發(fā)布時間：2018-10-08 09:12

【摘要】：目的:宮頸癌是嚴重威脅女性健康的惡性腫瘤。在發(fā)展中國家,宮頸癌居女性癌癥診斷率第二位,死亡率的第三位。近年來研究發(fā)現(xiàn),宮頸癌的表觀遺傳學變化,尤其是甲基化調(diào)控,在宮頸癌發(fā)生發(fā)展的病因?qū)W過程中占有重要地位。因此,去甲基化藥物或許能夠在一定程度上控制宮頸上皮病變的進展。前期研究提示micro RNA-203(mi R-203)是一種上皮特異性RNA,在宮頸癌前病變到宮頸癌的進展過程中起到了重要作用,且其啟動子在宮頸癌細胞系中呈高甲基化狀態(tài)。DAC(地西他濱,5-aza-2deoxycytidine)作為去甲基化藥物,或許能夠在一定程度上影響mi R-203的表達及啟動子甲基化,進而影響宮頸癌細胞的生物學功能。本研究通過細胞實驗,觀察去甲基化藥物DAC對宮頸癌細胞HELA侵襲、凋亡的影響,以及其對mi R-203甲基化的影響。探討DAC對宮頸癌細胞侵襲能力和凋亡水平的影響,在mi R-203表達及其甲基化調(diào)控在宮頸癌中的作用機制,對于宮頸癌的病因?qū)W及臨床處理提供一定思路。方法:利用不同濃度DAC處理宮頸癌HELA細胞,實驗分三組:藥物處理HELA細胞作為實驗組,Ha Ca T細胞作為陰性對照組,未藥物處理HELA細胞作為空白對照組;實驗組按照DAC藥物濃度分為0.1u M、0.5 u M、2 u M、10 u M、50 u M。藥物處理24h、48h,采用逆轉(zhuǎn)錄熒光實時定量PCR法檢測細胞中mi R-203 RNA水平相對表達量;甲基化特異性PCR(MSP)法檢測mi R-203基因啟動子Cp G島甲基化水平;Transwell法檢測細胞侵襲能力,流式細胞術(shù)檢測細胞凋亡程度。結(jié)果:1.實驗組mi R-203表達水平、甲基化水平、侵襲能力均低于空白對照組,高于陰性對照組,凋亡率低于陰性對照組,高于空白對照組;2.隨藥物處理濃度增加其mi R-203表達水平逐漸升高、甲基化水平升高、侵襲能力減弱、凋亡率明顯升高。結(jié)論:DAC可能通過誘導(dǎo)宮頸癌HELA細胞中mi R-203啟動子序列去甲基化,使其表達水平升高,從而抑制其細胞侵襲,誘導(dǎo)其發(fā)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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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