摘要
我国属肝癌高发国家,肝癌病死率占恶性肿瘤病死率第二位,占全球肝癌死亡人数的53%。尽管肝癌的治疗学近些年有了很大的进步,但在过去的三十多年,肝癌生存率并无明显改善。临床实践证明,肝癌的成功切除与预后,取决于早期诊断。在高危人群中,早期筛查和发现肝癌,可赢得手术治疗的时间,提高术后生存率。腹部超声、CT、MRI的检查,虽能发现部分小肝脏肿瘤,但其鉴别良、恶性病变较难,因此肿瘤标志物的研究开发和临床应用对肿瘤的诊断治疗预后具有十分重要的意义。
目前,AFP为肝癌较可靠的血清标志物,其灵敏度为60%~70%,特异性为90%左右,但近40%的肝癌AFP不升高,其原因可能是AFP含量与肿瘤体积大小有关。有文献报道,早期肝癌AFP的假阴性率可达40%以上,进展期肝癌AFP的假阴性率也可达15%~20%,因此,单凭AFP诊断肝癌易造成漏诊与误诊。寻找更加敏感、特异的早期诊断标记物显得十分迫切。
有学者认为,将一些敏感性和特异性较差的标志物联合检测,结合生物信息技术,可明显改善诊断的敏感性和特异性。人类基因组计划的完成及相关检测技术的进步,给大规模筛查并发现肿瘤和其他疾病的生物标记带来期望。作为标志物,肿瘤分泌蛋白无疑最具研究价值。
基于此,我们在前期研究中建立了大鼠早期肝癌模型,应用Affymetrix公司的Rat Genome 230.2.0基因芯片检测早期肝癌组织(T)和正常肝组织(N)中mRNA,构建差异表达谱,得到T与N Log R≥2和Log R≤-2差异表达基因共677个,其中表达上调409个,表达下调268个。经后续分析,上调和下调共涉及178种分子功能和204种生物学过程,并根据Gene ontology(GO)分类和应用EMBOSS程序TMAP和SIGLEAVE分别预测氨基酸序列的跨膜结构域和信号肽剪切位点以及Excel交集补集分析,按照其参与的生物学过程和具有分子功能进行聚类并筛选出可能的诊断标志物14个。经同源分析14个候选诊断标志物均与人具有同源性,随后采用RT-PCR及免疫组化对其在大鼠试验组与对照组及人正常肝脏组织、癌旁组织、小肝癌及大肝癌中mRNA水平和蛋白水平的表达进行鉴定,初步预测其在小肝癌诊断中的价值。
研究方法:
1.应用二乙基亚硝胺(diethylnitrosamine DEN)构建大鼠肝癌模型,并经常规病理学检测;
2.提取对照组大鼠正常肝脏组织与实验组早期肝癌组织总mRNA,逆转录cDNA,应用Rneasy Mini kit合成cRNA。cRNA与Affymetrix Rat Genenome 230.2.0基因芯片杂交,扫描芯片,筛选差异表达基因,并行数据归一化与和目标性去冗余处理;
3.应用GO注释结合序列分析筛选上调基因中编码分泌蛋白的基因后行Blast比对、HPRD数据库与文献查询,选择人同源基因作为候选诊断标志物;
4.应用RT-PCR、定量PCR及免疫组化对部分候选标志物在大鼠早期肝癌和人小肝癌中转录和蛋白表达水平进行验证。
结果
1.在DEN灌胃14-16周时,成功构建大鼠早期肝癌模型;
2.Affymetrix Rat Genenome 230.2.0基因芯片杂交后,扫描芯片原始数据经规一化处理筛选出差异表达基因677个,其中上调表达基因409个,下调表达基因268个,其中全长已知基因368个;
3.对上调基因中已知211个基因应用GO注释结合序列分析的二次筛选得到22个编码分泌蛋白的基因,再次经“PubMed”“Homogene”与“HPRD”检索,将同时具备人同源性、表达位点(Expression sits)中包括血清或血浆、文献显示与肿瘤发生有相关性的14个人同源的编码分泌蛋白基因作为候选标志物;
4.14个候选标志物经文献检索,其中AFP(alpha-fetoprotein),TGFBP1(transforming growth factor,beta1),GGT1(gamma-glutamyltransferase 1)已作为肝癌的诊断标志物在临床上应用,另4个:LOX(Lysyl oxidase),IGFBP3(insulin-like growth factor binding protein3),ENTPD 5(ectonudeoside triphosphate dipho sphohu drolase 5),PLAT(plasminogen activator,tissue)已有与肝癌相关的报道,本研究予以剔出。剩余的7个应用RT-PCR和免疫组化在大鼠正常组织和早期肝癌中行初步验证显示其中4个:CTGF(connective tissuegrowth factor),GDF15(growth differentiated factor 15),TXNRD 1(Thioredoxin reductase 1),SPP1(secreted phosphoperotein 1)在大鼠早期肝癌组织中高表达;
5.进一步在人正常肝脏组织、癌旁组织、小肝癌与大肝癌组织中对前述4个基因半定量RT-PCR显示小肝癌、大肝癌组织中mRNA表达明显上调;免疫组化显示,小肝癌和大肝癌组织中CTGF,GDF15,TXNRD1,SPP1的阳性表达面积比、积分灰度值与面积灰度整合值均明显高于正常肝组织与癌旁肝硬化组织。表达阳性部位均主要在胞浆和胞外基质。
结论:
1.商业化基因芯片结合合理的生物信息学数据处理是筛选肿瘤疾病基因差异表达的高效方法;
2.以动物模型为线索,利用基因芯片技术结合GO注释与针对性的序列分析是筛选分泌性蛋白作为人疾病早期诊断标志物的有效策略;
3.CTGF,GDF15,TXNRD1,SPP1初步鉴定显示其对人小肝癌筛选有潜在应用价值。
Hepatocellular carcinoma (HCC) has one of the worst prognoses among malignancies as the majority of this type of cancer at the early-stage is asymptomatic, and over three-quarter of the diagnoses are made at a time when the disease has already established regional or distant metastases. The 5-year survival is favorable for cases with an early diagnosis (80% approx), while survival with distant metastases over the same time period is less than 5%. The only validated serology tumor-marker in current use for diagnosis of HCC is AFP, which can be detected in the serum of more than 56.1% of cases with HCC. However, AFP is thought to be robust only in following the progression of the disease, but is not sensitive enough as an early stage diagnostic marker. Thus, there is an important need for additional diagnostic markers for this disease. Microarray technology allows researchers to simultaneously monitor the expression of thousands of genes, which provides molecular biomarkers to differentiate normal cells from malignant ones. For its capability to illustrate the cellular behavior on the genomic level, microarray technology has been applied in cancer research as one of the major topics in this field. Furthermore, effective application of this technique relies on well-organized data; because most existing microarray databases in cancer research are raw data sets and are hard-coded as columns in a table. It is difficult to draw directly supportive information for clinical cancer research from these. Gene expression profiling in the animal model holds great promise for our understanding of human pathogenesis. Here, we have generated and analyzed gene expression from Rat's liver cancer that was induced by DEN.
We used oligonucleotide microarrays that including over 30,000 well-substantiated rat genes to monitor the levels of expression within normal and early stage malignant liver tissues. Furthermore, we extended this analysis to investigate secreted protein by web resource that allows users to easily perform common queries on the data and to discover candidate diagnostic markers of early-stage HCC that can be detected in the blood. The overexpression of several genes that encode secreted protein were confirmed at transcript and protein expression level in the microtumors of rat models, as well as in the early-stage HCC patients. Our results show several of the genes as up-regulated in cancer and diagnostically useful. Other genes presented here, some of which we have validated, may find similar utility and suggest that many of these molecules may be relevant to tumorigenesis and may combination to form a simultaneous determination of early-stage HCC.
Methods
1. HCC model of rats were developed by feeding DEN (diethylnitrosamine) and early-stage HCC tissues were gathered at 14th -16th week.
2. Affymetrix Rat Genenome 230.2.0 GeneChip was used to detect the gene expression profile. Web-based bio-information and sequence analysis were used to screen target genes which significantly elevated expressed in tumor tissues and encode secreted proteins.
3. Transcription and protein expression levels of four target markers in both of animal model and human different stage HCC have subsequently been confirmed by RT-PCR and Immunohistochemistry. RT-PCR, Real-time quantitative PCR and immunohistochemistry were performed to identify the expression of candidate molecul in human HCC tissues.
Results
1. DEN successfully induced rat early-stage liver cancer
2. When compared with normal liver tissues, 677 genes(EST/Full length) in early-stage HCC were found to have different expression levels of more than two folds. Among those, 409 genes were up-regulated (log ratio>2), and 268 genes were down-regulated (log Ratio<-2).
3. According to the GO consortium annotations and the EMBOSS programs TMAP and SIGCLEAVE overlapping analysis, 22 genes were predicted to encode the secreted proteins in 211 highly expression known genes. After defining the query and gaining access to web-based resource , 14 retrieved genes were found to have the common features of H.sapiens orthologous gene, correlation with oncogenesis and expression sites including blood, serum, plasma. Of the 14 genes, Several have been applied to clinical diagnosis or reported to have relevant with tumorigenesis of HCC, such as AFP (α-fetoprotein), TGFβ1 (transforming growth factor, beta 1) and GGT1 (gamma-glutamyltransferase 1).
4. Primary validations were employed in four potential markers, CTGF, GDF15, SPP1 and TXNRD1. The results show a significant higher expression of the four markers in tumors than in normal tissues at both transcript and protein expression level.
Conclusions
Four candidate diagnostic markers were successfully screened and theidentification results show it can be a potential serology diagnosis markerof early-stage HCC.
引文
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