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Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

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World Journal of Gastroenterology 2004年第16期10卷 2318-2322页

作者： lihou jan-wutang xiao-nancui bowang bosong leisun DepartmentofPathology DalianMedicalUniversityDalian116027LiaoningProvinceChina

AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis ***: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver, cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual dories were randomly selected and used for PCR *** DNA from positive clones was isolated for ***: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes(mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel ***: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

关键词： cDNA图书馆肝癌老鼠癌细胞系统淋巴腺转移肿瘤

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Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip

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World Journal of Gastroenterology 2005年第10期11卷 1463-1472页

作者： bosong Jian-wutang bowang xiao-nancui lihou LuSun Li-MinMao Chun-HuiZhou YueDu Li-HuiWang Hua-XinWang Ren-ShuZheng leisun DepartmentofPathology DalianMedicalUniversityDalian116027LiaoningProvinceChina ShanghaiJingtaiBiotechnologyCorporationLtd Shanghai200000China

AIM: In order to obtain lymphogenous metastasisassociated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis ***: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip() MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by ***: Out of the 14 500 known genes investigated,110 (0.8%) were up regulated at least 23 fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110genes were further classified into two groups: differential biological process profile and molecular function ***: Using high-throughput gene chip method,a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription,chaperone activity, motor activity, protein kinase activity,receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further *** D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later *** (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic m

关键词： Hepatocarcinoma Lymphatic metastasis Cell lines Hca-F and Hca-P Gene chip

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