| In 2000 a cDNA(6A8) encoded a new humanα-mannosidase was cloned in our laboratory.The enzyme has been nominated as MAN2C1 by an international committee.Our previous experiments revealed that the in vitro growth of human nasopharyngeal carcinoma cell CNE-2L2,the tumorous growth of the cells inoculated into nude mice and the metastasis of the tumour into lungs were inhibited,if the expression of Man2cl gene was supressed.Considering that the malignant behavior of tumor cells was regulated by numbers of genes,I first tried to find the differentially displayed genes between the CNE-2L2 cell with suppressed expression of Man2cl gene and the CNE-2L2 cell with normal expression of Man2cl gene.The analysis was carried out on a DNA chip applied with DNA fragments of 1069 genes covering oncogenes,tumor supperssor genes, apoptosis related genes,immune associated genes,some other genes.In SAM analysis,when the threshold was set to 0.416,the number of the differential displayed genes was 182.When the threshold was set to 0.548,the number of the differential displayed genes was 70 with up-regulation of 26 genes and down-regulation of 44 genes in the cell with suppressed expression of Man2c/ gene.The up-regulated genes which might have suppressive effect on the malignant behavior of tumor cells included P130 mRNA for 130K protein, TGF-betalIR alpha GABBR1,TGFBR1,TNFAIP1,STANIN,E-CADHERIN,CTNNAI, CTNNA 2,RFX2 and TMPO etc.The down-regulated genes which might have suppressive effect on the malignant behavior of tumor cells included CD44, NDRG1,TGFB1,RPSS,LEGUMAIINCBS,CD59 and SNRPAl etc.CD44 mediates cell-matrix interation,which plays important roles in tumor invasion and metastasis.Therefore,the effect of down-regulation of CD44 gene on the malignant behavior of the CNE-2L2 cell was then analyzed. The expression of CD44 gene in wild type CNE-2L2 cells(called W cell in this thesis) was inhibited by using RNAi technique.The CNE-2L2 cell with suppressed expression of CD44 gene was called AS cell in this thesis. Suppression of CD44 gene expression was confirmed by means of Westerning blotting.AS cells were cloned.Four cell clones with profound suppression of CD44 gene were selected and named ASCI,ASC2,AS 3 and ASC4.The biological behaviors of ASCI and ASC2 were examined with the W cell and the CNE-2L2 cell transfected the retrovirus containing an empty vector(M cell) as controls.It was observed that in the first several days after being inoculated into culture bottle,W cells and M cells grew in disperse fasion,In contrast,ASC1 and ASC2 cells aggregated into cell mess rapidly.The growth of ASCI and ASC2 cell is much slower than that of W and M cell in culture.The anchor-independent colony formation rate of ASCI,ASC2,W and M cell in soft agar are 7.3±0.5, 8.5±0.5,21.3±0.7 and 22.1±0.4 respectively.The difference of the colony formation rate between ASCI cell,ASC2 cell and W cell,M cell was significant (P＜0.01).The colony formed by ASCI and ASC2 cell was much smaller than that of W and P cell.In addition,the former looks dark,the later was bright and refractive.Tumors grew from W cells-inoculated in 8/8 nude mice,from M cells in 8/8 mice,from ASCI cells in 3/8 mice and form ASC2 cells in 4/8 mice.The average weight of the tumors at the end of the 8th week was 1.56±0.65g,1.24±0.86g, 0.22±0.09g and0.28±0.13g for the W,M,ASCI and ASC2 cell respectively.The difference of the weight between the tumors from W and M cell and the tumors from ASCI and ASC2 cells was significant(P＜0.01).Tumor from CNE-2L2 cells metastizes to lungs frequently in nude mice.The lung metastasis of the tumors in my experiment is being analysed.The natural ligand for CD44 molecule is hyaluronan which is broadly expressed in matrix.Adhesion of tumor cell to matrix through interaction of CD44 molecule to hyaluronan plays important roles in the invasion and metastasis of tumor cells.My experiment showed that the number of cells adhered to a 96 well plate-coated with hyaluronan was much less when ASC1 and ASC2 cells were applied to the plate than W and M cells.In addition,W and M cell showed very weak expression of E-cadherin.In sharp contrast,the expression of E-cadherin was much stronger on ASC1 and ASC2 cell.It was reported that E-cadherin could down-regulate CD44 expression.My experiment showed a reverse phenomenon.That was a down-regulation of E-cadherin expression by CD44 molecule.My work demonstrates that down-expression of CD44 has suppressive effect on the malignant behaviors of CNE-2L2 cells.In the inhibition of the malignant behaviors of the CNE-2L2 cells with suppression of Man2cl gene expression, down-expression of CD44 in the cells is one of the factors in th inhibition.The CNE-2L2 cell with supressed expression of Man2cl gene and the CNE-21.2 cell with normal expression of Man2clgene also give us an opportunity to find noval genes related to the malignant behaviors of tumor cell.For this purpose, I constructed two cDNA liberaries from both the cells.The size of the liberaries were 4.5×105pfu/ml and 3.8×105 pfu/ml for the cell with supressed expression of Man2cl gene and the cell with normal expression of Man2cl gene respectively.Because of the time limitation for my thesis work,expreiments for cloning new genes are not completed. |
