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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 investigate the effect of miR-205 on the expression of parental 3 (PEG3) protein in endometrial carcinoma cell lines. Methods Immunohistochemistry was used to determine the difference of the expression of PEG3 protein between normal endometrium and high, moderate and poorly differentiated endometrial carcinoma. RT-PCR screened high, moderate and poorly differentiated endometrial carcinoma cell lines with high expression levels of miR-205 as compared with normal endometrial cell lines. CCK-8 and flow cytometry were used to detect cell proliferation and expression of miR-205 Apoptosis; Western blot, RT-PCR and dual luciferase reporter assay and other experimental methods to verify whether miR-205 has a direct target inhibition of PEG3. Results The expression of PEG3 protein in normal endometrium was significantly higher than that in high, moderate and poorly differentiated endometrial carcinoma. The expression of miR-205 was up-regulated in high, moderate and poorly differentiated endometrial carcinoma cell lines, and the proliferation and apoptosis of miR-205 were inhibited. The results of Western blot showed that the content of PEG3 protein was decreased, and the two key proteins of WNT pathway, catenin and c-myc were up-regulated, while the RT-PCR results showed that there was no significant difference in the content of PEG3mRNA. The results of dual luciferase reporter assay showed that miR-205 acts on the 3’UTR end of PEG3 gene. Conclusion MiR-205 acts on the 3’UTR of PEG3 gene and inhibits the expression of PEG3 at the post-transcriptional level, suggesting that PEG3 is a direct target of miR-205.