皮肤黑素瘤细胞IGFBP7基因启动子区CpG岛异常甲基化的研究
发布时间：2019-05-18 00:48
【摘要】： 皮肤黑素瘤是一种高度恶性的皮肤肿瘤,其发病机制仍未完全阐明。近来研究发现肿瘤抑制基因胰岛素样生长因子结合蛋白7(insulin-like growth factor binding protein 7,IGFBP7)的表达受抑制是黑素瘤发生的一个重要环节,抑癌基因的表达异常与DNA异常甲基化相关,本课题的目的是研究皮肤黑素瘤细胞中IGFBP7基因的表达调控机制,重点阐明启动子区CpG岛异常甲基化对IGFBP7基因表达调控的影响。 1 IGFBP7在皮肤黑素瘤组织和人黑素瘤细胞株中的表达 应用免疫组化法检测发现,IGFBP7蛋白在原发性皮肤黑素瘤和转移性黑素瘤组织中阳性表达率明显低于普通良性色素痣组织,有显著性差异。应用半定量RT-PCR和免疫细胞化学法检测发现,IGFBP7mRNA和蛋白在人黑素瘤细胞株A375、M14和SK-MEL-1中均阴性表达,在原代黑素细胞和人黑素瘤细胞株MV3中阳性表达。结果显示,IGFBP7在皮肤黑素瘤组织和人黑素瘤细胞株中表达抑制。 2人黑素瘤细胞株IGFBP7基因启动子区CpG岛异常甲基化的研究 应用亚硫酸氢钠修饰后测序法(bisulfite sequencing PCR, BSP)检测人黑素瘤细胞株A375、M14、SK-MEL-1、MV3和原代黑素细胞中IGFBP7基因启动子区CpG岛所有CpG位点的甲基化状态,结果显示,在IGFBP7表达阴性和阳性的细胞之间存在显著的甲基化差异。 35-aza-dC对人黑素瘤A375、M14细胞IGFBP7的表达及生物学行为的影响 应用去甲基化药物5-aza-dC处理阴性表达IGFBP7的A375和M14细胞,药物处理后,2株细胞IGFBP7mRNA和蛋白均恢复表达,且BSP检测证实了该基因启动子区发生了去甲基化改变,验证了IGFBP7在这2株细胞中的表达恢复是该基因去甲基化的直接作用,表明启动子区CpG岛DNA异常甲基化是黑素瘤细胞IGFBP7表达改变的直接调控机制。 经MTT实验、流式细胞术、TUNEL法和trans well小室侵袭实验检测发现,5-aza-dC在体外抑制A375和M14细胞增殖,促进凋亡,并导致细胞周期出现G1期阻滞,抑制细胞侵袭。结合药物处理后IGFBP7的表达变化,推测IGFBP7表达的恢复,很可能参与了5-aza-dC处理所引起的黑素瘤细胞上述生物学行为的改变。 以上研究结果说明,IGFBP7基因启动子区CpG岛DNA异常甲基化是黑素瘤细胞中IGFBP7表达改变的主要调控机制。去甲基化药物5-aza-dC还有抑制肿瘤细胞生长、阻滞细胞周期、促进细胞凋亡、抑制肿瘤细胞侵袭的作用,IGFBP7表达的恢复很可能在5-aza-dC引起的黑素瘤细胞生物学行为改变中发挥作用。
[Abstract]:Skin melanoma is a highly malignant skin tumor, the pathogenesis of which has not been fully clarified. Recently, it has been found that the inhibition of the expression of tumor inhibitor gene insulin-like growth factor binding protein 7 (insulin-like growth factor binding protein 7, IGFBP7) is an important link in the occurrence of melanoma. The abnormal expression of tumor inhibitor gene is related to the abnormal methylation of DNA. The purpose of this study was to study the regulatory mechanism of IGFBP7 gene expression in skin melanoma cells, and to elucidate the effect of abnormal methylation of CpG island in promoter region on the regulation of IGFBP7 gene expression. 1 the expression of 1 IGFBP7 in skin melanoma tissue and human melanoma cell line was detected by immunohistochemical method, and the results were as follows: (1) the expression of VEGF in skin melanoma tissue and human melanoma cell line was detected by immunohistochemistry. The positive expression rate of IGFBP7 protein in primary skin melanoma and metastatic melanoma was significantly lower than that in normal benign pigmented nevi. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the negative expression of IGFBP7mRNA and protein in human melanoma cell line A375, M14 and SK-MEL-1, and positive expression in primary melanocytes and human melanoma cell line MV3. The results showed that the expression of IGFBP7 was inhibited in skin melanoma tissue and human melanoma cell line. Abnormal methylation of CpG island in promoter region of IGFBP7 gene of two human melanoma cell lines (bisulfite sequencing PCR, BSP) was used to detect human melanoma cell line A375, M14, SK / MEL 鈮，
本文编号：2479531
[Abstract]:Skin melanoma is a highly malignant skin tumor, the pathogenesis of which has not been fully clarified. Recently, it has been found that the inhibition of the expression of tumor inhibitor gene insulin-like growth factor binding protein 7 (insulin-like growth factor binding protein 7, IGFBP7) is an important link in the occurrence of melanoma. The abnormal expression of tumor inhibitor gene is related to the abnormal methylation of DNA. The purpose of this study was to study the regulatory mechanism of IGFBP7 gene expression in skin melanoma cells, and to elucidate the effect of abnormal methylation of CpG island in promoter region on the regulation of IGFBP7 gene expression. 1 the expression of 1 IGFBP7 in skin melanoma tissue and human melanoma cell line was detected by immunohistochemical method, and the results were as follows: (1) the expression of VEGF in skin melanoma tissue and human melanoma cell line was detected by immunohistochemistry. The positive expression rate of IGFBP7 protein in primary skin melanoma and metastatic melanoma was significantly lower than that in normal benign pigmented nevi. Semi-quantitative RT-PCR and immunocytochemistry were used to detect the negative expression of IGFBP7mRNA and protein in human melanoma cell line A375, M14 and SK-MEL-1, and positive expression in primary melanocytes and human melanoma cell line MV3. The results showed that the expression of IGFBP7 was inhibited in skin melanoma tissue and human melanoma cell line. Abnormal methylation of CpG island in promoter region of IGFBP7 gene of two human melanoma cell lines (bisulfite sequencing PCR, BSP) was used to detect human melanoma cell line A375, M14, SK / MEL 鈮，
本文编号：2479531
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