Yi S-J, Li L-L, Tu W-B
General Surgery Department of Shenzhen University Affiliated Hospital, Shenzhen, China.
Eur Rev Med Pharmacol Sci. 2016 Dec;20(24):5148-5154.
To analyze the function and mechanism of miR-214 in regulating breast cancer cell proliferation.
MiR-214 expression level was measured by quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR). The protein level of β-catenin, PCNA and WNT targets (cyclin D1 and c-Myc) was evaluated by Western blot analysis. The effects of miR-214 on cell proliferation and cisplatin sensitivity were assessed by Cell Counting Kit-8 (CCK-8) assay and/or 5-bromo-2'-deoxyuridine (BrdU) assay. The effect of miR-214 on β-catenin and WNT signaling activity was tested by luciferase reporter assay.
MiR-214 was significantly downregulated in breast cancer tissues and was inversely correlated with β-catenin expression. Forced expression of miR-214 in breast cancer cell line MCF-7 led to a decrease in cell proliferation and an increase in cisplatin sensitivity. Moreover, forced expression of miR-214 decreased the activity of WNT luciferase reporter and the luciferase reporter containing the 3'-untranslated region (3'UTR) of β-catenin, whereas antisense inhibitor of miR-214 showed an opposite effect. Finally, miR-214 decreased the expression of β-catenin and multiple WNT target genes.
MiR-214 is downregulated and serves as a novel tumor suppressor in breast cancer. Forced expression of miR-214 in breast cancer cells diminished cancer cell survival possibly through inhibiting WNT signaling by direct repression of β-catenin.
分析miR-214在调节乳腺癌细胞增殖中的作用及机制。
采用定量逆转录聚合酶链反应（qRT-PCR）检测miR-214表达水平。通过蛋白质免疫印迹分析评估β-连环蛋白、增殖细胞核抗原（PCNA）和WNT靶标（细胞周期蛋白D1和c-Myc）的蛋白水平。采用细胞计数试剂盒-8（CCK-8）检测法和/或5-溴-2'-脱氧尿苷（BrdU）检测法评估miR-214对细胞增殖和顺铂敏感性的影响。通过荧光素酶报告基因检测法检测miR-214对β-连环蛋白和WNT信号活性的影响。
miR-214在乳腺癌组织中显著下调，且与β-连环蛋白表达呈负相关。在乳腺癌细胞系MCF-7中强制表达miR-214导致细胞增殖减少和顺铂敏感性增加。此外，强制表达miR-214降低了WNT荧光素酶报告基因以及含有β-连环蛋白3'-非翻译区（3'UTR）的荧光素酶报告基因的活性，而miR-214的反义抑制剂则表现出相反的效果。最后，miR-214降低了β-连环蛋白和多个WNT靶基因的表达。
miR-214在乳腺癌中表达下调，是一种新型肿瘤抑制因子。在乳腺癌细胞中强制表达miR-214可能通过直接抑制β-连环蛋白来抑制WNT信号，从而减少癌细胞存活。
