ABSTRACT
AIM: To reveal new tumor markers and target genes from differentially expressed genes of primary tumor samples using cDNA microarray. METHODS: The (33 )P labeled cDNAs were synthesized by reverse transcription of message RNA from the liver cancerous tissue and adjacent non-cancerous liver tissue from the same patient and used to hybridize to LifeGrid 1.0 cDNA microarray blot containing 8400 known and unique human cDNA gene targets, and an expression profile of genes was produced in one paired human liver tumor tissue. After a global analysis of gene expression of 8400 genes, we selected some genes to confirm the differential expression using Northern blot and RT-PCR. RESULTS: Parallel analysis of the hybridized signals enabled us to get an expression profile of genes in which about 500 genes were differentially expressed in the paired liver tumor tissues. We identified 4 genes, the expression of three (Beclin 1, RbAp48 and Pir51) were increased and one (aldolase b) was decreased in liver tumor tissues. In addition, the expression of these genes in 6 hepatoma cell lines was also showed by RT-PCR analysis. CONCLUSION: cDNA microarray permits a high throughput identification of changes in gene expression. The genes encoding Beclin 1, RbAp48, Pir51 and aldolase b are first reported that may be related with hepatocarcinoma.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carrier Proteins/genetics , DNA-Binding Proteins/genetics , Fructose-Bisphosphate Aldolase/genetics , Liver Neoplasms/genetics , Nuclear Proteins/genetics , Proteins/genetics , Apoptosis Regulatory Proteins , Beclin-1 , Carcinoma, Hepatocellular/physiopathology , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/physiopathology , Membrane Proteins , Oligonucleotide Array Sequence Analysis , RNA-Binding Proteins , Retinoblastoma-Binding Protein 4 , Up-RegulationABSTRACT
Primary hepatocellular carcinoma(HCC) is one of the common malignant tumors in China. In our previous work, a gene named fup1(function-unknown protein 1) was isolated that was expressed differently in HCC and in normal liver. We assumed that it might be a candidate oncogene for the HCC. The fup1 gene had a ORF of 1 233 bp, encoding a protein with M(r) of 46 kD and isoelectric point of 5.48. The sequence characteristics showed its possible localization in nuclei. Northern blots showed that this gene was weakly expressed in many types of human tissues, except in the heart, implying its tissue-specific expression pattern. MTT assay of the NIH 3T3 cells transfected with this gene in the form of recombinant eukaryotic expression plasmid showed its enhancing role to cellular proliferation.
ABSTRACT
GST fusion protein expression system combined with protein truncation test(PTT) protocol was used to detect gene frame shift mutation. The RT-PCR products of Lis1 genes from hepatocarcinoma samples were respectively cloned into a GST fusion protein expression vector pGEX-1, then expressed in E.coli. The results showed a truncated 33 kD fusion protein in SDS-PAGE, although the full-translated product of Lis1 gene should be of 71 kD. Sequencing revealed insertion of an A residue, causing the premature termination of translation, between the 163th and 164th nucleotide of Lis1 gene. This improved PTT assay was proved to be a fast and effective way in detecting gene frame shift mutation.
