| Hepatocellular carcinoma (HCC) is one of the most popular and important malignant neoplasm in China. To elucidate the molecular mechanism of the proliferation, differentiation and transforming of the liver cell is very helpful for the early diagnosis and efficient treatment of the HCC. In present study we combined DNA microarray and inhibition subtract hybridization to set up a powerful technique named Arrayed Library Subtraction Hybridization (ALSH) to investigate gene screening, expression and transcription regulation of hepatocellular carcinoma associated gene. We use the ALSH to large-scale gene screening of liver cDNA library and harvest a number of known genes, ESTs and a new gene. We used the ALSH in the analysis of HCC associated gene expression profile. We also combined the ALSH and the chromatin immunoprecipitation (ChIP) to study the gene transcription regulation in the scale of whole genome and harvested an arrayed library of DNA fragment immunoselected by anti- NFicB antibody from liver cells treated by mitomycin-C (MMC).PART I Investigation of gene screening and differentiate expression profiling of HCC associated gene using arrayed library and arrayed library subtraction hybridizationThe arrayed library was prepared from normal liver cDNA library purchased from Invitrogen company. Arrayed library subtraction hybridization was carried by the Biorobotics TAS multifuncton DNA microarray machine. The gene chip from the arrayed library and the genes screened from the arrayed library by ALSH were used to HCC expression profile analysis. The rat HCC and cirrhosis model were induced by DENA and TAA to further study of gene expression.A number of known genes and ESTs as well as a new gene fragment were screened. The full length of new gene named as myl was cloned by the method of single strain reverse nested PCR. The mxr7/gpc3 gene, which is an important member of glypican family, was found as an overexpressed gene in the HCC by the method of expression profile DNA microarray. The cyp2el gene, one memeber of cytochrome P450 family,was identified as a gene, which express highly and uniquely in. normal liver but do not express in HOC at all. The result was confirmed by the method of half-quantity HT-PCR and Northern Blot analysis. All the stages of occuireneee and development of the HOC was repeated in the chemical, induced model in. rat. The oci-5 gene hontologene of mxr7/gpc3 in rat, expressed hardly in normal not liver and. gradualy highly during the process of the development of HCC. Until the later phase of HCC oci-5 expressed very highly. The cyp2el gene expressed lower in the cirrhosis tissue than 'that: of normal liver tissue md did .not express in, HCC tissue. The change of the expression level of cyp2el during the development of HCC was opposite to that of mxr7/gpc3 gene.It was showed that the ALSH technique could screen the cDNA library to get all the grates in 'the library just as- the achievement of sequencing of whole library, We have cloned a new gene my1 through small scale test experiment and identified as a low expression gene. The mxr7/gpc3/oci-5 gene was found to be closely associated to the accuroence and development of HCC and may play a key role in. the early stage before the occurrence of HCC, It was suggested that mxr7/gpe3/oci-5 gene may not only be a diagnosis index of HCC but also the index, in the stage of cirrhosis to anticipate the occrareoce of HCC. The cyp2el gene slopped to express in HCC and express gradually low during the development of cirrhosis and HOC.PART II Combine the ALSH and ChIP to investigate thetranscription regulation in genome-wide scaleThe specific site of DMA binding and activation of Izasnscriptionai factor are key process wf gene expression regulation. Chcomotin. immunoprecipitatioii (ChIP)procedure was a powerful teeMiqu* to study the transcription factor prptdn-DNA ineractions in vivo, ChIP combined with ALSH could explore genome-wide searching of transcription factor spciilc DMA binding sites in the h...
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