Chuang Tsai-Der, Khorram Omid
1 Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center and LA-Biomed Research Institute, Torrance, CA, USA.
Reprod Sci. 2019 Feb;26(2):250-258. doi: 10.1177/1933719118768692. Epub 2018 Apr 11.
The objective of this study was to determine whether miR-93, miR-29c, and miR-200c, which we previously reported to be downregulated in leiomyomas, target cell cycle regulatory proteins that influence cell proliferation. Based on TargetScan algorithm 3 cell cycle regulatory proteins namely, E2F transcription factor 1 (E2F1), Cyclin D1 (CCND1) and CDK2 which were predicted to be targets of these miRNAs were further analyzed. In 30 hysterectomy specimens, we found the expression of E2F1 and CCND1 messenger RNA (mRNA) was increased in leiomyoma as compared to matched myometrium, with no significant changes in CDK2 mRNA levels. There was a significant increase in the abundance of all 3 proteins in leiomyoma in comparison with matched myometrium. Using luciferase reporter assay, we demonstrated E2F1 and CCND1 are targets of miR-93 and CDK2 is a target of miR-29c and miR-200c. We confirmed these findings through transfection studies in which transfection of primary leiomyoma cells with miR-93 resulted in a significant decrease in the expression of E2F1 and CCND1 mRNA and protein levels, whereas knockdown of miR-93 had the opposite effect. Similarly, overexpression of miR-29c and miR-200c in leiomyoma cells inhibited the expression of CDK2 protein and mRNA, whereas knockdown of this microRNAs (miRNA) had the opposite effect. Transfection of miR-29c, miR-200c, and miR-93 in primary leiomyoma cells resulted in a time-dependent inhibition of cell proliferation and cell motility. These results collectively indicate that the 3 miRNAs known to be downregulated in fibroid tumors are critical in regulation of cell proliferation because of their effects on 3 key cell cycle regulatory proteins, which are overexpressed in uterine leiomyomas.
本研究的目的是确定我们之前报道的在平滑肌瘤中表达下调的miR-93、miR-29c和miR-200c是否靶向影响细胞增殖的细胞周期调节蛋白。基于TargetScan算法，对3种细胞周期调节蛋白，即E2F转录因子1（E2F1）、细胞周期蛋白D1（CCND1）和细胞周期蛋白依赖性激酶2（CDK2）进行了进一步分析，这些蛋白被预测为这些miRNA的靶标。在30例子宫切除标本中，我们发现与配对的子宫肌层相比，平滑肌瘤中E2F1和CCND1信使核糖核酸（mRNA）的表达增加，而CDK2 mRNA水平无显著变化。与配对的子宫肌层相比，平滑肌瘤中所有3种蛋白的丰度均显著增加。使用荧光素酶报告基因检测，我们证明E2F1和CCND1是miR-93的靶标，而CDK2是miR-29c和miR-200c的靶标。我们通过转染研究证实了这些发现，即用miR-9进行原代平滑肌瘤细胞转染，导致E2F1和CCND1 mRNA及蛋白水平显著降低，而敲低miR-93则产生相反的效果。同样，在平滑肌瘤细胞中过表达miR-29c和miR-200c可抑制CDK2蛋白和mRNA的表达，而敲低这些微小核糖核酸（miRNA）则产生相反的效果。在原代平滑肌瘤细胞中转染miR-29c、miR-200c和miR-93可导致细胞增殖和细胞运动的时间依赖性抑制。这些结果共同表明，已知在纤维瘤肿瘤中表达下调的这3种miRNA对细胞增殖的调节至关重要，因为它们对子宫平滑肌瘤中过表达的3种关键细胞周期调节蛋白有影响。
