甲基化作用调节miR-200b表达及对胃腺癌细胞增殖、侵袭能力的影响
发布时间：2018-08-07 15:25
【摘要】：目的:基因异常甲基化在多种疾病发生发展机制中有着重要的作用。本研究旨在探讨miR-200b发生甲基化程度不同及其表达量的差异对于胃癌细胞MGC-803、BGC-823增殖、侵袭、凋亡及对上皮间质转化作用的影响。方法:本研究以正常人胃粘膜上皮细胞(GES-1)和胃癌细胞(MGC-803、BGC-823)为研究对象,miR-200b为研究指标。首先提取正常未处理人正常胃粘膜上皮细胞GES-1及胃癌MGC-803、BGC-823细胞总RNA,逆转录之后进行实时荧光定量PCR(RT-PCR)法检测miR-200b在三种细胞中表达的差别,而miR-200b启动子区甲基化水平通过亚硫酸氢钠修饰的PCR(BSP)法进行检测。其次,对MGC-803、BGC-823细胞进行不同浓度和不同时间点的去甲基化处理,即使用浓度为2.5、5.0、7.5、10μM的5氮杂胞苷(5’-Aza-CdR)处理细胞,每24h换液一次作用72小时后,用RT-PCR法检测miR-200b的表达量变化,BSP法检测miR-200b启动子区甲基化水平的差异。根据表达量的差异及甲基化的程度选择合适的药物处理浓度。最后,使用最合适的药物浓度(10μM)作用MGC-803、BGC-823细胞72h,transwell实验检测细胞侵袭能力,流式细胞计术检测细胞周期及凋亡的情况。Western Blot检测受miR-200b表达量差异影响的上皮间质转化(EMT)相关指标的变化。结果:正常胃粘膜上皮细胞(GES-1)中miR-200b的表达量较高于胃癌细胞MGC-803、BGC-823,差异有统计学意义(P0.05)。而miR-200b启动子区甲基化水平则与之相反,MGC-803、BGC-823细胞系均较高于GES-1,差异有统计学意义(P0.05)。选用不同浓度5’-Aza-CdR药物处理细胞,两种胃癌细胞系miR-200b的表达量有随着药物浓度逐渐升高的趋势,而浓度为10μM时miR-200b启动子区甲基化水平较空白对照组降低,差异有统计学意义(P0.05)。Transwell实验表明,处理组的胃癌细胞其侵袭能力较对照组明显减弱(P0.05)。流式细胞术表明实验组较对照组细胞周期进展减慢,主要停留在G1期而S期细胞数相对减少(P0.01)。与对照组相比实验组细胞晚期凋亡数量稍稍增多,差异有统计学意义(P0.05)。Western Blot实验表明,在表皮粘膜细胞中表达量较高的E-cadherin实验组较对照组表达量升高,而在间质细胞中表达量较高的N-cadherin,ZEB1,Slug,MMP9实验组较对照组表达量降低,即实验组细胞上皮间质转化(EMT)程度降低。结论:MiR-200b表达的下降可能在胃癌变过程中起到重要作用,其作用机制可能与miR-200b启动子区高度甲基化作用有关。miR-200b启动子区存在发生甲基化的位点并且甲基化程度的不同可以影响其表达量,而miR-200b表达量的差异又与胃癌细胞MGC-803、BGC-823增殖、侵袭能力密切相关。所以抑癌基因启动子区发生异常甲基化,在胃癌发生、发展及预后研究中有重要作用和深远影响。
[Abstract]:Objective: aberrant methylation plays an important role in the pathogenesis of many diseases. The purpose of this study was to investigate the effects of different levels of methylation and expression of miR-200b on the proliferation, invasion, apoptosis and epithelial mesenchymal transformation of gastric cancer cell line MGC-803BGC-823. Methods: normal human gastric mucosal epithelial cells (GES-1) and gastric cancer cells (MGC-803BGC-823) were used as the study targets. The total RNAs of normal untreated human gastric epithelial cells (GES-1) and gastric cancer cell line MGC-803 (BGC-823) were extracted. After reverse transcription, the difference of miR-200b expression in the three cells was detected by real-time fluorescence quantitative PCR (RT-PCR). The methylation level of miR-200b promoter was detected by PCR (BSP) modified with sodium bisulfite. Secondly, MGC-803BGC-823 cells were treated with demethylation at different concentrations and at different time points. The cells were treated with 5-azacytidine (5'-Aza-CdR) at a concentration of 2.5 渭 m or 7.5 渭 M, and then treated every 24 h for 72 hours. The expression of miR-200b was detected by RT-PCR method. The difference of methylation level in promoter region of miR-200b was detected by BSP method. The appropriate drug concentration was selected according to the difference of expression and the degree of methylation. Finally, the most suitable concentration (10 渭 M) was used to detect the invasion ability of MGC-803 BGC-823 cell line by 72 h transwell assay. Flow cytometry was used to detect cell cycle and apoptosis. Western Blot was used to detect the changes of (EMT) related indexes of epithelial interstitial transformation affected by the difference of miR-200b expression. Results: the expression of miR-200b in normal gastric mucosal epithelial cells (GES-1) was significantly higher than that in MGC-803BGC-823cells (P0.05). The methylation level of miR-200b promoter was significantly higher than that of GES-1 (P0.05). The expression of miR-200b in two gastric cancer cell lines increased with the concentration of 5'-Aza-CdR, and the methylation level of miR-200b promoter was decreased when the concentration of 10 渭 M was 10 渭 M compared with the control group. The difference was statistically significant (P0.05) .Transwell experiment showed that the invasive ability of gastric cancer cells in the treatment group was significantly lower than that in the control group (P0.05). Flow cytometry showed that the progress of cell cycle in the experimental group was slower than that in the control group, mainly in G1 phase, but the number of S phase cells was relatively decreased (P0.01). Compared with the control group, the number of late apoptosis in the experimental group increased slightly, the difference was statistically significant (P0.05). Western Blot experiment showed that the expression of E-cadherin in the epidermal mucosal cells was higher than that in the control group. However, the expression of N-cadherin 1 SlugMMP9 in the interstitial cells was lower than that in the control group, that is, the degree of epithelial interstitial transformation (EMT) in the experimental group was lower than that in the control group. Conclusion the decreased expression of MiR-200b may play an important role in the progression of gastric cancer. The mechanism may be related to the hypermethylation of the promoter region of miR-200b. There are sites of methylation in the promoter region of .miR-200b, and the different degree of methylation may affect the expression of miR-200b, while the difference of the expression of miR-200b is related to the proliferation of MGC-803BGC-823. Invasiveness is closely related. Therefore, abnormal methylation in the promoter region of tumor suppressor gene plays an important and far-reaching role in the study of carcinogenesis, development and prognosis of gastric cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2
本文编号：2170459
[Abstract]:Objective: aberrant methylation plays an important role in the pathogenesis of many diseases. The purpose of this study was to investigate the effects of different levels of methylation and expression of miR-200b on the proliferation, invasion, apoptosis and epithelial mesenchymal transformation of gastric cancer cell line MGC-803BGC-823. Methods: normal human gastric mucosal epithelial cells (GES-1) and gastric cancer cells (MGC-803BGC-823) were used as the study targets. The total RNAs of normal untreated human gastric epithelial cells (GES-1) and gastric cancer cell line MGC-803 (BGC-823) were extracted. After reverse transcription, the difference of miR-200b expression in the three cells was detected by real-time fluorescence quantitative PCR (RT-PCR). The methylation level of miR-200b promoter was detected by PCR (BSP) modified with sodium bisulfite. Secondly, MGC-803BGC-823 cells were treated with demethylation at different concentrations and at different time points. The cells were treated with 5-azacytidine (5'-Aza-CdR) at a concentration of 2.5 渭 m or 7.5 渭 M, and then treated every 24 h for 72 hours. The expression of miR-200b was detected by RT-PCR method. The difference of methylation level in promoter region of miR-200b was detected by BSP method. The appropriate drug concentration was selected according to the difference of expression and the degree of methylation. Finally, the most suitable concentration (10 渭 M) was used to detect the invasion ability of MGC-803 BGC-823 cell line by 72 h transwell assay. Flow cytometry was used to detect cell cycle and apoptosis. Western Blot was used to detect the changes of (EMT) related indexes of epithelial interstitial transformation affected by the difference of miR-200b expression. Results: the expression of miR-200b in normal gastric mucosal epithelial cells (GES-1) was significantly higher than that in MGC-803BGC-823cells (P0.05). The methylation level of miR-200b promoter was significantly higher than that of GES-1 (P0.05). The expression of miR-200b in two gastric cancer cell lines increased with the concentration of 5'-Aza-CdR, and the methylation level of miR-200b promoter was decreased when the concentration of 10 渭 M was 10 渭 M compared with the control group. The difference was statistically significant (P0.05) .Transwell experiment showed that the invasive ability of gastric cancer cells in the treatment group was significantly lower than that in the control group (P0.05). Flow cytometry showed that the progress of cell cycle in the experimental group was slower than that in the control group, mainly in G1 phase, but the number of S phase cells was relatively decreased (P0.01). Compared with the control group, the number of late apoptosis in the experimental group increased slightly, the difference was statistically significant (P0.05). Western Blot experiment showed that the expression of E-cadherin in the epidermal mucosal cells was higher than that in the control group. However, the expression of N-cadherin 1 SlugMMP9 in the interstitial cells was lower than that in the control group, that is, the degree of epithelial interstitial transformation (EMT) in the experimental group was lower than that in the control group. Conclusion the decreased expression of MiR-200b may play an important role in the progression of gastric cancer. The mechanism may be related to the hypermethylation of the promoter region of miR-200b. There are sites of methylation in the promoter region of .miR-200b, and the different degree of methylation may affect the expression of miR-200b, while the difference of the expression of miR-200b is related to the proliferation of MGC-803BGC-823. Invasiveness is closely related. Therefore, abnormal methylation in the promoter region of tumor suppressor gene plays an important and far-reaching role in the study of carcinogenesis, development and prognosis of gastric cancer.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2
【共引文献】
相关期刊论文 前3条
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2 黄毕林;胡世莲;沈干;沈国栋;孙玉蓓;徐维平;黄大兵;王海;方中良;;地西他滨联合化疗药物对胃癌SGC-7901细胞增殖的抑制作用[J];实用医学杂志;2012年01期
3 冷俊;翁文浩;李智;许闪闪;李晶华;;5-Aza-CdR对膀胱癌EJ细胞系生长及Apaf-1基因异常甲基化的影响[J];同济大学学报(医学版);2009年03期
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