| Objective: Lung carcinoma is the most common malignancy tumor in theworldwide, and its morbidity is still increasing. About60%of lung cancer have been inthe advanced stage when patients visit the doctor. Although many therapeutic measureswere taken such as surgery operarion, radiotherapy, chemotherapy and biotherapy,five-year survival rate of non-small cell lung cancer (NSCLC) is less than10%, andfive-year survival rate of small cell lung cancer (SCLC) is less than3%. The etiology oflung cancer is multi-factorial, including environmental factor, smoking habits, familialheredity and so on. A huge number of research shows that inactivation ordown-regulated expression of tumor suppressor gene (TSG) is an important mechanismin lung cancer. Aberrant DNA methylation of CpG islands plays an important role inepigenetic inactivation of TSGs. Therefore, it is urgent to find new diagnostic criteriaand effective therapeutic method. RASSF10(Ras association domain family10) is anovel TSG, has been found to be epigenetically inactivated in childhood leukaemias,thyroid cancer, gliomas and Malignant Melanoma of the Skin. However, there is noresearch indicates the relationship between RASSF10promoter methylation and lungcancer. The purpose of this study is to detect the methylation of RASSF10gene in6lung cancer cell lines, to investigate the reexpression of RASSF10after the treatmentwith5-aza-dC and cell apoptosis changes, to provide a new theory evidence for clinicalprognosis and therapy targets of lung cancer.Methods:We use COBRA(Combined Bisulfite Restriction Analysis) method to examine themethylation status of the RASSF10gene in lung cancer cell lines (A549、H157、SPCA-1、H460、H446and QG56). RASSF10mRNA expression was determined byRT-PCR; MTT assay and flow cytometry (FCM) were used to detect the growth ratioand apoptosis of the cell lines which were found to have been methylated. SPSS17.0was applied to analyze the results of experiment. Results:1. RASSF10gene was methylated in67%(4/6) lung cancer cell lines. It`scompletely methylated in SPCA-1cell line, partially methylated in H446, H157, H460,and unmethylated in A549, QG56.2. RT-PCR analysis showed that there were two straps of244bp (RASSF10) and230bp (β-actin) respectively. RASSF10mRNA was lost in SPCA-1and stronglyreduced in H446. The other groups treated with different concentrations of5-aza-dC, asubstance that inhibits de novo DNA methylation, resulted in increased RASSF10mRNA levels. Expression of β-actin was determined as a control of RNA integrity.There was significant difference between the different concentration groups (p<0.05).MTT results indicated that5-aza-dC could inhibit the proliferation of SPCA-1andH446significantly in a dose and time dependent manner. Apoptosis was confirmed byflow cytometry, when0、5、10μmol/L of5-aza-dC were given to the two kinds of celllines for72h, the apoptosis rate were0.23%±0.10、15.09%±0.26and34.9%±0.32inH446group and0.77%±0.10,、14.82±0.23and35.4±0.30in SPCA-1group,respectively. There was significant difference between the different concentrationgroups (p<0.05).Conclusion:1. This is the first experiment which detected the methylation of the novelcandidate tumor suppressor gene RASSF10in lung cancer cell lines.2. We further confirmed that5-aza-dC can restore the transcription of RASSF10inSPCA-1and H446；3.5-aza-dC can influence the proliferation, and induce apoptosis；... |
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