修回日期: 2010-11-17
接受日期: 2010-12-01
在线出版日期: 2011-03-18
目的: 探索大肠腺瘤与大肠腺癌差异表达的肿瘤相关基因.
方法: 利用基因芯片技术筛选大肠腺瘤与大肠腺癌差异表达的肿瘤相关基因, 比较两组基因特点, 寻找大肠腺癌重要致病基因, 并以RT-PCR对部分差异表达基因进行检测来验证芯片结果.
结果: (1)大肠腺瘤差异表达的肿瘤相关基因9个, 均上调; (2)大肠腺癌差异表达的肿瘤相关基因47个, 其中上调29个, 下调18个; (3)大肠腺瘤与大肠腺癌共同差异表达的肿瘤相关基因有17个, 其中14个基因均上调; (4)CAPN1、JUNB、ELF3、IER3等基因在大肠腺癌与大肠腺瘤中差异表达上调, 但在大肠腺癌中显著表达, PDGFRA及PLAGL1基因在大肠腺瘤中表达上调, 而在大肠腺癌相反.
结论: 大肠腺瘤差异表达的肿瘤相关基因较少, 大肠腺癌则明显增多; CAPN1、JUNB、ELF3、IER3、PDGFRA及PLAGL1等基因可能为大肠腺癌重要致病基因.
引文著录: 曾伟, 南清振, 戴益琛, 朱小三, 陈章兴, 谢军培, 傅育卡, 林园园. 大肠腺瘤与大肠腺癌肿瘤相关基因的差异表达. 世界华人消化杂志 2011; 19(8): 868-873
Revised: November 17, 2010
Accepted: December 1, 2010
Published online: March 18, 2011
AIM: To identify differentially expressed tumor-related genes between colorectal adenoma and adenocarcinoma.
METHODS: Gene chip technology was used to screen differentially expressed tumor-related genes between colorectal adenoma and adenocarcinoma. RT-PCR was then performed to validate microarray results.
RESULTS: Nine differentially expressed tumor-related genes were expressed only in colorectal adenoma, all of which were up-regulated. Forty-seven differentially expressed tumor-related genes were expressed only in adenocarcinoma, of which 29 were up-regulated and 18 down-regulated. Seventeen differentially expressed tumor-related genes were expressed in both colorectal adenoma and adenocarcinoma, of which 14 were up-regulated. CAPN1, JUNB, ELF3 and IER3 genes were up-regulated in both colorectal adenoma and adenocarcinoma, but the up-regulation was more significant in colorectal adenocarcinoma. PDGFRA and PLAGL1 genes were up-regulated in colorectal adenoma but down-regulated in adenocarcinoma.
CONCLUSION: Colorectal adenoma expresses less differentially expressed tumor-related genes than adenocarcinoma. CAPN1, JUNB, ELF3, IER3, PDGFRA and PLAGL1 genes may be involved in the pathogenesis of colorectal adenocarcinoma.
- Citation: Zeng W, Nan QZ, Dai YC, Zhu XS, Chen ZX, Xie JP, Fu YK, Lin YY. Differential expression of tumor-related genes between colorectal adenoma and adenocarcinoma. Shijie Huaren Xiaohua Zazhi 2011; 19(8): 868-873
- URL: https://www.wjgnet.com/1009-3079/full/v19/i8/868.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v19.i8.868
大肠癌是最常见的恶性肿瘤之一, 本研究利用基因芯片技术分别筛选大肠腺瘤与大肠腺癌差异表达的肿瘤相关基因, 分析两者基因表达的特点, 寻找大肠腺癌可能的致病基因[1].
大肠腺癌合并大肠腺瘤患者5例, 内镜下留取组织标本均经病理检查证实为大肠腺瘤及大肠腺癌, 标本用RNase-Free生理盐水漂洗, 锡纸包裹并标记后迅速冻存于液氮中备用. 含有8 064个人类靶基因的基因表达谱芯片1张, 由深圳微芯生物公司提供.
按TRIzol法提取样品组织总RNA[2], 将5例大肠腺癌的RNA样品等量混合, 进行分光光度计样品质检及反转录荧光标记, 用Cy3-dUTP标记组织的cDNA; 同理将5例大肠腺瘤的RNA等量混合, 再逆转录荧光标记, 用Cy5-dUTP标记组织的cDNA. 将已标记的样品cDNA探针与深圳微芯生物公司提供的含有8 064个人类靶基因的基因表达谱芯片进行杂交, 经清洗、扫描仪扫描荧光图像、提取杂交信号、经转换后以数据形式输出、对数据进行标准化处理后用生物信息学软件进行生物信息学分析. 以芯片中密度值在5×108以上的数据点为有效数据, 同时把Ratio(Cy3/Cy5)比值>2或<0.5的数据点作为存在表达差异基因点的筛选标准, 筛选各组间差异表达基因. 为验证芯片的结果, 应用半定量RT-PCR的方法检测SFN、JUNB、IER3、CAPN1基因在大肠腺癌和腺瘤组织中的表达情况.
实验芯片杂交扫描图像荧光信号强度高, 背景均一, 且分光光度计结果提示两组样本总RNA完整性好(表1). 同时用RT-PCR技术对其中基因表达的验证结果与基因芯片检测结果一致, 符合重复性和可靠性的要求, 说明本研究结果真实、可靠(表2).
| 样品 | A260 | A280 | A260/A280 | RNA浓度(mg/L) | RNA总量(μg) |
| 大肠腺瘤 | 1.602 | 0.796 | 2.02 | 6.410 | 192 |
| 大肠腺癌 | 0.456 | 0.244 | 1.97 | 1.824 | 18.24 |
| 基因功能 | 基因标签 | Ratio | |
| 芯片技术 | RT-PCR技术 | ||
| Stratifin | SFN | 4.22 | 4.18 |
| jun B proto-oncogene | JUNB | 12.25 | 11.86 |
| immediate early response 3 | IER3 | 16.83 | 15.19 |
| calpain 1, (mu/I) large subunit | CAPN1 | 14.75 | 15.30 |
LMNA、PDCD4、TU3A、ZAP70、MAD4、IFI30、RARRES3及SGK2基因在大肠腺瘤组织中表达下调, 仅LCN2基因表达上调, 这些基因在大肠腺癌中未表达(表3).
| GenBank序列号 | 基因功能 | 基因标签 | Ratio | |
| 大肠腺瘤 | 大肠腺癌 | |||
| AA496997 | lamin A/C | LMNA | 0.24 | - |
| N71003 | programmed cell death 4 | PDCD4 | 0.21 | - |
| AA436401 | TU3A protein | TU3A | 0.45 | - |
| AI817942 | zeta-chain associated protein kinase (70 000 Da) | ZAP70 | 0.30 | - |
| AA447515 | Mad4 homolog | MAD4 | 0.31 | - |
| AA630800 | interferon, gamma-inducible protein 30 | IFI30 | 0.47 | - |
| W47350 | retinoic acid receptor responder 3 | RARRES3 | 0.18 | - |
| AI650283 | serum/glucocorticoid regulated kinase 2 | SGK2 | 0.45 | - |
| AA400973 | lipocalin 2 (oncogene 24p3) | LCN2 | 3.16 | - |
下面这部分的基因在大肠腺瘤中正常表达, 而在大肠腺癌组织中显著差异表达, 同时发现ERBB3、ERBB2及KRAS2、HRAS2表达相反(表4).
| GenBank序列号 | 基因功能 | 基因标签 | Ratio | |
| 大肠腺瘤 | 大肠腺癌 | |||
| NM001170341 | myeloid leukemia factor 2 | MLF2 | - | 2.05 |
| NM005438 | FOS-like antigen-1 | FOSL1 | - | 2.09 |
| BC059522 | ribosomal protein S30 | FAU | - | 2.12 |
| BC08072 | v-raf murine sarcoma 3611 viral oncogene homolog 1 | ARAF1 | - | 2.20 |
| NM020531 | chromosome 20open reading frame 3 | C20ORF3 | - | 2.23 |
| NM008583 | multiple endocrine neoplasia I | MEN1 | - | 2.23 |
| NM204434 | cyclin-dependent kinase inhibitor 2A | CDKN2A | - | 2.27 |
| NM133862 | fibrinogen, gamma polypeptide | FGG | - | 2.75 |
| BC162533 | GRO2 oncogene | GRO2 | - | 2.93 |
| NM011492 | serine/threonine kinase 11 (Peutz-Jeghers syndrome) | STK11 | - | 3.02 |
| NM205510 | fibroblast growth factor receptor 1 | FGFR1 | - | 3.52 |
| NM001040403 | prohibitin | PHB | - | 3.66 |
| NM00109824 | v-Ha-ras Harvey rat sarcoma viral oncogene homolog | HRAS | - | 4.03 |
| NM001950 | E2F transcription factor 4,p107/p130-binding | E2F4 | - | 4.08 |
| NM001082478 | insulin-like growth factor 2 receptor | IGF2R | - | 4.17 |
| NM004448 | v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 | ERBB2 | - | 4.57 |
| NM001107159 | matrix metalloproteinase 19 | MMP19 | - | 4.60 |
| NM010658 | v-maf musculoaponeurotic fibrosarcoma oncogene family | MAFG | - | 4.85 |
| NM033158 | hyaluronoglucosaminidase 2 | HYAL2 | - | 5.26 |
| NM022012 | mitogen-activated protein kinase kinase kinase 11 | MAP3K11 | - | 5.34 |
| NM023983 | melanoma adhesion molecule | MCAM | - | 5.48 |
| NM001170716 | breast cancer anti-estrogen resistance 1 | BCAR1 | - | 6.16 |
| NM000535 | postmeiotic segregation increased (S. cerevisiae) 2 | PMS2 | - | 6.18 |
| BC046375 | P53-induced protein | PIG11 | - | 7.66 |
| NM001142573 | IMP (inosine monophosphate) dehydrogenase 1 | IMPDH1 | - | 7.67 |
| NM005380 | neuroblastoma, suppression of tumorigenicity 1 | NBL1 | - | 7.69 |
| NM003542 | H4 histone family, member G | H4FG | - | 8.13 |
| NM002466 | v-myb avian myeloblastosis viral oncogene homolog-like 2 | MYBL2 | - | 10.76 |
| NM022588 | metastasis associated 1 | MTA1 | - | 12.03 |
| NM194359 | v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3 | ERBB3 | - | -6.02 |
| L36953 | MAD (mothers against decapentaplegic) homolog 4 | MADH4 | - | -5.03 |
| NM005638 | ADP-ribosylation factor GTPase activating protein 1 | ARFGAP | - | -4.76 |
| NM032415 | mucosa associated lymphoid tissue lymphoma translocation gene 1 | MALT1 | - | -3.27 |
| NM008284 | v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog | KRAS2 | - | -3.26 |
| XM002190948 | v-ral simian leukemia viral oncogene homolog B | RALB | - | -3.21 |
| NM003766 | coiled-coil, myosin-like BCL2-interacting protein | BECN1 | - | -3.15 |
| NM001005765 | RAP1A, member of RAS oncogene family | RAP1A | - | -3.08 |
| NM002439 | phosphatase and tensin homolog | PTEN | - | -3.05 |
| XM002084629 | TRK-fused gene | TFG | - | -3.03 |
| AY805747 | ras homolog gene family, member E | ARHE | - | -2.95 |
| NM001099373 | SET translocation | SET | - | -2.93 |
| NM053455 | fibrinogen-like 2 | FGL2 | - | -2.70 |
| NM012680 | tuberous sclerosis 1 | TSC1 | - | -2.67 |
| NM001146216 | transforming acidic coiled-coil containing protein 1 | TACC1 | - | -2.59 |
| NM017045 | retinoblastoma 1 (including osteosarcoma) | RB1 | - | -2.42 |
| NM133250 | mutS (E. coli) homolog 2 | MSH2 | - | -2.24 |
| NM005805 | cadherin 1, type 1, E-cadherin (epithelial) | CDH1 | - | -2.10 |
CAPN1、FLT3LG、GADD34、SFN、MAF、PDGFRA、JUNB、LENG4、LOC51329、ELF3、FOS、FOSB、IER3基因在大肠腺癌中高表达, 在腺瘤中低表达, 仅有MGST1、PLAGL1、PDGFRA在腺瘤中高表达, 而在腺癌中低表达, MYC、PTTG1基因则同为高表达(表5).
| GenBank序列号 | 基因标签 | 基因标签 | Ratio | |
| 大肠腺瘤 | 大肠腺癌 | |||
| H15456 | calpain 1, (mu/I) large subunit | CAPN1 | 0.36 | 14.75 |
| AI677994 | fms-related tyrosine kinase 3 ligand | FLT3LG | 0.30 | 2.09 |
| AA464600 | v-myc avian myelocytomatosis viral oncogene homolog | MYC | 2.85 | 3.90 |
| AA460168 | growth arrest and DNA-damage-inducible 34 | GADD34 | 0.25 | 3.70 |
| AA495936 | microsomal glutathione S-transferase 1 | MGST1 | 2.18 | -2.87 |
| AA191692 | stratifin | SFN | 0.36 | 4.22 |
| AA043501 | v-maf musculoaponeurotic fibrosarcoma (avian) oncogene homolog | MAF | 0.36 | 4.85 |
| H23235 | platelet-derived growth factor receptor | PDGFRA | 0.44 | -3.24 |
| N94468 | jun B proto-oncogene | JUNB | 0.33 | 12.25 |
| AA426216 | malignant cell expression-enhanced gene/tumor progression-enhanced gene | LENG4 | 0.49 | 5.80 |
| AA486219 | SRp25 nuclear protein | LOC51329 | 0.26 | 9.77 |
| AA434373 | E74-like factor 3 (epithelial-specific) | ELF3 | 0.33 | 13.54 |
| AA485377 | v-fos FBJ murine osteosarcoma viral oncogene homolog | FOS | 0.32 | 2.09 |
| T61948 | FBJ murine osteosarcoma viral oncogene homolog B | FOSB | 0.28 | 11.31 |
| AA463204 | pleiomorphic adenoma gene-like 1 | PLAGL1 | 2.54 | -2.47 |
| AA430032 | pituitary tumor-transforming 1 | PTTG1 | 2.64 | 15.76 |
| AA457705 | immediate early response 3 | IER3 | 0.21 | 16.83 |
目前认为多数的大肠腺癌来源于大肠腺瘤, 大肠腺瘤发展为大肠腺癌与多个癌基因及抑癌基因有关, Kinzler等[3]提出结肠癌发生及演进模式图, 并阐述APC→K-ras→p53→DCC等基因的变化, 本研究试图寻找其他由大肠腺瘤发展为大肠腺癌的其他少见重要致病基因, 因肿瘤发生、发展是多步骤、多因素的复杂过程, 涉及多种基因及产物相互作用, 故利用芯片技术的高通量、高特异性、快速等优点全面研究大肠腺瘤及腺癌的肿瘤相关基因, 进一步分析差异表达的基因, 从而了解大肠腺瘤发展为腺癌的可能致病基因, 为临床治疗提供基础[4,5].
肿瘤的发生、发展主要是癌基因激活及抑癌基因失活的结果, 本研究发现大肠腺瘤肿瘤相关差异表达基因共9个(均上调), 而大肠腺癌差异表达肿瘤相关基因47个(表达上调的基因29个), 其中两者共同差异表达的肿瘤相关基因17个, 表达上调14个, 同时发现PDGFRA、PLAGL1、CAPN1、JUNB、ELF3、IER3等基因在大肠腺瘤及腺癌中均差异表达, 同时发现较为熟悉的STK11、PTEN等基因仅在大肠腺癌中差异表达, 而在大肠腺瘤正常表达, 推测这些基因可能是从大肠腺瘤进展为大肠腺癌的关键基因.
从上述结果中可以看出并非在癌组织或细胞中癌基因必定表达上调, 如癌基因ERBB3、KRAS2、RALB、RAP1A等表达下调, 然而ERBB2、HRAS2则表达上调, 大部分抑癌基因均表达下调, 个别上调抑癌基因如STK11等. 针对这种现象我们提出可能的假说: 癌变早期或超早期抑癌基因均表达下调, 癌基因表达上调, 但到一定阶段癌基因及抑癌基因表达错乱(癌基因表达下调、抑癌基因表达上调的现象), 目前国内外无类似观点的文献报道, 具体机制需要进一步研究证实. 下面介绍常见癌基因及抑癌基因生物学特点及在大肠腺癌中的作用. PDGFRA基因突变常见于胃肠道间质瘤(gastro intestinal stromal tumor, GIST), Hirota等[6]研究GIST中发现PDGFRA基因功能获得性突变, PDGFRA信号转导途径与c-Kit相似, 其突变后可限制和激活野生型c-Kit在转染细胞中的共表达. 该癌基因在大肠癌中的作用国内外报道甚少, 本研究发现其在大肠腺瘤表达上调, 而大肠腺癌表达下调(Ratio = -3.24), 他与MGST1均为癌基因, 但在大肠癌中表达下调, 故并非所有癌基因均在癌组织中表达上调. 而抑癌基因PLAGL1在大肠腺癌中表达下调, 故推测PDGFRA基因可能在大肠腺癌中的作用与在GIST中不一样, 可能为大肠腺癌特殊的致病基因, 具体机制有待进一步研究. 癌基因junB编码蛋白JUNB常与Fos蛋白形成同源或异源二聚体, 再与DNA结合后可在转录水平调节下游靶基因的表达, 参与肿瘤细胞增殖及转化[7,8], 从表5中发现JUNB在大肠腺癌中高表达(Ratio = 12.25), 而在大肠腺瘤正常表达, 很可能是细胞不断增殖的触发点, 并通过影响其他基因的表达(如ELF3、IER3、CAPN1等), 使细胞最终增殖失控, 发生癌变. STK11基因是一种抑癌基因, 又称LKB1基因, 定位于人染色体19p13.3区, 最先在Peutz-Jeghers综合征中被发现, 他编码丝-苏氨酸激酶(STK11), 对肿瘤的作用是阻断其细胞周期, 从而抑制其生长, 同时有研究发现STK11基因能在P53诱导的细胞凋亡中发挥作用[9,10]. STK11基因为肿瘤抑制基因, 但本研究中其为差异上调表达(Ratio = 3.02), 可能与不同抑癌基因在不同肿瘤发生中的作用不同有关. PTEN基因又称MMA1基因, 是到目前为止发现的首个具有磷酸酶活性的抑癌基因, 可编码具有双重特异性磷酸酶活性的蛋白, 他和STK11基因一样, 不仅仅是个抑癌基因, 也是肿瘤易感基因, 在大肠腺癌的发生和发展中发挥作用[11,12]. 研究证实, 在大肠癌、乳腺癌、子宫内膜癌及非小细胞肺癌等均存在PTEN失活及表达的异常现象, 发病机制: 使PI-3K信号途径中的主要信号分子PIP3等去磷酸化,从而发挥重要的对肿瘤抑制作用[13,14]. 本研究显示, 大肠腺癌组织中PTEN表达水平明显低于大肠腺瘤(Ratio = -3.05), 故PTEN表达下调可能与大肠腺癌关系密切.
本研究发现大肠腺瘤差异表达的肿瘤相关基因较少, 大肠腺癌则明显增多, 较为常见的大肠癌肿瘤相关基因在本研究未筛选到, 如MCC、APC、DCC及p53等, 这些抑癌基因在大肠腺癌组织中表达缺失, 考虑可能与基因表达具有时间性及空间性有关, 即基因表达具有时序性, 不同细胞周期上述基因表达情况不同, 同时可能与不同大肠癌病理类型有关, 也反应了解大肠癌完备的发生、发展机制十分困难, 同时筛选出的MYBL2、H4FG等肿瘤相关基因与大肠癌的关系国内外研究报道甚少[15], 我们所筛选CAPN1、JUNB、ELF3、IER3、PDGFRA及PLAGL1等基因可能为大肠腺癌关键致病基因, 但具体机制有待进一步研究证实.
目前关于大肠癌发病机制仍不清, 仍围绕单个基因的表达调控阐述大肠癌发生及演变模式.
崔莲花, 副教授, 青岛大学医学院公共卫生系; 周素芳, 教授, 广西医科大学科技处
Hirota等研究GIST中发现PDGFRA基因功能获得性突变, PDGFRA信号转导途径与c-Kit相似, 其突变后可限制和激活野生型c-Kit在转染细胞中的共表达.
本研究主要了解大肠腺瘤及大肠腺癌差异表达基因, 同时从单个基因生物学特点, 了解其在大肠腺癌中的作用, 同时创新性提出肿瘤相关基因表达错乱的概念.
本文筛选大肠腺癌关键致病基因, 同时寻找结肠癌是否存在特异性靶点, 为大肠腺癌的早期诊治提供依据.
本文创新性尚可, 对探讨大肠癌发病分子机制有重要参考作用.
编辑: 李薇 电编:李薇
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