甲基化作用調(diào)節(jié)miR-200b表達(dá)及對胃腺癌細(xì)胞增殖、侵襲能力的影響
發(fā)布時間：2018-08-07 15:25
【摘要】：目的:基因異常甲基化在多種疾病發(fā)生發(fā)展機(jī)制中有著重要的作用。本研究旨在探討miR-200b發(fā)生甲基化程度不同及其表達(dá)量的差異對于胃癌細(xì)胞MGC-803、BGC-823增殖、侵襲、凋亡及對上皮間質(zhì)轉(zhuǎn)化作用的影響。方法:本研究以正常人胃粘膜上皮細(xì)胞(GES-1)和胃癌細(xì)胞(MGC-803、BGC-823)為研究對象,miR-200b為研究指標(biāo)。首先提取正常未處理人正常胃粘膜上皮細(xì)胞GES-1及胃癌MGC-803、BGC-823細(xì)胞總RNA,逆轉(zhuǎn)錄之后進(jìn)行實(shí)時熒光定量PCR(RT-PCR)法檢測miR-200b在三種細(xì)胞中表達(dá)的差別,而miR-200b啟動子區(qū)甲基化水平通過亞硫酸氫鈉修飾的PCR(BSP)法進(jìn)行檢測。其次,對MGC-803、BGC-823細(xì)胞進(jìn)行不同濃度和不同時間點(diǎn)的去甲基化處理,即使用濃度為2.5、5.0、7.5、10μM的5氮雜胞苷(5’-Aza-CdR)處理細(xì)胞,每24h換液一次作用72小時后,用RT-PCR法檢測miR-200b的表達(dá)量變化,BSP法檢測miR-200b啟動子區(qū)甲基化水平的差異。根據(jù)表達(dá)量的差異及甲基化的程度選擇合適的藥物處理濃度。最后,使用最合適的藥物濃度(10μM)作用MGC-803、BGC-823細(xì)胞72h,transwell實(shí)驗(yàn)檢測細(xì)胞侵襲能力,流式細(xì)胞計術(shù)檢測細(xì)胞周期及凋亡的情況。Western Blot檢測受miR-200b表達(dá)量差異影響的上皮間質(zhì)轉(zhuǎn)化(EMT)相關(guān)指標(biāo)的變化。結(jié)果:正常胃粘膜上皮細(xì)胞(GES-1)中miR-200b的表達(dá)量較高于胃癌細(xì)胞MGC-803、BGC-823,差異有統(tǒng)計學(xué)意義(P0.05)。而miR-200b啟動子區(qū)甲基化水平則與之相反,MGC-803、BGC-823細(xì)胞系均較高于GES-1,差異有統(tǒng)計學(xué)意義(P0.05)。選用不同濃度5’-Aza-CdR藥物處理細(xì)胞,兩種胃癌細(xì)胞系miR-200b的表達(dá)量有隨著藥物濃度逐漸升高的趨勢,而濃度為10μM時miR-200b啟動子區(qū)甲基化水平較空白對照組降低,差異有統(tǒng)計學(xué)意義(P0.05)。Transwell實(shí)驗(yàn)表明,處理組的胃癌細(xì)胞其侵襲能力較對照組明顯減弱(P0.05)。流式細(xì)胞術(shù)表明實(shí)驗(yàn)組較對照組細(xì)胞周期進(jìn)展減慢,主要停留在G1期而S期細(xì)胞數(shù)相對減少(P0.01)。與對照組相比實(shí)驗(yàn)組細(xì)胞晚期凋亡數(shù)量稍稍增多,差異有統(tǒng)計學(xué)意義(P0.05)。Western Blot實(shí)驗(yàn)表明,在表皮粘膜細(xì)胞中表達(dá)量較高的E-cadherin實(shí)驗(yàn)組較對照組表達(dá)量升高,而在間質(zhì)細(xì)胞中表達(dá)量較高的N-cadherin,ZEB1,Slug,MMP9實(shí)驗(yàn)組較對照組表達(dá)量降低,即實(shí)驗(yàn)組細(xì)胞上皮間質(zhì)轉(zhuǎn)化(EMT)程度降低。結(jié)論:MiR-200b表達(dá)的下降可能在胃癌變過程中起到重要作用,其作用機(jī)制可能與miR-200b啟動子區(qū)高度甲基化作用有關(guān)。miR-200b啟動子區(qū)存在發(fā)生甲基化的位點(diǎn)并且甲基化程度的不同可以影響其表達(dá)量,而miR-200b表達(dá)量的差異又與胃癌細(xì)胞MGC-803、BGC-823增殖、侵襲能力密切相關(guān)。所以抑癌基因啟動子區(qū)發(fā)生異常甲基化,在胃癌發(fā)生、發(fā)展及預(yù)后研究中有重要作用和深遠(yuǎn)影響。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.2
本文編號：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.2
【共引文獻(xiàn)】
相關(guān)期刊論文 前3條
1 王賀玲;張建;李巖;王學(xué)清;;5-Aza-CdR對胃癌細(xì)胞系p27kip1基因異常甲基化的影響[J];實(shí)用藥物與臨床;2011年02期
2 黃畢林;胡世蓮;沈干;沈國棟;孫玉蓓;徐維平;黃大兵;王海;方中良;;地西他濱聯(lián)合化療藥物對胃癌SGC-7901細(xì)胞增殖的抑制作用[J];實(shí)用醫(yī)學(xué)雜志;2012年01期
3 冷俊;翁文浩;李智;許閃閃;李晶華;;5-Aza-CdR對膀胱癌EJ細(xì)胞系生長及Apaf-1基因異常甲基化的影響[J];同濟(jì)大學(xué)學(xué)報(醫(yī)學(xué)版);2009年03期
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