摘要
背景与目的
目前已经知道,Hedgehog(Hh)信号通路在胚胎的正常发育中起着非常重要的作用。近年来的研究发现,Hh信号通路可能在成体组织中再度激活,从而在肿瘤发生中起重要作用。
Hh有三种同分子体配体:Sonic hedgehog(SHH),Indian hedgehog(IHH)和Desert hedgehog(DHH),其中对SHH的研究最多,主要是因为传统观点认为:SHH在各种组织中均有表达,SHH的实验结果通常适用于Hh的其它同分子体(IHH、DHH)。但是,最近的研究结果对Hh三种同分子体的功能重叠性提出了质疑,有研究表明,在肿瘤形成过程中,IHH并不是SHH的功能同系物。SHH和IHH在胃肠道早期发育的几个关键过程中是必需的,结肠中主要表达这两种形式的配体。
Hh配体结合跨膜受体Patched(PTCH)后,释放PTCH介导抑制的Smoothened(SMO)受体。SMO阻遏解除使GLI锌指转录因子激活并转入核内,促进Hh靶基因的转录。脊椎动物有3种GLI,即GLI-1、GLI-2和GLI-3,是Hh信号通路的最终效应分子,其中GLI-1激活大部分Hh信号通路靶基因的转录,其本身也是Hh信号通路的靶基因。
肿瘤中Hh信号通路的异常激活主要是通过基因突变(配体非依赖性)或配体高表达(配体依赖性)实现的。文献报道,PTCH、SMO、Suppressor of Fused(SUFU)基因突变与基底细胞癌、成神经管细胞瘤、横纹肌肉瘤中Hh信号通路的激活有关。另一方面,在一些消化道肿瘤中发现了该通路的配体依赖性激活,包括:肝癌、胰腺癌、食管癌、胃癌、胆管癌。此外,消化道肿瘤中很少发现Hh通路组成成分的突变,提示消化道肿瘤中Hh信号通路的激活是配体依赖性的。
有关Hh信号通路在结直肠癌(colorectal cancer,CRC)中的确切作用,目前已作了大量研究。但是,有关该通路在CRC中是否激活的研究结果互相矛盾,Hh通路阻滞剂对结肠癌细胞活性的影响也存在争议。此外,Hh信号通路在结直肠肿瘤发生中的调节机制尚未完全阐明。因此,阐明Hh信号通路在结直肠癌中的作用及其调节机制,可望为其治疗措施带来新的发现。
表观遗传修饰是基因表达调控的最重要机制之一,尤其是DNA甲基化在肿瘤发病机制的表观修饰异常中研究得最为广泛。DNA甲基化检测方法众多,选择合适的方法对于甲基化研究至关重要,但目前国内尚缺乏对常用甲基化检测方法的系统评价。
本研究目的在于研究结直肠肿瘤中Hh信号通路的配体依赖性活性,并试图阐明结直肠肿瘤发生过程中Hh配体表达的调控方式。另外,本研究对常用甲基化检测方法进行初步评价,为甲基化研究提供参考。
材料和方法
一、Hh信号通路在原发性结直肠肿瘤中的活性研究。
1、收集21例结直肠腺瘤(adenoma,AD)和18例CRC标本。每例标本分为两部分:一部分立即液氮冷冻,-80℃保存备用;另一部分10%福尔马林固定,石蜡包埋。
2、采用半定量RT-PCR方法,检测结直肠肿瘤组织中Hh信号通路关键分子SHH、IHH、GLH mRNA的表达水平。
3、采用免疫组织化学方法,检测结直肠肿瘤组织中SHH、IHH、GLI1蛋白质的表达,并采用半定量方法对组织化学染色进行评分。
二、Hh信号通路在结肠癌细胞株中的活性研究。
采用半定量RT-PCR方法,检测HCT8、SW1116、SW480和Lovo 4株结肠癌细胞株中Hedgehog信号通路关键分子SHH、IHH、PTCH、SMO、GLH mRNA的表达水平。
三、Hh信号通路在CRC中配体依赖性活性调控机制的研究。
1、去甲基化试验在4株结肠癌细胞株中进行去甲基化试验,10μM 5-氮-2-脱氧胞苷(5-aza-dCyd)去甲基化处理4株结肠癌细胞株5天,对照组仅给予PBS处理,同样条件下培养。每个细胞株均进行3次重复独立试验。观察去甲基化处理对4株结肠癌细胞株SHH、IHH、PTCH、SMO、GLI1 mRNA表达的影响,筛选可能被高甲基化沉默的基因。
2、亚硫酸氢盐修饰后测序法(bisulfite sequencing,BSP)采用BSP方法,检测细胞株中SHH、IHH基因启动子的甲基化状态。从细胞株及组织中提取基因组DNA,取2μg进行亚硫酸氢盐修饰后,半巢式PCR扩增,将扩增产物亚克隆入pGMT-T载体。然后对克隆进行测序分析,细胞株中至少检测5个克隆,组织至少检测10个克隆。
3、甲基化特异性PCR(methylation specific PCR,MSP)采用MSP方法,检测原发性肿瘤组织中SHH、IHH基因的甲基化状态。亚硫酸氢盐修饰后的DNA作为MSP的模板,采用对甲基化或非甲基化序列特异的引物进行扩增。扩增产物在2%的琼脂糖凝胶上分离,EB染色,紫外灯下成像观察。
四、甲基化检测方法的初步评价。
1、以含SHH全甲基化克隆序列和全非甲基化克隆序列质粒为模板,对常用甲基化检测方法,包括BSP、MSP、combined bisulfite restriction analysis(COBRA)、PCR产物直接测序进行初步评价。
2、BSP:TA克隆试剂盒将BSP扩增产物亚克隆进pGMT-T载体,采用下游引物测序分析。
3、MSP:以含不同比例甲基化序列的质粒混合物作为模板,检测MSP方法对甲基化序列检出的敏感性。
4、COBRA:对含不同比例甲基化序列的模板进行PCR扩增,随后采用限制性内切酶HhaⅠ对PCR产物进行酶切消化。
5、传统亚硫酸氢盐修饰后直接测序法:采用BSP引物扩增不同比例甲基化序列模板,随后采用下游引物对PCR产物进行直接测序分析。
6、标记引物亚硫酸氢盐修饰测序法:采用标记引物扩增不同比例甲基化序列模板,随后采用下游tagged引物对产物进行直接测序分析。
五、统计学处理
计量资料以(?)±SD表示,计数资料以百分比(%)表示。采用SPSS13.0对数据进行分析,连续变量两组间比较采用独立t检验或Mann-Whitney U检验。分类变量两组间比较采用x~2检验或Fisher精确概率法,P＜0.05认为差异有统计学显著性,所有检验均为双侧。
结果
1.原发性CRC中存在Hh信号通路的SHH配体依赖性激活。
SHH mRNA在CRC组和AD组中无显著差异(t＝-0.225,P＝0.823);与AD组相比,CRC组IHH mRNA水平(Mann-Whitney U检验,Z＝-3.197,P＝0.001)及GLI1 mRNA水平(t＝2.787,P＝0.008)均显著降低。但是,SHH蛋白(Mann-Whitney U检验,Z＝-4.407,P＜0.001)和GLI1蛋白(t＝-2.213,P＝0.033)在CRC组的表达显著高于AD组;IHH蛋白在CRC及AD组中均为阴性或弱阳性表达,两组间无显著差异(t＝0.696,P＝0.491),而在腺瘤旁正常组织常常有IHH的阳性表达。
2.结肠癌细胞株中Hh配体转录物表达缺失,去甲基化处理能诱导其重新表达。
除了SHH mRNA在HCT8有表达外,SHH、IHH mRNA在大部分结肠癌细胞株中均没有表达,大部分Hh通路组成成分在低分化的Lovo细胞中均没有表达。5-氮-2-脱氧胞苷去甲基化处理细胞5天后,能诱导SHH和IHH mRNA在HCT8、SW1116和SW480的重新表达,但在Lovo细胞中没有表达。此外,去甲基化处理后,没有观察到PTCH和SMO mRNA表达的显著增加。
3.结肠癌细胞株中SHH和IHH基因启动子高甲基化。
我们在Lovo细胞株中未能扩增出BSP产物。HCT8、SW1116和SW480结肠癌细胞株中,SHH基因5'端CpG岛为高甲基化,IHH基因5'端CpG岛部分甲基化。
4.原发性CRC中SHH基因的低甲基化以及IHH基因的高甲基化。
50.0%的AD和70.6%的CRC中检出IHH甲基化;80.0%的AD中检出SHH高甲基化,而SHH在CRC中的甲基化率仅为23.5%,显著低于AD组(P＝0.001)。
5.常用甲基化检测方法中,每种方法各有其优缺点。
(1)BSP法能精确分析目的序列中每个CpG位点的甲基化状况;
(2)MSP检测甲基化具有高灵敏度的优点,提高MSP退火温度能克服假阳性结果的产生;
(3)COBRA容易出现非甲基化偏性;
(4)PCR产物直接测序定量分析存在信号质量差、C峰过度代偿的问题。
结论
1.SHH配体依赖性Hh信号通路激活可能在原发性CRC发生中起重要作用。
2.IHH表达缺失或下调可能是结直肠肿瘤进展过程中的一个早期事件。
3.结肠癌细胞株中不存在Hh信号通路的配体依赖性活性。
4.SHH、IHH在原发性结直肠肿瘤和结肠癌细胞株中的表达调控主要与其启动子异常甲基化有关。
5.常用甲基化检测方法中,每种方法各有其优缺点。
(1)BSP法能精确分析每个CpG位点的甲基化状况,但较昂贵和繁琐;
(2)MSP法灵敏度高,但要注意优化退火温度,防止产生假阳性结果;
(3)COBRA法简便易行,但容易出现非甲基化偏性;
(4)PCR产物直接测序适用于大规模检测的需要,但目前技术仍有待改进。
Background and aims
The Hedgehog(Hh) signaling pathway is known to have a paramount role in the proper development of the embryo.In the past few years,it has become clear that the Hh pathway can have a crucial role in tumorigenesis when reactivated in adult tissues.
There are 3 vertebrate homologues of Hh ligands:Sonic hedgehog(SHH),Indian hedgehog(IHH),and Desert hedgehog(DHH).Of the three Hh-family genes in mammals,SHH has been the most studied,mainly because of the prevailing paradigm that SHH is expressed in various tissues and experiments with SHH protein are generally also applicable to other Hh homologues.However,recent studies challenge the functional redundancy of three Hh molecules by showing that IHH is not a functional homologue of SHH activity during tumor formation.SHH and IHH,required in early endoderm and several essential aspects of gastrointestinal development,seem to be the only Hh ligands expressed in the colon.
Briefly,when Hh ligands bind the transmembrane receptor Patched(PTCH), PTCH-mediated suppression of Smoothened(SMO) receptor is relieved,leading to GLI entering the nucleus and activating transcription of Hh target genes. Vertebrate cells contain 3 Gli genes,Gli-1,-2 and -3.Gli1,the final effector molecule of Hh signaling,activates transcription of most downstream target genes and is itself a transcriptional target of the pathway.
Aberrant activation of the Hh pathway in cancers is driven by either mutations (ligand independent) or ligand overexpression(ligand dependent).Mutations of PTCH,SMO and Suppressor of Fused(SUFU) have been reported to be responsible for ligand independent activation of the pathway in basal cell carcinoma, medulloblastoma,and rhabdomyosarcoma.On the other hand,ligand-dependent activation of Hh pathway has been shown in a number of digestive tract malignancies, including liver cancer,pancreatic adenocarcinoma,oesophageal,stomach and biliary tract cancers.Moreover,rare mutations of pathway components in digestive tract tumors also suggest that the Hh pathway is activated ligand dependently in these tumors.
Much work has been done to understand the exact role of this pathway in colorectal cancer(CRC).However,the published data on the Hh pathway activation in CRC are conflicting,and the effect of Hh pathway inhibitor on the viability of colon cancer cells is controversial.Furthermore,the details of Hh pathway regulation in CRC are still being unravelled.Thus,understanding the roles and mechanisms of Hh signaling in CRC should give novel insights into potential treatments for this disease.
Epigenetic modification is one of the most important mechanisms in gene expression regulation.The most widely studied epigenetic abnormality in tumorigenesis is DNA methylation.There are a range of widely used methodologies in methylation studies,and each has its own advantages and disadvantages.The choice of proper technique is crucial in methylation analysis.However,an overall assessment for most commonly used techniques in methylation studies is lacking, especially in China.
The present study was undertaken to investigate the ligands dependent activity of Hh pathway in colorectal tumors,and to attempt to clarify the regulatory mechanism of the Hh ligands expression during colorectal carcinogenesis.In addition, we assessed the commonly used methods for methylation measurement,in an attempt to provide information on methylation analysis.
Materials and Methods
The activity of the Hh signaling pathway in primary colorectal tumors.
1.21 ADs and 18 CRCs were used in the study.Each specimen was divided into two parts:one was frozen immediately in liquid nitrogen and stored at -80℃until required,the other was fixed in 10%formalin and embedded in paraffin.
2.We investigated the mRNA levels of key molecules(SHH,IHH,GLI1) of the Hh signaling by semi-quantitative RT-PCR in tumor specimens.
3.We also examined the protein expression of these molecules by immunostaining in neoplastic colonic tissues.Immunoreactivity was estimated semiquantitatively
The activity of the Hh signaling pathway in colon cancer cell lines.
The mRNA levels of key molecules of Hedgehog sinaling,including SHH,IHH, PTCH,SMO,and GLI1 were examined by semi-quantitative RT-PCR in 4 colon cancer cell lines.
The regulatory mechanism of the ligands dependent activity of the Hh pathway during colon carcinogenesis.
1.Demethylation treatment We screened for epigenetically silenced genes of the Hh pathway in 4 colon cancer cell lines by demethylation assay.For demethylation experiments,all the 4 colon cancer cells were treated with 10μM of 5-aza-2'-deoxycytidine(5-aza-dCyd) for 5 days.PBS-treated cells were cultured similarly and used as a control.All experiments were done in triplicates.
2.Bisulfite sequencing(BSP) We investigated the methylation status of the SHH and IHH promoters in the colon cancer cell lines by BSP.Genomic DNA was extracted from the colon cancer cell lines and tissue samples.Bisulfite modification of 2μg of genomic DNA was performed.The bisulfite-modified DNA was amplified by seminested PCR.For sequence analysis,the PCR products were subcloned into the pGMT-T vector using a TA cloning kit.At least 5 clones were sequenced for each cell line and 10 clones for each primary tumor tested.
3.Methylation specific PCR(MSP) We also examined the methylation status of SHH and IHH in primary colorectal tumors by methylation specific PCR(MSP). Bisulfite modified DNA was used as a template for MSP using primers specific for either the methylated or unmethylated sequences.The amplification products were separated on a 2%agarose gel and visualized by ethidium bromide staining and UV transillumination.
The preliminary assessment of commonly used methods for methylation measurement.
1.Plasmids derived from hypermethylated clone and unmethylated clone for SHH gene were served as template,to assess commonly used methods for methylation measurement,including BSP,MSP,combined bisulfite restriction analysis(COBRA) and direct sequencing of PCR products.
2.BSP:The PCR products were subcloned into the pGMT-T vector using a TA cloning kit.Clones were sequenced using reverse primers.
3.MSP:Variant proportion of methylated sequence was used as template,to determine the sensitivity of MSP for detecting methylated sequence.
4.COBRA:Variant proportion of methylated sequence was amplified by BSP primers,followed by restriction enzyme digestion using Hha I.
5.Conventional bisulfite genomie sequencing:Variant proportion of methylated sequence was amplified by BSP primers,and the products were sequenced directly using reverse primers.
6.Tagged bisulfite genomie sequencing:Variant proportion of methylated sequence was amplified by tagged primers,and the products were sequenced directly using tagged primers.
Statistical analysis
Results for continuous variables were presented as Mean±SD.Results for categorical variables were presented as Number(%).Two-group differences in continuous variables were assessed by independent-samples T test or Mann-Whitney U-test.Two-group differences in categorical variables were determined by the x~2 or Fisher exact tests.Differences were considered significant if P＜0.05.All significance tests are two-tailed.All statistical tests were performed using the software SPSS Version 13.0.
Results
1.The Hh pathway is activated SHH-dependently in primary CRCs.
There was no significant difference.in mRNA expression of SHH between CRCs and ADs(t＝-0.225,P＝0.823).Both IHH transcripts(Mann-Whitney U test, Z＝-3.197,P＝0.001) and GLI1 transcripts(t＝2.787,P＝0.008) were significantly decreased in CRCs compared with those in ADs.However,Protein expressions of SHH and GLI1 were increased significantly in primary CRCs compared with those in colorectal adenomas(ADs)(Mann-Whitney U test,Z＝-4.407,P＜0.001;t＝-2.213, P＝0.033,respectively).IHH was not expressed or expressed at a very low level in primary colorectal tumors,and no significant difference was found between the 2 groups(t＝0.696,P＝0.491).In addition,positive staining for IHH was frequently observed in the adjacent morphologically normal tissue of ADs.
2.Absence of Hh ligands transcriptions and their induction after DNA demethylation treatment in colon cancer cell lines.
SHH and IHH mRNA showed a complete absence of expression in all of the colon cancer cells,except expression of SHH in HCT8.Most components were silenced in poorly differentiated Lovo cells.After treatment with 5-aza-dCyd for 5 days,induction of SHH and IHH mRNA expression occurred in HCT8,SW1116,and SW480 cells,but not in Lovo cells.In addition,significant increase of PTCH and SMO expression was not observed after demethylation treatment.
3.Promoters of SHH and IHH were hypermethylated in colon cancer cell lines.
We failed to amplify BSP products from Lovo cells.In HCT8,SW1116,and SW480 cells,the 5'CpG island of SHH was densely methylated,and the island of IHH was partially methylated.
4.Hypomethylation of SHH promoter and hypermethylation of IHH promoter in primary CRCs.
IHH hypermethylation was frequently observed in ADs(50.0%) and CRCs (70.6%);SHH was hypermethylated in ADs(80.0%),but hypomethylated in CRCs (23.5%,P＝0.001).
5.In the range of currently widely used methodologies in methylation studies, each method has its own advantages and disadvantages.
a.BSP revealed the methylation status of each CpG dinuclotide for a gene of interest.
b.MSP had the advantage of high sensitivity for methylated sequences,and elevating annealing temperature overcame false positivity in MSP analysis.
c.There was a bias towards the amplification of unmethylated DNA in COBRA analysis.
d.Quantitative analysis by direct sequencing of the PCR products from bisulfite-treated DNA implicated several challenges:poor signal quality and overscaled cytosine signals.
Conclusions
1.The SHH dependent activation of the Hh pathway may play a role in the carcinogenesis of primary CRCs.
2.Absence or downregulation of IHH expression may be an early event in the course of colonic tumor progression.
3.Ligand dependent activity of the Hh pathway is lost in colon cancer cell lines.
4.Aberrant methylation of promoter plays a major role in the expression regulation of SHH and IHH in primary colorectal tumors and colon cancer cell lines.
5.In the range of currently widely used methodologies in methylation studies, each method has its own advantages and disadvantages.
a.BSP can reveal the methylation status of each CpG dinuclotide.Nevertheless, this technique is relatively expensive and elaborate.
b.MSP had the advantage of high sensitivity for methylated sequences,but the annealing temperature should be optimized to overcome false positivity.
c.There was a bias towards the amplification of unmethylated DNA in COBRA analysis,although this technique is relatively easy to perform.
d.Direct sequencing of the PCR products from bisulfite-treated DNA implied a potential high-throughput sequencing,but this technology need to be improved according to our experience.
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