尿路上皮癌相关1基因通过竞争性抑制miR-18a增强乳腺癌细胞的他莫昔芬治疗耐药

发布时间：2018-03-24 09:31

本文选题：受体　切入点：雌激素　出处：《北京大学学报(医学版)》2017年02期

【摘要】：目的:探讨尿路上皮癌相关1(urothelial carcinoma-associated 1,UCA1)基因及miR-18a在雌激素受体(estrogen receptor,ER)阳性的乳腺癌细胞他莫昔芬耐药中的作用和调控机制。方法:通过反转录实时定量PCR测定乳腺癌细胞中UCA1和miR-18a的表达,用双荧光素酶报告实验验证miR-18a和UCA1 3'UTR区域结合。在他莫昔芬敏感的MCF-7细胞中过表达UCA1或敲减miR-18a,在他莫昔芬耐药的LCC9和BT474细胞中敲减UCA1或过表达miR-18a,CCK-8方法测定细胞活力,软琼脂克隆形成实验测定细胞克隆形成能力,流式细胞术测定细胞周期。结果:他莫昔芬耐药的LCC2、LCC9和BT474细胞中,UCA1表达量显著高于他莫昔芬敏感的MCF-7细胞。MCF-7细胞在0.1μmol/L他莫昔芬处理后,UCA1的表达水平显著升高。UCA1过表达提高了MCF-7细胞在他莫昔芬处理后的活力,而UCA1 siRNA则显著降低了LCC9和BT474细胞在他莫昔芬处理后的活力。在MCF-7细胞中,相对于质粒对照+他莫昔芬组,UCA1+他莫昔芬组的克隆数目多19.0%,克隆平均大小增加29.0%,G1期细胞比例减少7.3%,S期细胞增加6.7%。在BT474细胞中,相对于siRNA对照+他莫昔芬组,si-UCA1+他莫昔芬组的克隆数目少54.0%,克隆平均大小减少42.0%,G1期细胞比例增加9.0%,S期细胞减少6.2%。UCA1有一个miR-18a结合位点,敲减miR-18a提高了MCF-7细胞在他莫昔芬处理后的活力,过表达miR-18a则显著降低了BT474细胞在他莫昔芬处理后的活力。在MCF-7细胞中,相对于对照+他莫昔芬组,miR-18a抑制+他莫昔芬组的克隆数目多15.0%,克隆平均大小增加33.0%,G1期细胞比例减少8.8%,S期细胞增加5.3%。在BT474细胞中,相对于对照+他莫昔芬组,miR-18a过表达+他莫昔芬组的克隆数目少47.0%,克隆平均大小减少25.0%,G1期细胞比例增加13.3%,S期细胞减少7.9%。结论:UCA1可以通过竞争性抑制miR-18a增强乳腺癌细胞的他莫昔芬治疗耐药。
[Abstract]:Objective: to investigate the role and regulatory mechanism of 1(urothelial carcinoma-associated 1 UCA1) gene and miR-18a in tamoxifen resistance in breast cancer cells with estrogen receptor estrogen receptor ER1 positive. Methods: reverse transcription real-time quantitative PCR was used to detect the fine breast cancer cells. Expression of UCA1 and miR-18a in cells, Double luciferase report assay was used to verify the binding of miR-18a and UCA1 3'UTR region. UCA1 or miR-18a was overexpressed in tamoxifen sensitive MCF-7 cells, and UCA1 or miR-18aCCK-8 was detected in tamoxifen resistant LCC9 and BT474 cells. The ability of cell clone formation was determined by soft Agar clone forming assay. Results: the expression of UCA1 in tamoxifen resistant LCC2 + LCC9 and BT474 cells was significantly higher than that in tamoxifen sensitive MCF-7 cells. MCF-7 cells treated with tamoxifen 0.1 渭 mol/L. Expression increased the activity of MCF-7 cells after tamoxifen treatment. UCA1 siRNA significantly decreased the activity of LCC9 and BT474 cells after tamoxifen treatment. Compared with the plasmid control group of tamoxifen group, the number of clones in tamoxifen group was 19.0. The average size of clone increased 29.0% and the proportion of cells in G1 phase decreased by 7.3%. Compared with the siRNA control group, the number of clones in tamoxifen group was 54.0 less than that in tamoxifen group, and the average clone size was reduced by 42.0 and 42.0g / G _ 1 phase cell proportion increased by 9.0g / L 6.2%.UCA1. There was a miR-18a binding site in 6.2%.UCA1. Knockout miR-18a increased the activity of MCF-7 cells after tamoxifen treatment, and overexpression of miR-18a significantly decreased the activity of BT474 cells after tamoxifen treatment. Compared with the control tamoxifen group, the number of clones in tamoxifen group inhibited by miR-18a was 15.0%, and the average clone size increased by 33.0g / g phase cells. The percentage of cells in G1 phase decreased by 8.8g / L and increased by 5.3% in BT474 cells. Compared with the control tamoxifen group, the number of clones in the tamoxifen group was 47.0 less, the average clone size was reduced by 25.0 and the proportion of cells in G1 phase was increased by 13.3and the cells in S phase were decreased by 7.9.The conclusion is that the weight UCA1 can enhance breast cancer fineness through competitive inhibition of miR-18a. Cytosolic tamoxifen is used to treat drug resistance.
【作者单位】：首都医科大学附属北京妇产医院乳腺科;
【基金】：北京市自然科学基金资助项目(7142055)资助~~
【分类号】：R737.9

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