| Part I--Study of MXR7 methylation in HCC: Liver cancer, a major public health problem in China, is the third most common malignancy in men and the fifth in women. Just like many other cancer, there are many factors involved in the process of HCC. One of them is methylation of cancer-related genes. MXR7 gene is highly expressed in HCC cell lines, but not in other 21 non-HCC tumor cell lines. Recent study of MXR7 gene have suggested that it is only expressed in cancerous tissue of HCC and serum of HCC patients, low in paracancerous tissue, and not in normal liver, which demonstrate that MXR7 is a gene overexpressed specifically in HCC and a novel tumor marker. However, the mechanism is not clear by now. This study was undertaken to obtain information at the molecular level on the possible mechanism of MXR7 gene overexpressed in HCC and also to provide a clue for further study. We adopted the method which is used commonly in methylation analysis. Firstly, we selected 20 samples of hepatoma and paired non-HCC liver tissues, 2 cases of hepatic hemangiomas and 1 type of HCC cell (HepG2). Genomic DNA was isolated from each sample according to the method described in 'Molecular Cloning: A Laboratory Manual'. And then they were digested with two kinds of endonucleases (EcoRI and EagI which is methylation sensitive endonuclease). Secondly, to prepare a probe for MXR7 gene promoter region, PCR was performed using genomic DNA as a template with a forward primer, 5'- CTC TCT CTG GAC ATT AGG AC - 3', and a reverse primer, 5'- ACC ATC ATG ATG ACC GCT TG - 3'. And the probe was labeled with a-32P-dCTP. Finally, we examined the condition of MXR7 gene methylation by Southern blot and analyzed the relationship between methylation of MXR7 and reopening of this gene. We found that MXR7 was unmethylated in most tumorous liver samples and HepG2. In addition, the same result was obtained in pairednon-HCC liver tissues and 2 hepatic hemangiomas samples. There were only two paired samples having different methylation outcome, one is unmethylated and the other is partly methylated. To confirm these results, MCF-7, a breast cancer cell, was used in this study and the same result can be also found in the essay of Xiang YY, et al. In all cases, the results above demonstrate that MXR7 is unmethylated in HHC, suggesting methylation of MXR7 may not have direct relation with its expression and regulation.Part II - Study of killing effect of MXR7 antibody on HCC cells by cooperation of chemotherapeutic drug: Chemotherapeutic drugs are commonly used to treat patients in HCC. However, the side effect of these drugs may cause serious harms to patients. So to improve the treatment efficacy and reduce the side effect of chemotherapeutic drugs, the cooperation of MXR7 polyclonal antibody was raised. In this part, we examined two kinds of chemotherapeutic drugs, MMC(mitomycin C) and 5-FU(5-fluorouracil), and choosed two sorts of HCC cells, HepG2 and Huh7. These cells were incubated in different cell number, 5.0x104 cells/ml (HepG2), 2x106 cells/ml (Huh7) and under same condition in 96 wells boards. Choosing different concentration of MXR7 polyclonal antibody and chemotherapeutic drugs, we put them in the serum culture medium and then these cells were incubated by dissimilar days. Setting up control was very prominent. Finally, We detected absorbance of 2 kinds of cells by MTT assay. Obtained date indicated that at 72h, the effect of 0.1ug/ml MXR7 polyclonal antibody was higher than other concentration (P<0.05) . In addition, under 0.1ug/ml MXR7 polyclonal antibody, 10ug/ml 5-FU had the same effect with 100ug/ml 5-FU when killing Hun7 (P<0.05) . These results suggested that the cooperative effect of MXR7 polyclonal antibody may cause the death of HCC cells. Therefore, MXR7 in controlling cell survival could be potential targets for screening chemotherapeutic drugs. |
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