CAJ | 학술논문

目的:通过比较小鼠不同细胞类型之间Pdx-1基因转录起始区的表观遗传修饰差异,探讨表观遗传修饰对Pdx-1基因转录表达的作用.方法:采用免疫共沉淀-实时定量PCR法检测小鼠胚胎干细胞(mES)、小鼠成纤维细胞株NIH3T3细胞和小鼠β细胞株 NIT-1细胞Pdx-1和MLH1基因转录起始区DNA甲基化和组蛋白修饰(H3K4m3、H3K9m3和 H3乙酰化)的状况.同时采用实时定量RT-PCR检测上述3种细胞各基因mRNA表达水平.分析基因的DNA甲基化水平、H3K4m3、H3K9m3和 H3乙酰化修饰与基因表达之间的相互关系.结果:(1)以mES细胞为对照,NIT-1细胞的Pdx-1基因转录起始区呈低DNA甲基化和高H3K4m3修饰(P<0.05),NIH 3T3细胞的Pdx-1基因的转录起始区的DNA甲基化、H3乙酰化、H3K4m3和H3K9m3修饰水平明显增高(P<0.05);(2)Pdx-1基因仅在NIT-1细胞表达,其表达与DNA甲基化存在等级负相关(r=-0.802,P<0.01),与H3K4m3修饰存在直线相关(r=0.997,P<0.01),与H3K9m3修饰存在等级负相关(r=-0.879,P<0.01);(3)管家基因MLH1的表达与所检测的表观遗传修饰无相关性.结论:DNA甲基化、H3K9m3与H3K4m3修饰能相互协调,共同调控Pdx-1基因的表达,对胚胎干细胞向β细胞分化具有重要意义.
목적:통과비교소서불동세포류형지간Pdx-1기인전록기시구적표관유전수식차이,탐토표관유전수식대Pdx-1기인전록표체적작용.방법:채용면역공침정-실시정량PCR법검측소서배태간세포(mES)、소서성섬유세포주NIH3T3세포화소서β세포주 NIT-1세포Pdx-1화MLH1기인전록기시구DNA갑기화화조단백수식(H3K4m3、H3K9m3화 H3을선화)적상황.동시채용실시정량RT-PCR검측상술3충세포각기인mRNA표체수평.분석기인적DNA갑기화수평、H3K4m3、H3K9m3화 H3을선화수식여기인표체지간적상호관계.결과:(1)이mES세포위대조,NIT-1세포적Pdx-1기인전록기시구정저DNA갑기화화고H3K4m3수식(P<0.05),NIH 3T3세포적Pdx-1기인적전록기시구적DNA갑기화、H3을선화、H3K4m3화H3K9m3수식수평명현증고(P<0.05);(2)Pdx-1기인부재NIT-1세포표체,기표체여DNA갑기화존재등급부상관(r=-0.802,P<0.01),여H3K4m3수식존재직선상관(r=0.997,P<0.01),여H3K9m3수식존재등급부상관(r=-0.879,P<0.01);(3)관가기인MLH1적표체여소검측적표관유전수식무상관성.결론:DNA갑기화、H3K9m3여H3K4m3수식능상호협조,공동조공Pdx-1기인적표체,대배태간세포향β세포분화구유중요의의.
AIM: To investigate the role of epigenetic modification in Pdx-1 gene transcription and expression, and to compare the differences between epigenetic modifications of Pdx-1 gene promoter in various cell types of mice. METHODS: The promoter DNA methylation and histone modification status of Pdx-1 and MLH1 genes in NIT-1 cells, NIH3T3 cells and mouse embryonic stem cells were measured by chromatin immunoprecipitation-real time PCR method. The expression levels of these genes in the three cell lines were measured by real time RT-PCR. The relation between epigenetic modifications and gene expression was analyzed. RESULTS: (1) Compared to mES cells, there was lower DNA methylation and higher H3K4m3 modification levels in the promoter of Pdx-1 gene in NIT-1 cells (P<0.05). DNA methylation, H3 acetylation, H3K4m3 and H3K9m3 modification levels in the promoter of Pdx-1 gene in NIH3T3 cells were distinctly increased (P<0.05). (2) Pdx-1 gene transcription expressed only in NIT-1 cells. The Spearman's rho between Pdx-1 gene expression and DNA methylation (r=-0.802,P<0.01) was observed. The Pearson correlation between Pdx-1 gene expression and H3K4m3 modification (r=0.997,P<0.01) was also found. The Spearman's rho between Pdx-1 gene expression and H3K9m3 modification (r=-0.879,P<0.01) was observed. (3) No correlation between housekeeper MLH1 gene expression and epigenetic modification was found. CONCLUSION: DNA methylation, H3K4m3 and H3K9m3 modification coordinated participate to regulate and control the expression of Pdx-1 gene. It is of great significance to the differentiation of β cells from ES cells.