DAC对宫颈癌HELA细胞miR-203表达及细胞侵袭、凋亡过程的影响

发布时间：2018-10-08 09:12

【摘要】：目的:宫颈癌是严重威胁女性健康的恶性肿瘤。在发展中国家,宫颈癌居女性癌症诊断率第二位,死亡率的第三位。近年来研究发现,宫颈癌的表观遗传学变化,尤其是甲基化调控,在宫颈癌发生发展的病因学过程中占有重要地位。因此,去甲基化药物或许能够在一定程度上控制宫颈上皮病变的进展。前期研究提示micro RNA-203(mi R-203)是一种上皮特异性RNA,在宫颈癌前病变到宫颈癌的进展过程中起到了重要作用,且其启动子在宫颈癌细胞系中呈高甲基化状态。DAC(地西他滨,5-aza-2deoxycytidine)作为去甲基化药物,或许能够在一定程度上影响mi R-203的表达及启动子甲基化,进而影响宫颈癌细胞的生物学功能。本研究通过细胞实验,观察去甲基化药物DAC对宫颈癌细胞HELA侵袭、凋亡的影响,以及其对mi R-203甲基化的影响。探讨DAC对宫颈癌细胞侵袭能力和凋亡水平的影响,在mi R-203表达及其甲基化调控在宫颈癌中的作用机制,对于宫颈癌的病因学及临床处理提供一定思路。方法:利用不同浓度DAC处理宫颈癌HELA细胞,实验分三组:药物处理HELA细胞作为实验组,Ha Ca T细胞作为阴性对照组,未药物处理HELA细胞作为空白对照组;实验组按照DAC药物浓度分为0.1u M、0.5 u M、2 u M、10 u M、50 u M。药物处理24h、48h,采用逆转录荧光实时定量PCR法检测细胞中mi R-203 RNA水平相对表达量;甲基化特异性PCR(MSP)法检测mi R-203基因启动子Cp G岛甲基化水平;Transwell法检测细胞侵袭能力,流式细胞术检测细胞凋亡程度。结果:1.实验组mi R-203表达水平、甲基化水平、侵袭能力均低于空白对照组,高于阴性对照组,凋亡率低于阴性对照组,高于空白对照组;2.随药物处理浓度增加其mi R-203表达水平逐渐升高、甲基化水平升高、侵袭能力减弱、凋亡率明显升高。结论:DAC可能通过诱导宫颈癌HELA细胞中mi R-203启动子序列去甲基化,使其表达水平升高,从而抑制其细胞侵袭,诱导其发生凋亡。
[Abstract]:Objective: cervical cancer is a malignant tumor that seriously threatens the health of women. In developing countries, cervical cancer is the second highest rate of cancer diagnosis in women and the third highest in mortality. In recent years, it has been found that epigenetic changes, especially methylation, play an important role in the etiology of cervical cancer. Demethylated drugs may thus be able to control the progression of cervical epithelial lesions to some extent. Previous studies suggest that micro RNA-203 (mi R-203) is an epithelial-specific RNA, that plays an important role in the progression from precancerous lesions to cervical cancer. Its promoter was hypermethylated in cervical cancer cell line. DAC (desitabine 5-aza-2deoxycytidine) as demethylating drug might affect the expression of mi R-203 and methylation of promoter to some extent, and then affect the biological function of cervical cancer cells. The effects of demethylating drug DAC on the invasion and apoptosis of HELA and the methylation of mi R-203 in cervical cancer cells were observed by cell experiments. To investigate the effect of DAC on the invasive ability and apoptosis of cervical cancer cells, the expression of mi R-203 and the mechanism of its methylation regulation in cervical cancer, which provide some ideas for the etiology and clinical management of cervical cancer. Methods: Cervical cancer HELA cells were treated with different concentrations of DAC. The cells were divided into three groups: drug treated HELA cells as the negative control group, and untreated HELA cells as the blank control group. According to the concentration of DAC, the experimental group was divided into 0. 1 u MX 0. 5 u MX 2 u MX 10 u MU 50 u M0. The relative expression of mi R-203 RNA was detected by reverse transcription-fluorescence real-time PCR assay and methylation specific PCR (MSP) assay was used to detect the invasion ability of mi R-203 gene promoter Cp G island methylation level. Apoptosis was detected by flow cytometry. The result is 1: 1. The expression level, methylation level and invasion ability of mi R-203 in the experimental group were lower than those in the blank control group, higher than those in the negative control group, and the apoptotic rate was lower than that in the negative control group, and higher than that in the blank control group. With the increase of drug concentration, the expression level of mi R-203 increased gradually, the methylation level increased, the invasiveness decreased, and the apoptosis rate increased significantly. Conclusion the expression level of mi R-203 promoter is increased by inducing mi R-203 promoter demethylation in cervical cancer HELA cells, thus inhibiting its invasion and inducing apoptosis.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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