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目的研究抑癌基因p16INK4A在卵巢上皮性癌(卵巢癌)组织及细胞系中的表达变化,分析其表达变化与甲基化的关系。方法选取7种卵巢癌细胞系、18份卵巢癌组织和10份正常卵巢组织为研究对象。采用甲基化特异性PCR方法检测p16INK4A基因甲基化状态;RT-PCR技术检测p16INK4A基因的mRNA表达;蛋白印迹(western blot)法检测P16INK4A蛋白的表达。5-杂氮-2′-脱氧胞苷对p16INK4A基因甲基化的卵巢癌细胞进行去甲基化处理,再次进行p16INK4A基因的mRNA和蛋白表达的检测,以及p16INK4A基因甲基化的分析。检测5-杂氮-2′-脱氧胞苷处理前后卵巢癌细胞的生长情况;并将处理前后的细胞接种于裸鼠,观测肿瘤的体积、重量。结果3种卵巢癌细胞系(Anglne、SW626和OVCAR3细胞)、6份卵巢癌组织中存在p16INK4A基因甲基化,卵巢癌细胞和卵巢癌组织中的甲基化率分别为3/7和33%(6/18)。卵巢癌细胞系、卵巢癌组织和正常卵巢组织中,p16INK4A基因的mRNA相对含量的平均值分别为0·34±0·11、0·81±0·13、1·52±0·12,蛋白相对含量的平均值分别为0·56±0·14、1·32±0·12、2·09±0·11,卵巢癌细胞系、卵巢癌组织分别与正常卵巢组织相比,差异均有统计学意义(P<0·05)。有甲基化表现的卵巢癌细胞和组织中p16INK4A基因的mRNA和蛋白表达均下降。5-杂氮-2′-脱氧胞苷处理能使p16INK4A基因甲基化的卵巢癌细胞中的p16INK4A基因的mRNA和蛋白重新表达或表达增高。与去甲基化处理前比较,去甲基化处理后Anglne、SW626和OVCAR3细胞的生长速度均减慢;接种去甲基化处理的OVCAR3细胞的裸鼠中,肿瘤体积和重量明显减小,分别为(0·243±0·022)cm3、(0·035±0·004)g。结论p16INK4A基因的表达下降或缺失在卵巢癌的发生中起重要作用,DNA甲基化是其表达缺陷的原因,去甲基化处理可以恢复p16INK4A基因的表达并抑制卵巢癌细胞的增殖。
Objective To investigate the expression of tumor suppressor gene p16INK4A in ovarian epithelial carcinoma (ovarian cancer) tissues and cell lines, and to analyze the relationship between its expression and methylation. Methods Seven ovarian cancer cell lines, 18 ovarian cancer tissues and 10 normal ovarian tissues were selected as research objects. The methylation status of p16INK4A gene was detected by methylation-specific PCR. The mRNA expression of p16INK4A was detected by RT-PCR. The protein expression of P16INK4A was detected by western blot. 5-Aza-2’-deoxycytidine on p16INK4A gene methylation in ovarian cancer cells were demethylated, again p16INK4A gene mRNA and protein expression detection, and p16INK4A gene methylation analysis. The growth of ovarian cancer cells before and after 5-aza-2’-deoxycytidine treatment was detected. The cells before and after the treatment were inoculated into nude mice to observe the tumor volume and weight. Results The methylation of p16INK4A gene was found in 3 ovarian cancer cell lines (Anglne, SW626 and OVCAR3) and 6 ovarian cancer tissues. The methylation rates in ovarian cancer cells and ovarian cancer tissues were 3/7 and 33% (6/18). Ovarian cancer cell lines, ovarian cancer and normal ovary tissues, the average relative mRNA levels of p16INK4A gene were 0.34 ± 0.11, 0.81 ± 0.13 and 1.52 ± 0.12, respectively The average relative content was respectively0.56 ± 0.14, 1.32 ± 0.12, 2.09 ± 0.11. The difference between ovarian cancer cell lines and ovarian cancer tissues was Statistical significance (P <0.05). The expression of p16INK4A mRNA and protein in ovarian cancer cells and tissues with methylation decreased. The 5-aza-2’-deoxycytidine treatment can re-express or express the p16INK4A gene mRNA and protein in methylated ovarian cancer cells with p16INK4A gene. Compared with that before demethylation, the growth rate of Anglne, SW626 and OVCAR3 cells were slowed down after demethylation treatment. The volume and weight of tumor in nude mice inoculated with demethylated OVCAR3 cells were significantly decreased, (0 · 243 ± 0 · 022) cm3 and (0 · 035 ± 0 · 004) g, respectively. Conclusions The decrease or loss of p16INK4A gene plays an important role in the development of ovarian cancer. DNA methylation is the cause of its expression defect. Demethylation can restore the expression of p16INK4A gene and inhibit the proliferation of ovarian cancer cells.