叶酸调控miR-375甲基化在宫颈癌发生中的作用研究

发布时间：2018-04-26 23:34

本文选题：宫颈癌 + 宫颈上皮内瘤变(CIN)　；参考：《山西医科大学》2014年硕士论文

【摘要】：目的：宫颈癌是一种严重危害女性健康的恶性肿瘤,在我国女性恶性肿瘤死亡排名中占第二位,且山西省为宫颈癌的高发区。而宫颈癌是一个连续发生的过程,宫颈上皮内瘤变(cervical intraepithelial neoplasias, CIN)为宫颈癌的癌前病变,如何阻断CIN进展为宫颈癌成为目前亟待解决的问题。众所周知,宫颈癌的发生受多种因素的影响,目前最新研究表明microRNA及叶酸缺乏均可导致宫颈癌的发生,且是HPV感染的协同因素,而他们的作用机制如何目{前尚无定论,因此本研究的目的是：1.检测宫颈上皮内瘤变及宫颈癌组织标本中,miR-375的表达水平,探讨其在宫颈癌变中的作用；2.检测在不同浓度叶酸干预下宫颈癌细胞中 miR-375的表达水平,研究叶酸与miR-375表达水平的关系,并进一步研究其在宫颈癌发生发展中的作用。3.检测在不同浓度叶酸干预下宫颈癌细胞中 miR-375启动子区的甲基化状态,探讨叶酸与miR-375启动子区甲基化状态的关系,研究其在宫颈癌中的作用,进一步了解宫颈癌的发生机制,为宫颈癌的治疗提供新思路。方法：1.标本来源收集2013年5月-2014年1月,在我院妇科门诊确诊为CIN及宫颈鳞癌(Ⅰ a-Ⅱa期)患者的病变组织各30例,取宫颈病变组织离体后,立即用生理盐水冲洗血液,置于组织冻存管,将标本在液氮桶内放置1h后,放入-80℃冰箱保存待实验用；另取确诊为粘膜慢性炎患者的宫颈组织30例作为对照组。2.检测miR-375的表达水平取上述所选90例患者的宫颈组织标本,根据病理结果分为CIN组、宫颈癌组和正常对照组,提取总RNA,采用荧光定量PCR技术检测miR-375的表达水平。3.检测miR-375启动子区的甲基化状态宫颈癌细胞株CaSki常规体外培养,不同浓度叶酸干预后,提取基因组DNA,采用甲基化特异性PCR(methylation specific PCR, MSP)技术,检测miR-375启动子区的甲基化状态。4.结果判定取10μL PCR产物在2%琼脂糖凝胶上电泳,Ethidium Brmide染料染色,成像仪观察并摄像。获得甲基化产物,无论是否出现非甲基化产物,均判定为甲基化阳性；只出现非甲基化产物时记为非甲基化；既未获得甲基化产物也未获得非甲基化产物则标志本次实验失败。6.统计学分析采用SPSS16.0软件包进行数据分析,正态分布资料采用均数±标准差描述；两组资料的比较采用两独立样本的t检验,多组资料的比较采用one-wayANOVA和LSD-t检验；相关性分析采用Spearman等级相关；P0.05为差异有统计学意义。结果：1.miR-375的表达水平在三组之间进行两两比较,差异均有统计学意义(P0.05),且在CIN及宫颈癌组织的表达水平与宫颈正正常上皮组织相比显著下调,差异有统计学意义(P0.05)。2.在不同浓度叶酸干预的宫颈癌细胞中,随着叶酸浓度的升高,miR-375启动子区CpG岛的甲基化阳性率分别为56.67%(17/30)、56.67%(17/30)、53.33%(16/30)、46.67%(14/30)、36.67%(11/30)、20.00%(6/30)、6.67%(2/30),呈现出递减趋势,差异有统计学意义(P0.05)3.在不同浓度叶酸干预的宫颈癌细胞中,随着叶酸浓度的升高,miR-375的表达水平逐渐升高,差异有统计学意义(P0.05)。4.在不同浓度叶酸干预下,宫颈癌细胞中miR-375的表达水平与甲基化状态呈负相关。结论：1.与正常宫颈组织相比,CIN及宫颈癌组织中miR-375的表达水平明显下调。2.叶酸缺乏可能通过诱导miR-375启动子区的异常高甲基化,使miR-375表达水平下调,在宫颈癌变的过程中发挥一定作用。
[Abstract]:Objective : Cervical cancer is a kind of malignant tumor which is harmful to women ' s health . It is the second place in the death rank of female malignant tumor in China , and Shanxi is the high incidence area of cervical cancer . It is well known that cervical cancer has been affected by many factors . It is well known that the lack of microRNA and folic acid can lead to cervical cancer , and how to block CIN is the co - factor of HPV infection . So the aim of this study is to : 1 . To detect the expression level of miR - 375 in cervical epithelial neoplasia and cervical cancer tissue specimen , and to explore its role in cervical cancer .
2 . To investigate the expression level of miR - 375 in cervical cancer cells at different concentrations of folic acid , investigate the relationship between folic acid and miR - 375 expression level , and further study its role in the development of cervical cancer .
The expression level of miR - 375 was determined by fluorescence quantitative polymerase chain reaction ( MSP ) and methylation specific PCR ( MSP ) .
Only non - methylated products appear as non - methylated ;
No non - methylated products were obtained from methylated products , indicating that the experiment was failed . 6 . SPSS 16.0 software was used for data analysis , and the normal distribution data was described by mean 卤 standard deviation .
Two groups of data were compared with t - test of two independent samples , one - way ANOVA and LSD - t test were used for the comparison of multiple sets of data .
The correlation analysis was correlated with man ' s rank .
Results : 1 . The expression level of miR - 375 in cervical cancer cells was significantly lower than that of normal cervical tissue ( P0.05 ) .

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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