHIP gene in leukemia cell lines and its influence on the expression of Gli
2.1Using DNA Extraction Kit by silica gel column extraction of genomic DNA, Select specimen of purity between1.70-1.90.Using DNA CpGenome DNA Methylation kit sample kit for DNA was modified, then using methylation-specific PCR (MS-PCR) to detect Hedgehog signal pathway of HHIP gene promoter region methylation of4leukemia cell lines.
2.2Using siRNA closed HHIP gene expression, then use RT-PCR to detect the expression of Glil HHIP gene.
3、 Demethylation mechanism of arsenic trioxide
3.1Treat BALL-1cell line With different concentrations of As2O3, in accordance with the concentration of As2O3is divided into0.0,2.5,5.0, 10.0umol/L group, after treatment for24,48,72,96hours, add lOul CCK-8solution, the microplate reader on450nm single wavelength measuring absorbance0D value, calculate the inhibition rate, and take the time as the horizontal axis,0D value for the cell growth curve.
3.2As203treatment after48hours, collecte the cells, washing2times by PBS, add cold ethanol fixation for more than24hours, washing cells by PBS,37℃magazine incubated for30minutes, PI staining, avoid the light for30minutes, detect changes of cell cycle after arsenic trioxide treatment by flow cytometry.
3.3As2O3influence HHIP gene methylation pattern of BALL-1cell.BALL-1cells were treated with As2O3for24hours,48hours,72hours, DNA Extraction Kit by silica gel column extraction of genomic DNA, using DNA CpGenome DNA Methylation kit to modify1g DNA sample, then using MS-PCR to detect HHIP gene promoter region methylation of BALL-1cell line.3.4Gli is not only the Hh pathway members, but also the target genes of the pathway, therefore may reflect the activity of the pathway. As2O3act on BALL-1cells after48h, Total RNA extracting reagent RNAiso extracted from BALL-1cell line, using RT-PCR to detect the expression of Gli mRNA.
4.5How does As2O3influence DNA methyltransferase dnmt1、 dnmt3b mRNA and mbd2mRNA expression:We detect dnmt1、 dnmt3b and mbd2mRNA expression by RT-PCR.
Result
1、 The activity level of Hh pathway in leukemia cell lines
The important components of Hh signaling pathways such as signal peptide SHH, membrane receptor PTCH, downstream of the transcription factor Gli and HHIP mRNA have different levels of expression. In the majority of acute leukemia cell lines, Gli or PTCH mRNA expression were increased(P<0.05), the expression of SHH had no obvious change, except for K562cell lines, the rest of leukemic cell lines HHIP expression is reduced (P<0.05). The expression of HHIP protein in BALL-1、Jurkat、 HL60was lower than that of control group, and protein expression of Gli in BALL、HL60is higher than that of Control group.
2、 Methylation status of HHIP gene in leukemia cell lines
2.1BALL-1、 Jurkat cells showed hypermethylation,HL60、 K562showed partial methylation, and HHIP gene were not detected methylation in normal lymphocyte.
2.2Incubation BALL-1by HHIPsiRNA for48h, HHIP mRNA transcription is inhibited, HHIP/GAPDH decreased from0.983to0.409; GlimRNA transcription levels are elevated, Gli/GAPDH increased from1.045to1.383. This indicate that HHIP is probably the upstream negative regulatory factor of Glil.
3、Demethylation mechanism of arsenic trioxide
3.1After treated with arsenic trioxide, the inhibition rate of cell proliferation is increased remarkably with time and concentration, compared with the negative control group the differences were statistically significant (P<0.05), the inhibition rate was time and concentration dependent.3.2After treated with Different concentrations of As2O3, BALL-1cells wre blocked in G0, G1in different degree, percentage of G0/G1phase cells increased, and percentage of S、 G2/M phase decreased (P<0.05).
3.3HHIP-M amplification is positive, while the HHIP-U amplification results is negative, suggesting HHIP gene is highly methylated in BALL-1cells. As the As2O3concentration increased, HHIP-M bands gradually weakened, while the HHIP-U strip increases gradually, suggesting that HHIP gene methylation degree is reduced gradually, but the degree of unmethylation increased gradually. The above results indicate:As2O3can reverse HHIP gene promoter hypermethylation status, and the effect depended on the concentration of
3.4As2O3influence the expression of HHIP and Gli mRNA
Detected the expression of Gli and HHIP mRNA by RT-PCR before and after treated with As2O3, the results showed that HHIP mRNA expression was significantly upregulated after treated with5.0umol/L As2O3for72h. HH pathway target genes GlimRNA expression were reduced.
3.5As2O3influence the expression of dnmt1、 dnmt3b mRNA
As2O3can downregulate DNA methyltransferase(dnmtl、 dnmt3b) mRNA. experimental groups with As2O3increasing the concentration of dnmt1、 dnmt3b gene expression gradually weakened(P<0.05). But the expression of mbd2mRNA did not change significantly after treated with As2O3(p>0.05).
Conelution
1The expression of Gli and PTCH were increased in some leukemic cell lines, which suggest that Hedgehog pathway is aberrant activate in leukemia. Hh pathway activation may be associated with the pathogenesis of leukemia.
2Promoter methylation induced the silence of HHIP gene, and HHIP lost its function on the Hh pathway. It may be the aberrant activation mechanism of Hedgehog pathway.
3As2O3can demethylation HHIP gene in BALL-1cells in a concentration dependent manner.
4As2O3demethylation effect mechanism for the inhibition of S-methionine dependence of the methyltransferase, such as dnmtl、dnmt3b,thereby restoring gene expression.
抑癌基因启动子甲基化导致转录失活是一个可逆的表观遗传学修饰过程,恢复或改变基因的高甲基化状态,从而改变基因组表达,对细胞的功能状态进行调节,是去甲基化治疗的理论基础。去甲基化治疗作为一种新的抗肿瘤方法已在白血病及骨髓增生异常综合征(MDS)的治疗中展示了良好的应用前景,特别是为难治复发白血病的治疗开辟了一条新的途径。目前临床上常用的去甲基化药物是5-氮杂-2’-脱氧胞苷(5-aza-2'deoxyeytidine,地西他滨)等。三氧化二砷(As203)是一种传统中药,最初用于治疗急性早幼粒细胞白血病(APL)并取得良好效果,As2O3治疗APL的主要机制与诱导白血病细胞的凋亡有关,随着研究的深入,发现砷剂对其他类型白血病细胞株及实体肿瘤细胞亦具有广泛的抑制增殖,诱导凋亡的作用。最近又有不可思议的新发现,As2O3可逆转骨髓瘤细胞及淋巴瘤细胞中某些抑癌基因的高甲基化状态,也就是说As2O3还具有去甲基化作用。As2O3对白血病细胞除诱导凋亡外,是否还有其他作用机制?As2O3对白血病发病过程中起重要作用的抑癌基因是否也有去甲基化作用?目前尚未见报道。
Hedgehog (Hh)信号通路是体内重要信号传导通路之一,它在人类和动物的正常胚胎发育和器官形成过程中发挥重要作用,但相对于WNT、NOTCH等其他信号通路,该通路的研究较少,也不够深入。通常情况下,该信号通路在机体发育成熟后进入失活状态,但其通路成员的突变或错误表达会激活该通路,导致肿瘤的发生和发展。目前已在多种肿瘤中发现Hh通路的异常激活,但其激活的具体机制尚不明确。在Hh通路被激活的肿瘤中,HHh激活的机制也是不同的,部分是由于Hh信号通路某个成员的突变,但约有一半并没有发现通路成员的突变,因而推测Hh通路的激活还有其他调控机制。研究发现肝癌、脑膜瘤等实体瘤中存在Hh信号通路成员HHIP启动子甲基化现象,提示表观遗传学可能参与了Hh通路的调控。
为进一步探讨Hh信号通路在白血病中的作用及激活的机制,Hh通路抑制因子HHIP的甲基化是否也参与了Hh通路的激活及白血病的发生,我们从RNA水平及蛋白水平上对As2O3处理前后该通路成员进行了检测,用甲基化特异性PCR检测Hh通路抑癌基因HHIP的甲基化状态,对进一步阐明白血病发病机制的未知领域,发现新的治疗靶点有重要意义。
目的
一、分别从mRNA水平及蛋白水平检测Hh信号通路成员及靶基因G1i、PTCH表达,了解白血病细胞系Hh通路的活性水平。
二、用甲基化特异性PCR检测白血病细胞系Hh通路抑制因子HHIP的甲基化状态,并探讨HHIP的高甲基化是否与Hh通路的异常激活有关。
三、观察三氧化二砷(As2O3)能否通过逆转HHIP基因甲基化而阻断Hh异常激活,从而明确Hh通路是否存在去甲基化药物的作用靶点。
四、探讨As2O3去甲基化作用的最佳浓度及具体机制。
方法
选用急性髓系白血病细胞系HL60、慢性粒细胞白血病细胞系K562、急性B淋巴细胞白血病细胞系BALL-1、急性T淋巴细胞白血病细胞系Jurkat作为研究对象,对照组为正常人外周血淋巴细胞。细胞株用含10%小牛血清的RPM I1640培养液、在37℃和5%C02培养箱中培养传代。
一、白血病细胞系Hh通路的活性水平
1、 RT-PCR检测SHH、PTCH、Gli、HHIP基因mRNA表达
用Total RNA提取试剂RNAiso提取细胞系总RNA,用逆转录聚合酶链反应(RT-PCR)检测各白血病细胞系及正常人外周血淋巴细胞Hedgehog通路成员SHH.PTCH、Gli、HHIP基因mRNA表达。
2、Western-blot检测Hh通路成员蛋白表达水平
采用蛋白裂解液提取蛋白,SDS-PAGE聚丙烯酰胺凝胶电泳,电泳后将蛋白转移到硝酸纤维素膜上,加一抗和酶标的山羊抗兔的IgG,最后加底物显色5min,暗室发光并显影定影。使用凝胶分析系统测定条带的面积和灰度值,以积分灰度值与内参的比值代表蛋白的相对表达量。
二、白血病细胞HHIP基因甲基化状态检测及对G1i表达的影响
1、采用甲基化特异性PCR (MS-PCR)检测4种白血病细胞系及正常人外周血淋巴细胞Hedgehog信号通路HHIP基因启动子区域甲基化情况。
2、用siRNA技术封闭HHIP基因表达,然后用RT-PCR检测Gli表达变化,进一步验证HHIP基因对Gli的调控作用。
三、As2O3去甲基化作用机制研究
用不同浓度的As2O3处理BALL-1细胞系,按照As2O3浓度分为0、2.5、5.0、10.0umol/L组,药后常规培养,培养过程不换液。
1、CCK-8检测细胞增殖
于药物处理后24、48、72、96小时加入1Oul CCK-8溶液,继续孵育2-3小时,置酶标仪上450nm单波长测吸光度OD值,计算抑制率,并以时间为横轴,OD值为纵轴绘制生长曲线。
2、流式细胞术(FCM)检测细胞周期变化
As2O3处理48小时后,收集各组细胞,FCM检测As2O3处理后细胞周期变化。
3、As2O3对BALL-1细胞HHIP基因甲基化模式的影响
As203对BALL-1细胞作用24小时、48小时、72小时后,提取细胞基因组DNA,采用CpGenome DNA Methylation kit试剂盒对DNA进行修饰,然后用MS-PCR检测BALL-1细胞系HHIP基因启动子区域甲基化情况。
4、As2O3对BALL-1细胞HHIP基因及Hh通路靶基因Gli、下游基因cyclinD2表达的影响
As2O3作用于BALL-1细胞48小时后,用Total RNA提取试剂RNAiso提取BALL-1细胞系总RNA,用RT-PCR检测HHIP、Gli、cyclinD2基因mRNA表达。用Western-blot检测药物处理后HHIP蛋白表达。
5、As2O3对DNA甲基转移酶dnmtl.dnmt3b mRNA及去甲基化酶mbd2mRNA表达的影响
As2O3作用于BALL-1细胞48小时后,用Total RNA提取试剂RNAiso提取BALL-1细胞系总RNA,用RT-PCR检测dnmtl.dnmt3b.mbd2mRNA表达。
结果
一、白血病细胞Hh通路的活性水平
PTCH.Gli既是Hh信号通路的成员,也是该通路的靶基因,通常用PTCH. G1i表达来代表Hh通路的活性。
4种白血病细胞系中,BALL-1.JUrkat细胞系Gli mRNA表达均明显高于对照组(1.163±0.066vs0.828±0.067,1.123±0.024VS0.828±0.067,P<0.05):HL60细胞PTCH表达高于对照组(0.783±0.045vs0.553±0.064,P<0.05),所有白血病细胞系中SHH配体表达与正常人淋巴细胞相比,无明显差异(P>0.05)；除K562细胞系外,HL60(0.638±0.130vs0.744±0.061).BALL-1(0.416±0.064vs0.744±0.061).Jurkat(0.544±0.098vs0.744±0.061中HHIP表达都明显减低(P<0.05).BALL-l细胞Gli蛋白表达高于对照组(1.223±0.102vs0.830±0.046,P<0.05),以上结果提示在BALL-1、Jurkat.HL60细胞中,Hh通路是激活的。
二、HHIP基因的MS-PCR检测结果及HH工P甲基化对Gli表达的影响
1、在4种白血病细胞系中检测到HHIP发生不同程度甲基化,其中BALL-1、 Jurkat中HH工P基因启动子区域CpG岛高甲基化,HL6O、K562细胞HHIP基因检测到部分甲基化,而正常人淋巴细胞HHIP基因未发生甲基化。
2.HHIPsiRNA孵育BALL细胞48h后,HHIP的mRNA转录受抑制,电泳条带变暗,HHIP/GAPDH从0.983降至0.409;GlilmRNA转录水平升高,条带变亮,Gli/GAPDH从1.045增至1.383,提示在BALL细胞中HHIP可能Gli的上游负性调控因子,可抑制Gli的转录。
三、As2O3去甲基化机制研究
1、As2O3对BALL-1细胞生长的抑制作用
各药物处理组的细胞增殖抑制率随时间和浓度增加而显著升高,与阴性对照组相比差异有统计学意义(P<0.05),其抑制率呈时间和浓度依赖性。
2、As2O3对细胞周期的影响
不同浓度As2O3处理后,BALL-1细胞不同程度阻滞于G0、G1期,G0/G1期细胞百分比升高,S、G2/M期百分比下降(P<0.05)。提示As2O3能引起BALL-1细胞的G0/G1期停滞,从而抑制细胞增殖。
3、As2O3对HHIP基因甲基化模式的影响
未加药组细胞HHIP-M扩增结果阳性,HHIP-U扩增结果为阴性,提示BALL-1细胞常态下HHIP基因呈高度甲基化；随着As2O3浓度的升高,HHIP-M条带逐渐减弱,而HHIP-U条带逐渐增强,提示BALL-1细胞HHIP基因甲基化程度逐渐降低,而非甲基化程度逐渐升高。以上结果表明：As2O3晚可逆转HHIP基因启动子区的高甲基化状态,且呈浓度依赖性。
4、As2O3对HHIP基因及Hh通路靶基因Gli、下游基因cyclinD表达的影响
5.Oumol/L As2O3处理BALL-1细胞72小时后,HHIP mRNA及蛋白表达明显上调(0.826±0.045vs0.253±0.012,0.328±0.021vs0.024±0.005, P<0.05)。说明5.Oumol/L以上浓度As2O3对BALL-1细胞有去甲基化作用。Hh通路靶基因Gli mRNA表达较药物处理前减低(1.030±0.088vs1.328±0.071P<0.05)。而下游基因cyclinDmRNA表达在药物处理自矿后无明显差异(P>0.05)。
5、As2O3对DNA甲基转移酶及去甲基化酶表达的影响
未加药组BALL-1细胞dnmt1、 dnmt3b基因呈强表达；加药组随As2O3浓度的增加,dnmt1、 dnmt3b基因的表达逐渐减弱,与未加药组差异有显著性(0.548±0.044vs0.886±0.028,0.463±0.043vs0.877±0.039,P<0.05). As2O3处理前后mbd2mRNA的表达无明显变化(P>0.05)。提示As2O3可下调DNA甲基转移酶dnmt1、 dnmt3bmRNA的表达,从而发挥去甲基化作用。
结论
一、BALL-1、Jurkat、HL60细胞系Gli或PTCH表达增加,Hedgehog信号通路异常激活,提示白血病发生可能与Hedgehog信号通路的异常激活有关。
二、HHIP基因启动子甲基化使HHIP基因沉默,失去对Hh通路的调控作用,可能是Hedgehog信号通路的异常激活的机制之一。
三、As2O3对BALL-1细胞HHIP基因有去甲基化作用,且呈浓度依赖性,5.0umol/L以上浓度的As2O3对BALL-1细胞HHIP基因有明显的去甲基化作用。
四、As2O3能抑制DNA甲基化转移酶的活性,从而逆转抑癌基因的甲基化水平,使其重新激活,发挥肿瘤抑制作用。
Background
The etiology and pathogensis of leukemia is very complex, and it is not entirely clear. Epigenetic regulation is a kind of control mode which based on non sequential genetic alterations induced heritable gene expression level changes, including DNA methylation, histone acetylation, small RNA (miRNA) silence. In leukemia development process, it plays a very important role. Epigenetic regulation has led to the tumor suppressor gene expression of silence is one of the important factors inducing leukemia. In recent years, some studys have proved that DNA methylation was a common phenomenon in human leukemia, and closely related to the onset of leukemia.
Demethylation therapy as a new antitumor method has been shows a good application prospect in leukemia treatment, especially opened up a new way in the treatment of refractory and relapsed leukemia.The clinical application of demethylation drugs have begun to hematological malignancies such as acute leukemia and myelodysplastic syndrome(MDS) treatment, such as5-aza-2'-deoxycytidine (deoxyeytidine) etc. As2O3is a kind of traditional Chinese medicine, initially for the treatment of acute promyelocytic leukemia and achieved good results. The main mechanism of As2O3in treatment of acute leukemia is inducing leukemia cell apoptosis. Recent studies found that, As2O3can reverse the methylation of certain tumor suppressor gene in myeloma cells and lymphoma cells, so in induction to apoptosis, As2O3may be has other mechanisms of action.
The Hedgehog signaling pathway plays an important role in human and animal normal embryonic development and organ formation.Its channel members including hedgehog, PTCH, SMO, and Gli.The channel member mutations or error expression will activate the pathways, ultimately leading to cancer occurrence and development. Recent research demonstrated that the pathways relevant to tumors including basal cell carcinoma, pancreatic cancer, colon cancer, stomach cancer, small cell lung cancer, melanoma, breast cancer, ovarian cancer and other. In the tumors that Hh pathway is activated, approximately half did not find the channel member mutation. so we hypothesized that Hh pathway activation may have other regulation mechanism. Although the existing research has proved that Hh pathway has close relationship with tumor, but Hh pathway through what mechanisms contribute to tumorigenesis remain unclear. Recent studies found that some tumor in the presence of Hh signal pathway members promoter methylation. There are also reports indicate that Hh pathway is activate in some leukemia.
For the further study of Hh signaling role in leukemia and activation mechanism, Whether aberrant methylation of tumor suppressor genes are involved in the Hh pathway activation and the occurrence of leukemia, we detect the channel member from the RNA levels and protein level before and after As2O3treatment, detect Hh pathway tumor suppressor gene HHIP methylation status by methylation specific PCR. The study will be have important significance on the further elucidation of the pathogenesis and the discovery of new therapeutic targets in leukemia.
Objective
The study was designed to investigate whether Hh pathway is activate in acute leukemia, and whether the activation of Hedgehog pathway is related to HHIP methylation. To explore the effect of DNA methylation on the pathogenesis of leukemia. Through the research of arsenic trioxide's demethylation effect mechanism on HHIP gene, to explore whether Hh pathway exists target of demethylation drugs.
Methods
Selection of acute myeloid leukemia cell line HL60, chronic myeloid leukemia cell line K562, acute T lymphocytic leukemia cell lines Jurkat, B cell acute lymphoblastic leukemia cell line BALL-1, cell lines containing10%calf serum RPMI1640culture medium, at37degrees and5%C02culture box culture.
1、 The activity level of Hh pathway in leukemia cell lines
1.1using reverse transcription polymerase chain reaction (RT-PCR) to detecte Hedgehog pathway members SHH, PTCH, Gli, HHIP gene expression of the leukemia cell lines and leukemia patients.
1.2Using Western-blot to detect the corresponding protein expression.
2、 Methylation status of HHIP gene in leukemia cell lines and its influence on the expression of Gli
2.1Using DNA Extraction Kit by silica gel column extraction of genomic DNA, Select specimen of purity between1.70-1.90.Using DNA CpGenome DNA Methylation kit sample kit for DNA was modified, then using methylation-specific PCR (MS-PCR) to detect Hedgehog signal pathway of HHIP gene promoter region methylation of4leukemia cell lines.
2.2Using siRNA closed HHIP gene expression, then use RT-PCR to detect the expression of Glil HHIP gene.
3、 Demethylation mechanism of arsenic trioxide
3.1Treat BALL-1cell line With different concentrations of As2O3, in accordance with the concentration of As2O3is divided into0.0,2.5,5.0, 10.0umol/L group, after treatment for24,48,72,96hours, add lOul CCK-8solution, the microplate reader on450nm single wavelength measuring absorbance0D value, calculate the inhibition rate, and take the time as the horizontal axis,0D value for the cell growth curve.
3.2As203treatment after48hours, collecte the cells, washing2times by PBS, add cold ethanol fixation for more than24hours, washing cells by PBS,37℃magazine incubated for30minutes, PI staining, avoid the light for30minutes, detect changes of cell cycle after arsenic trioxide treatment by flow cytometry.
3.3As2O3influence HHIP gene methylation pattern of BALL-1cell.BALL-1cells were treated with As2O3for24hours,48hours,72hours, DNA Extraction Kit by silica gel column extraction of genomic DNA, using DNA CpGenome DNA Methylation kit to modify1g DNA sample, then using MS-PCR to detect HHIP gene promoter region methylation of BALL-1cell line.3.4Gli is not only the Hh pathway members, but also the target genes of the pathway, therefore may reflect the activity of the pathway. As2O3act on BALL-1cells after48h, Total RNA extracting reagent RNAiso extracted from BALL-1cell line, using RT-PCR to detect the expression of Gli mRNA.
4.5How does As2O3influence DNA methyltransferase dnmt1、 dnmt3b mRNA and mbd2mRNA expression:We detect dnmt1、 dnmt3b and mbd2mRNA expression by RT-PCR.
Result
1、 The activity level of Hh pathway in leukemia cell lines
The important components of Hh signaling pathways such as signal peptide SHH, membrane receptor PTCH, downstream of the transcription factor Gli and HHIP mRNA have different levels of expression. In the majority of acute leukemia cell lines, Gli or PTCH mRNA expression were increased(P<0.05), the expression of SHH had no obvious change, except for K562cell lines, the rest of leukemic cell lines HHIP expression is reduced (P<0.05). The expression of HHIP protein in BALL-1、Jurkat、 HL60was lower than that of control group, and protein expression of Gli in BALL、HL60is higher than that of Control group.
2、 Methylation status of HHIP gene in leukemia cell lines
2.1BALL-1、 Jurkat cells showed hypermethylation,HL60、 K562showed partial methylation, and HHIP gene were not detected methylation in normal lymphocyte.
2.2Incubation BALL-1by HHIPsiRNA for48h, HHIP mRNA transcription is inhibited, HHIP/GAPDH decreased from0.983to0.409; GlimRNA transcription levels are elevated, Gli/GAPDH increased from1.045to1.383. This indicate that HHIP is probably the upstream negative regulatory factor of Glil.
3、Demethylation mechanism of arsenic trioxide
3.1After treated with arsenic trioxide, the inhibition rate of cell proliferation is increased remarkably with time and concentration, compared with the negative control group the differences were statistically significant (P<0.05), the inhibition rate was time and concentration dependent.3.2After treated with Different concentrations of As2O3, BALL-1cells wre blocked in G0, G1in different degree, percentage of G0/G1phase cells increased, and percentage of S、 G2/M phase decreased (P<0.05).
3.3HHIP-M amplification is positive, while the HHIP-U amplification results is negative, suggesting HHIP gene is highly methylated in BALL-1cells. As the As2O3concentration increased, HHIP-M bands gradually weakened, while the HHIP-U strip increases gradually, suggesting that HHIP gene methylation degree is reduced gradually, but the degree of unmethylation increased gradually. The above results indicate:As2O3can reverse HHIP gene promoter hypermethylation status, and the effect depended on the concentration of
3.4As2O3influence the expression of HHIP and Gli mRNA
Detected the expression of Gli and HHIP mRNA by RT-PCR before and after treated with As2O3, the results showed that HHIP mRNA expression was significantly upregulated after treated with5.0umol/L As2O3for72h. HH pathway target genes GlimRNA expression were reduced.
3.5As2O3influence the expression of dnmt1、 dnmt3b mRNA
As2O3can downregulate DNA methyltransferase(dnmtl、 dnmt3b) mRNA. experimental groups with As2O3increasing the concentration of dnmt1、 dnmt3b gene expression gradually weakened(P<0.05). But the expression of mbd2mRNA did not change significantly after treated with As2O3(p>0.05).
Conelution
1The expression of Gli and PTCH were increased in some leukemic cell lines, which suggest that Hedgehog pathway is aberrant activate in leukemia. Hh pathway activation may be associated with the pathogenesis of leukemia.
2Promoter methylation induced the silence of HHIP gene, and HHIP lost its function on the Hh pathway. It may be the aberrant activation mechanism of Hedgehog pathway.
3As2O3can demethylation HHIP gene in BALL-1cells in a concentration dependent manner.
4As2O3demethylation effect mechanism for the inhibition of S-methionine dependence of the methyltransferase, such as dnmtl、dnmt3b,thereby restoring gene expression.
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