摘要
目的探讨子宫内膜癌细胞系中miR-205对父性表达基因3(PEG3)蛋白的作用。方法免疫组化方法确定PEG3蛋白在正常子宫内膜与高、中、低分化子宫内膜癌组织中表达的差异。RT-PCR筛选出miR-205较正常子宫内膜细胞株表达量高的高、中、低分化子宫内膜癌细胞系,CCK-8及流式细胞学实验检测上调miR-205前后细胞增殖及凋亡;Western blot、RT-PCR和双荧光素酶报告基因分析等实验方法验证miR-205对PEG3是否有直接的靶抑制作用。结果 PEG3蛋白在正常子宫内膜组织中表达明显高于高、中、低分化子宫内膜癌组织。miR-205在高、中、低分化子宫内膜癌细胞系中上调后,细胞增殖促进,凋亡抑制;Western blot结果示PEG3蛋白含量降低,PEG3抑制的WNT通路中的两个关键蛋白β-catenin及c-myc也相应的表达量上调,而RT-PCR结果显示PEG3mRNA含量变化差异无统计学意义;双荧光素酶报告基因分析结果显示miR-205作用于PEG3基因的3’UTR端。结论 MiR-205作用于PEG3基因的3’UTR端,在转录后水平抑制PEG3的表达,提示PEG3是miR-205的直接靶基因。
Objective To explore the effect of miR-205 on PEG3 （ Paternally Expressed Gene 3 ） in endometrial cancer celllines. Methods Use immunohistochemical method to detect the different expressions of PEG3 in normal endometri- al tissue and high, moderately, poorly differentiated endometrial carcinoma tissues. Three different differtiation kinds of endometrial cancer cell lines which had higher expressions of miR-205 than normal endometrial cell line were chosen by RT-PCR. Then, CCK8 and flow cytometry analysis were used to detect the cell proliferation and apoptosis before and after the up-regulation of miR-205. Western blot, RT-PCR, and the dual luciferase assay were used to explore whether miR-205 had a target suppression effect on PEG3. Results The expression level of PEG3 in normal endometrial tissue was obviously higher than those in endometrial carcinoma tissues. After miR-205 was up-regulated in the three endome- trial cancer cell lines, the cell proliferation was improved and the cell apoptosis decreased. Western blot showed that the PEG3 expression level decreased and the expressions of two key proteins in WNT pathway, [3-catenin and c-myc, increased correspondingly. To the contrary, PEG3 mRNA showed no statistical significance change by RT-PCR. Dual luciferase assay demonstrated that miR-205 directly down-regulate PEG3 by targeting at the 3＇ UTR of PEG3 mRNA.Conclusion MiR-205 acts on the 3＇ UTR of PEG3 mRNA and suppresses the expression of PEG3 at the post-transcriptional level. That prompts PEG3 is the direct target gene of miR-205.
出处
《山东大学学报（医学版）》
CAS
北大核心
2013年第4期81-86,共6页
Journal of Shandong University:Health Sciences
基金
山东省科技攻关项目(2008GG10002058)
