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目的研究抑癌基因p16INK4A在卵巢癌中的表达变化,分析这种差异表达与甲基化的关系。方法逆转录聚合酶链技术(RT-PCR)检测7种卵巢癌细胞系、18例卵巢癌组织和10例正常卵巢组织中p16INK4A基因的表达, Western Blot对p16INK4A蛋白进行定量检测,分析蛋白水平上的P16INK4A表达的变化,并与正常卵巢组织中的p16INK4A表达情况进行对照。同时用甲基化特异性的PCR技术(MSP)对细胞系和组织的p16INK4A DNA进行甲基化分析。5-脱氧杂氮胞苷对细胞进行脱甲基化处理后检测细胞中p16INK4A基因的表达;体外体内检测细胞增殖变化。结果卵巢癌细胞系和卵巢癌组织中p16INK4A表达下降或缺失,与正常卵巢组织中的p16INK4A表达相比,差异具有显著意义(P <0．05)。蛋白的表达检测也得到相同的结果。MSP检测发现3种卵巢癌细胞株、6例卵巢癌组织p16INK4A第1外显子甲基化表现,细胞和组织中的甲基化率分别为42．86％和33．33％,甲基化者p16INK4A表达均下降。5-脱氧杂氮胞苷处理能使p16INK4A基因重表达,细胞增殖减慢,裸鼠荷瘤体积和重量明显下降。结论 p16INK4A作为一种抑癌基因,它的表达缺失或下降在卵巢癌的发生中起重要作用,外显子甲基化是导致这种表达的主要原因,脱甲基化处理可以抑制细胞的增殖。
Objective To study the expression of tumor suppressor gene p16INK4A in ovarian cancer and to analyze the relationship between the differential expression and methylation. Methods The expression of p16INK4A gene in 7 ovarian cancer cell lines, 18 ovarian cancer tissues and 10 normal ovarian tissues was detected by RT-PCR. The protein level of p16INK4A protein was detected by Western Blot P16INK4A expression changes, and with normal ovarian tissue p16INK4A expression control. Methylation analysis of p16INK4A DNA in cell lines and tissues was performed using methylation-specific PCR (MSP). 5-deoxy-aza-cytidine demethylation of cells after the detection of p16INK4A gene expression in vitro; in vitro detection of cell proliferation changes. Results The expression of p16INK4A in ovarian cancer cell lines and ovarian cancer tissues was decreased or absent, which was significantly different from that of p16INK4A in normal ovarian tissues (P <0.05). The expression of the protein was also tested for the same result. Methylation of p16INK4A exon 1 in 3 ovarian cancer cell lines and 6 ovarian cancer tissues was found by MSP assay. The methylation rates in cells and tissues were 42.86% and 33.33%, respectively. Methylation P16INK4A expression decreased. 5-deaza-azacytidine treatment can make p16INK4A gene overexpression, slow cell proliferation, tumor volume and weight of nude mice decreased significantly. Conclusion As a tumor suppressor gene, p16INK4A plays an important role in the development of ovarian cancer. Exon methylation is the main reason leading to this expression. Demethylation can inhibit cell proliferation .