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

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0.1 μmol/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。

Urothelial carcinoma-associated 1 enhances tamoxifen resistance in breast cancer cells through competitively inhibiting miR-18a

Objective:To investigate how urothelial carcinoma-associated 1 (UCA1) and miR-18a modulates acquired tamoxifen resistance and the relevant mechanisms in estrogen receptor (ER) positive cancer cells.Methods: qRT-PCR was performed to detect UCA1 and miR-18a expression in breast cancer cells.Dual luciferase assay was performed to detect the binding between miR-18a and UCA1 3′UTR.Tamoxifen sensitive MCF-7 cells were transfected with UCA1 expression vector or miR-18a inhi-bitors.Tamoxifen resistant LCC9 and BT474 cells were transfected with UCA1 siRNA or miR-18a mi-mics.CCK-8 assay was performed to detect cell viability.Soft agar assay was performed to assess cell colony formation.Flow cytometric analysis was performed to check cell cycle distribution.Results: UCA1 was significantly upregulated in tamoxifen resistant LCC2,LCC9,and BT474 cells than in tamoxifen sensitive MCF-7 cells.UCA1 expression was significantly upregulated in MCF-7 cells after treatment with 0.1 μmol/L tamoxifen.UCA1 overexpression enhanced cell viability of MCF-7 cells after tamoxifen treatment,while UCA1 siRNA significantly suppressed viability of LCC9 and BT474 cells after tamoxifen treatment.In MCF-7 cells,compared with vector control+tamoxifen group,the average cell colony number and colony size of the UCA1+tamoxifen group was 19.0％ more and 29.0％ larger respectively,while the proportions of the cells in G1 phase and in S phase were 7.3％ lower and 6.7％ higher respectively.In BT474 cells,compared with siRNA control+tamoxifen group,the average cell colony number and colony size of the si-UCA1+tamoxifen group were 54.0％ less and 42.0％ smaller respectively,while the proportions of the cells in G1 phase and in S phase were 9.0％ higher and 6.2％ lower respectively.UCA1 directly interacted with miR-18a and reduced its expression in ER positive breast cancer cells.Knockdown of miR-18a increased viability of MCF-7 cells after tamoxifen treatment,while miR-18a overexpression significantly reduced viability of BT474 cells after tamoxifen treatment.In MCF-7 cells,compared with miRNA inhibitor control+tamoxifen group,the average cell colony number and colony size of the miR-18a inhibitor+tamoxifen group were 15.0％ more and 33.0％ larger respectively,while the proportions of the cells in G1 phase and in S phase were 8.8％ lower and 5.3％ higher respectively.In BT474 cells,compared with miRNA control+tamoxifen group,the average cell colony number and colony size of the miR-18a mimics+tamoxifen group were 47.0％ less and 25.0％ smaller respectively,while the proportions of the cells in G1 phase and in S phase were 13.3％ higher and 7.9％ lower respectively.Conclusion: UCA1 can increase tamoxifen resistance of ER positive breast cancer cells via competitively inhibiting of miR-18a.