Identification Of Metastasis-associated Genes In Mice Histiocytosarcoma L-Ⅱ Using CDNA Microarray Of Tumor Metastasis-associated Genes

Objective:To analyze the differences in gene expression between mice histiocytosarcoma L-Ⅱof parent and offspring cell lines using microarray technology and to screen lymphoma metastasis-associated genes by bioinformaties.Methods:We subcutaneously injected histiocytosarcoma L–Ⅱcells in the right flank of mice, then isolate the cells in pulmonary metastasis and injected them in another mouse by the same methods. We analyze the two kinds of strain of distinct metastasis ability by morphlogy analysis,mean life span with cancer,cell growth curve and immunohistochemistry .Total RNA was isolated from the two generation mice histiocytosarcoma L-Ⅱand reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP. cDNA was hybridized to the Tumor Metastasis Gene Array of Superarray Bioscience, containing 113 key genes involved in apoptosis, cell cycle and adhesion,et al. The gene expression profile was analyzed by GEArray Analyzer software. The microarray results were confirmed by RT-PCR and Western-blot.Results: The present study was declined to screening the high invasive metastatic strain of histiocytosarcoma L–Ⅱby constant subculturing of the cells from the pulmonary metastasis in vivo.Now we have screening the high invasive metastatic strain of histiocytosarcoma L–Ⅱfrom the 7th to the 9th generation. Morphology observation:There are same morphous and construction between the two generation solid tumor under light microscope.The tumor appear polygon,disparity size,round,fusiform or anomalism of nucleus,stronger basophilia,bigger karyoplasmic ratio,lots of karyokinesis and pathological karyokinesis.Bionomics observation:Mean life span with cancer between parent and offspring cell lines were 29.23±2.90/23.66±2.97 days,respectively ( P﹤0.05 ) ,there were prominently differentia between the two generations. Tumor growth curve: offspring cell lines has more growth velocity than parent cell lines.Immunohistochemistry:IGF-1 and MMP-10 expression were more strong in offspring than parent cell lines. The gene chip analysis showed that 21 genes were screened out for differences between the two generations solid tumor,which was involved in several aspects of tumor metastasis. There were 4 genes down-regulated and 17 up-regulated.There were 2 extracellular matrix proteins;2 genes about cell adhesion;3 genes about cell cycle; 5 genes about cell growth and proliferation; 1 genes about apoptosis; 2 genes about transcription factors and regulators and others 6 genes. Four genes among them were further confirmed by RT-PCR and Western-blot respectively and the expression of them was coincided well with the gene chip results.Conclusion:1 We have screened the high invasive metastatic strain of histiocytosarcoma L–Ⅱsuccessfully and offspring cell lines are more invasive and metastatic than parent cell lines.2 The invasion and metastasis mechanism of histiocytosarcoma L–Ⅱis such a complex process that in which many important genes like Kras-2,IGF-1,MMP-10, MMP-13,Brms-1, as well as other relative genes are involved.