| In 2000 a cDNA (6A8) encoding a new humanα-mannosidase was cloned in our laboratory. The enzyme has been nominated as MAN2C1 internationally. My purpose in the thesis was to study effect of siMan2c1 on malignant activities of tumor cells and potentiality of Man2c1 gene in tumor gene therapy as a target, pXSN-hU6+27-siMan2c1 was first constructed and then packaged into retrovirus in GP-293 cell. Human esophageal carcinoma cell EC9706 with high expression of Man2c1 gene was infected with the virus and then cloned by limiting dilution. Real-time RT-PCR and immunofluorescence staining with the mAb to MAN2C1 developed by me showed a profound inhibition of Man2c1 gene expression in the cloned cells detected in comparison with that in controls, the wild type cell (W) and the cell infected with the virus containing the mock (M). 15 out of the 28 cloned cells were died in culture. The remaining grew very slow. Flowcytometric analysis showed an increase of the percentage of cells on apoptosis in the 2 cloned cells detected. The percentage was 7.30% (clone 1), 17.6% (clone 2), 1.55% (W) and 1.44% (M), respectively. Analysis of cell cycling for clone 2 cell, W cell and M cell showed that the percentage of cells in G1 phase was 28.5%, 59.6% and 58.2%, in S phase was 43.9%, 26.2% and 27.0%, and in G2 phase was 27.6%, 14.2% and 14.7% respectively. The data suggest a promotion of apoptosis of the EC9706 cell with inhibition of Man2c1 gene expression. In being inoculated into nude mice, tumor from the cells with inhibition of Man2c1 gene expression grew slower than those from W cells or M cells. On week 8, the average weight of tumors was 1.52±0.53g (clone 1), 1.47±0.62 g(clone 2), 3.033±0.65g (W cell) and 2.74±1.06g (M cell), respectively. The difference between clone 1 or clone 2 and W cell or M cell was statistically significant (t-test, p＜0.05)。The data indicate that the siMan2c1 used inhibits Man2c1 gene expression profoundly and inhibition of Man2c1 gene expression reduces malignant activities of EC9706 cell. Protein N-glycosylation has been found to be closely associated with tumor progression. Reduction of Con A binding to the EC9706 cell with reduced expression of Man2c1 gene which shows N-glycosylation modification suggests that N-glycosylation modification might play important roles in the reduction of malignant activities of EC9706 cell.In my study, recombinant adenovirus plasmid containing siMan2c1 sequence was then constructed and packaged into Ad5-siMan2c1. Owing to no effect of Ad5-siMan2c1 infection was observed on EC9706 cell (it might be associated with the low infection efficacy of Ad5 virus on the cell), human nasopharyngeal carcinoma cell CNE-2L2 was used to be as target cell in the study, which showed since had high efficacy of Ad5 virus infection. In addition, Ad5-siMan2c1 infection showed profound inhibition on growth of CNE-2L2 cell in culture. Ad5-siMan2c1 was injected into the tumors grown from inoculated CNE-2L2 cells in nude mice. Growth of tumors was inhibited in comparison with that of the tumors injected with controls, PBS or Ad5-EGFP (t-test, p＜0.05). Since BCSC-1 was up-regulated most and CD44 was down-regulated most in genes expressed in the cell with inhibited expression of Man2c1 in comparison with those expressed in W cell. Ectopic expression of BCSC-lgene or suppression of CD44 gene expression by siCD44 was studied on the malignant activities of CNE-2L2 cell. Reduction of malignant activities of the cell was observed. Therefore, effect of tumor inside injection of Ad5-BCSC-1 or with Ad5-siCD44 on growth of the tumor grown from CNE-2L2 cells was studied. The data showed inhibitive effect on the tumor growth. Thus, all the data suggest that Man2c1 gene might be a target in gene therapy on some tumors and so might be BCSC-1 gene and CD44 gene. |
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