Identification Of Differentially Expressed Genes In Mouse Hepatocarcinoma Cell Lines With Different Metastasis Potential Using Gene Chip

Background: Metastasis is the major biological marker in differentiating themalignant tumor, and most deaths from cancer are due to metastasis. So understandingthe molecular mechanisms of metastasis and improving the prognosis of cancer patientsare the most important and urgent issues in the field of oncology research. Becausemajority of malignant tumors are carcinomas and lymph node metastases oftenrepresent the first step in the metastatic process, it is very important to make themolecular mechanisms of lymphatic metastasis clearly. At present, various genes andsignal pathways which are involved in this complicated process have been widelyaccepted. However, traditional gene analysis could only study one or a few genes at atime, and is costly and time-consuming, hardly explaining the whole process accuratelyand completely. The recent development of DNA microarray technology, a type ofhigh-density analysis for gene expression, has opened a new era in medical sciences. Itcan analyze the expression of many genes simultaneously. cDNA microarray is the mostpopular one, it detects the cDNA synthesized from cellular mRNA. Hca-F and Hca-Pare a pair of synogenetic mouse hepatocarcinoma ascites cell lines, presenting a specificpotential of lymphogenetic metastasis when inoculated subcutaneously in 615 mice,Hca-F showing a highly metastatic potential(>70%), while Hca-P a low one(<30%).Synogenetic and specific for lymphogenetic metastasis, with significant differentiationonly in metastatic potential, differentially expressed genes obtained maybe invovled inthe lymphatic process. Object: In order to obtain lymphatic metastasis-associated genes, thetranscriptional profiles of mouse hapatocarcinoma ascites cell lines Hca-F(highlymetastatic) and Hca-P(low metastatic) were compared using cDNA microarray. Method: Total RNA was isolated from F and P cells respectively using TRIZOL?reagent, cDNA was synthesized using the T7-Oligo(dT)24 primer(5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3').Double-stranded cDNA was purified with Phase Lock Gel(PLG)-Phenol/chloroformextraction. Then in vitro transcription labeling was performed using the Enzo RNATranscript Labeling Kit (Affymetrix). The biotin-labeled cRNA was fragmentedrandomly to an average size of approximately 50-200 bases by mild alkaline treatment.Fragmented cRNA was hybridized to Affymetrix MOE430A array(containing 22,690transcripts, almost 14,500 known genes and 4,371 ESTs) for 16 hours in Affymetrix?Hybridization Oven 640. After washing and staining, the arrays were scanned using theAffymetrix?G2500AgeneArray Scanner. The data obtained through GeneChip?scanning was analyzed using Affymetrix? Microarray Suit Software 5.0. Before thetwo arrays were compared, the GeneChip? software conducted normalization andscaling of the data for each array. The mRNA expression level of a transcript is directlyrelated to the signal which is a quantitative metric calculated for each probe set andmeasures the mean difference of fluorescence intensity between perfect match andcentral mismatch oligonucleotides of a probe set. Signal Log Ratio, which estimates themagnitude and direction of change of a transcript when two arrays are compared, of atleast 1.0 or –1.0 (that indicates an increase or decrease of the transcript level by 2 foldchange) and Change p-value, which measures the probability that the expression levelsof a probe set in two different arrays are the same or not, ≤0.05( that means theexpression level in the experiment array is higher than that of the baseline array) werechoosen to select differentially expressed genes. The results were analyzed bybioinformatics. In order to confirm the quality, the cRNA probe was first hybridized toa "test chip" before to the MOE430A array. In addition, the hybridization solutioncontained a mixture of four control cRNAs for bacterial and phage genes(bioB, bioC,bioD and cre) to serve as comparison tools for hybridization efficiency between arrays.A biotinylated oligonucleotide B2, that specifically hybridized to features at the centerand co...