| 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. Methods1. 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.Results1. DEN successfully induced rat early-stage liver cancer2. 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.ConclusionsFour candidate diagnostic markers were successfully screened and theidentification results show it can be a potential serology diagnosis markerof early-stage HCC.
|
PDF Full Text Request