食管癌染色体易位和基因重排的鉴定
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摘要
染色体易位和基因重排的形成是人类恶性肿瘤发生发展的重要机制之一,目前只在少数几种实体肿瘤中发现了一些融合基因,尚无食管癌中基因重排的报道。本研究通过筛选和鉴定食管癌中由染色体易位引起的融合基因、截短基因以及异常拼接基因,旨在进一步探索可能与食管癌发生发展相关的特异性基因重排。
本实验室前期工作中,通过重复多色荧光原位杂交(M-FISH)分析在食管癌细胞系KYSE180和KYSE450中检测到一些染色体易位。本研究利用24色M-FISH技术对另外6个食管癌细胞系YES2、EC9706、KYSE30、KYSE140、KYSE150和KYSE510进行了检测。8个细胞系的M-FISH结果表明,各细胞系均存在众多染色体易位,涉及的染色体以1,2,3,5,7,8,9,11,12,13,15号居多,其中还包含一些复杂的多重易位,如KYSE30中的t(13;17;13;17)、YSE510中的t(8；9；18；9；8)等。
对8个细胞系进行了4×44 K寡核苷酸比较基因组杂交芯片(Oligo arrayCGH)检测,根据基因内部片段之间拷贝数的差别,并结合M-FISH结果,初步筛选出可能同时涉及染色体易位和断裂的基因。利用其中30个基因两侧BAC探针进行FISH分析,验证出23个基因在对应细胞系中确实发生断裂。然后运用多种分子生物学方法对其融合和断裂连接点进行鉴定。
利用3'RACE技术分别在KYSE450和KYSE30细胞系中鉴定出2个形成了融合转录本的基因和1个截短基因。序列比对结果表明,NTRK3基因(15q25.3)与5q32上的基因组DNA序列在KYSE450细胞系中由于t(5；15)从而发生拼接,BAC-FISH结果证实了二者的融合。形成的融合转录本5’端为NTRK3基因的Exonl-Exonl3,3’端由5q32上的两段序列拼接组成。进一步利用FISH技术验证了NTRK3在食管癌组织标本中的断裂频率为14.7%(10/68)。MYH10基因(17p13.1)与13q14.3上的序列在KYSE30细胞系中通过t(13；17；13；17)形成融合基因,包含2个融合转录本,二者5’端均为MYH10基因Exonl-Exon4,3’端来自于13q14.3的基因组DNA序列,在两个转录本中具有不同的长度,且与MYH 10 cDNA的连接点不同。MCTP1基因(5q14.4)在KYSE30细胞中形成了截短转录本,只具有Exon1-Exon17的部分。基因组DNA PCR结果显示,其3’端序列纯合缺失,断裂点位于Intronl7内一段505 bp的序列中。
针对arrayCGH吉果显示断点两侧拷贝数差异比较明显的基因,我们首先采用多轮半定量PCR方法将其断裂点范围缩小至1 kb以内,然后利用基因组步移技术扩增跨越断裂连接点的序列。结果显示,KYSE150细胞中PTPRD基因发生自身拼接,断裂连接点区域由两段相距约78 kb的序列正向连接形成。
以上结果表明,食管癌中存在众多染色体易位和基因重排,为进一步在食管癌组织中鉴定出特异的融合、截短以及异常拼接的基因提供了重要线索。
Chromosomal translocation is one of the most common genetic alterations in human malignant tumors. So far, a few gene rearrangements, especially fusion genes, have been identified in several solid tumors, but no such evidence was found in esophageal carcinoma. This research aims to identify specific fusion, truncated and abnormally joining genes caused by chromosomal translocations in esophageal tumors.
Several chromosomal translocations have been observed from esophageal cell line KYSE180 and KYSE 450 in previous studies. Here, we tested another six cell lines, including YES2, EC9706, KYSE30, KYSE140, KYSE150 and KYSE510, by using 24-color multiplex-fluorescent in-situ hybridization (M-FISH). M-FISH results show that many chromosomal translocations exit in eight cell lines, and even more complex translocations, such as t(13; 17; 13; 17) and t(8;9;18;9;8), could also be detected.
Gene breaks were analyzed according to the different copy numbers within the same gene, which could be tested by oligo array based comparative genomic hybridization (oligo array CGH). With the combination of M-FISH and array CGH, we screened genes with breakpoints which may caused by chromosomal translocations.
Two fusion genes and one truncated gene were discovered from 3'RACE and its sequencing results. NTRK3 (15q25.3) in KYSE450 fused to 5q32 as a result of t(5;15), which was validated by BAC FISH. The 5'side of the fusion transcript includes Exonl-Exon13 of NTRK3, and 3'side was composed of two genomic DNA fragments in 5q32, which were apart from each other. FISH results indicated that break of NTRK3 exist in 14.7%(10/68) tissue samples of esophageal carcinoma. Another fusion gene was between MYH10 (17p13.1) and 13q14.3 in KYSE30, which was caused by t(13;17;13;17). The fusion gene formed two transcripts, with the same 5' fragment, which was Exonl-Exon4 of MYH10, while 3'sides come from two different fragments inl3q14.3.
One truncated gene MCTPl (5q14.4) in KYSE30 was also identified by 3'RACE. The transcript contained only Exonl-Exon17, and subsequent genomic DNA PCR results indicated that the breakpoint was located in Intron17, with a homozygous deletion from the downstream of the breakpoint.
Genome walking was used in this study to amplify the junctions. Sequencing results showed that PTPRD in KYSE150 formed an abnormal self-joining gene, composed of two fragments with 78 kb apart from each other.
In conclusion, our studies indicated that the karyotypes of esophageal carcinoma cell lines were complex, and various gene rearrangements have been identified.
本实验室前期工作中,通过重复多色荧光原位杂交(M-FISH)分析在食管癌细胞系KYSE180和KYSE450中检测到一些染色体易位。本研究利用24色M-FISH技术对另外6个食管癌细胞系YES2、EC9706、KYSE30、KYSE140、KYSE150和KYSE510进行了检测。8个细胞系的M-FISH结果表明,各细胞系均存在众多染色体易位,涉及的染色体以1,2,3,5,7,8,9,11,12,13,15号居多,其中还包含一些复杂的多重易位,如KYSE30中的t(13;17;13;17)、YSE510中的t(8；9；18；9；8)等。
对8个细胞系进行了4×44 K寡核苷酸比较基因组杂交芯片(Oligo arrayCGH)检测,根据基因内部片段之间拷贝数的差别,并结合M-FISH结果,初步筛选出可能同时涉及染色体易位和断裂的基因。利用其中30个基因两侧BAC探针进行FISH分析,验证出23个基因在对应细胞系中确实发生断裂。然后运用多种分子生物学方法对其融合和断裂连接点进行鉴定。
利用3'RACE技术分别在KYSE450和KYSE30细胞系中鉴定出2个形成了融合转录本的基因和1个截短基因。序列比对结果表明,NTRK3基因(15q25.3)与5q32上的基因组DNA序列在KYSE450细胞系中由于t(5；15)从而发生拼接,BAC-FISH结果证实了二者的融合。形成的融合转录本5’端为NTRK3基因的Exonl-Exonl3,3’端由5q32上的两段序列拼接组成。进一步利用FISH技术验证了NTRK3在食管癌组织标本中的断裂频率为14.7%(10/68)。MYH10基因(17p13.1)与13q14.3上的序列在KYSE30细胞系中通过t(13；17；13；17)形成融合基因,包含2个融合转录本,二者5’端均为MYH10基因Exonl-Exon4,3’端来自于13q14.3的基因组DNA序列,在两个转录本中具有不同的长度,且与MYH 10 cDNA的连接点不同。MCTP1基因(5q14.4)在KYSE30细胞中形成了截短转录本,只具有Exon1-Exon17的部分。基因组DNA PCR结果显示,其3’端序列纯合缺失,断裂点位于Intronl7内一段505 bp的序列中。
针对arrayCGH吉果显示断点两侧拷贝数差异比较明显的基因,我们首先采用多轮半定量PCR方法将其断裂点范围缩小至1 kb以内,然后利用基因组步移技术扩增跨越断裂连接点的序列。结果显示,KYSE150细胞中PTPRD基因发生自身拼接,断裂连接点区域由两段相距约78 kb的序列正向连接形成。
以上结果表明,食管癌中存在众多染色体易位和基因重排,为进一步在食管癌组织中鉴定出特异的融合、截短以及异常拼接的基因提供了重要线索。
Chromosomal translocation is one of the most common genetic alterations in human malignant tumors. So far, a few gene rearrangements, especially fusion genes, have been identified in several solid tumors, but no such evidence was found in esophageal carcinoma. This research aims to identify specific fusion, truncated and abnormally joining genes caused by chromosomal translocations in esophageal tumors.
Several chromosomal translocations have been observed from esophageal cell line KYSE180 and KYSE 450 in previous studies. Here, we tested another six cell lines, including YES2, EC9706, KYSE30, KYSE140, KYSE150 and KYSE510, by using 24-color multiplex-fluorescent in-situ hybridization (M-FISH). M-FISH results show that many chromosomal translocations exit in eight cell lines, and even more complex translocations, such as t(13; 17; 13; 17) and t(8;9;18;9;8), could also be detected.
Gene breaks were analyzed according to the different copy numbers within the same gene, which could be tested by oligo array based comparative genomic hybridization (oligo array CGH). With the combination of M-FISH and array CGH, we screened genes with breakpoints which may caused by chromosomal translocations.
Two fusion genes and one truncated gene were discovered from 3'RACE and its sequencing results. NTRK3 (15q25.3) in KYSE450 fused to 5q32 as a result of t(5;15), which was validated by BAC FISH. The 5'side of the fusion transcript includes Exonl-Exon13 of NTRK3, and 3'side was composed of two genomic DNA fragments in 5q32, which were apart from each other. FISH results indicated that break of NTRK3 exist in 14.7%(10/68) tissue samples of esophageal carcinoma. Another fusion gene was between MYH10 (17p13.1) and 13q14.3 in KYSE30, which was caused by t(13;17;13;17). The fusion gene formed two transcripts, with the same 5' fragment, which was Exonl-Exon4 of MYH10, while 3'sides come from two different fragments inl3q14.3.
One truncated gene MCTPl (5q14.4) in KYSE30 was also identified by 3'RACE. The transcript contained only Exonl-Exon17, and subsequent genomic DNA PCR results indicated that the breakpoint was located in Intron17, with a homozygous deletion from the downstream of the breakpoint.
Genome walking was used in this study to amplify the junctions. Sequencing results showed that PTPRD in KYSE150 formed an abnormal self-joining gene, composed of two fragments with 78 kb apart from each other.
In conclusion, our studies indicated that the karyotypes of esophageal carcinoma cell lines were complex, and various gene rearrangements have been identified.
引文
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