上调的 miR-1258 通过直接靶向 CRC 中的 E2F8 来调节细胞周期并抑制细胞增殖。
Upregulated miR-1258 regulates cell cycle and inhibits cell proliferation by directly targeting E2F8 in CRC.
机构信息
Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China.
出版信息
Cell Prolif. 2018 Dec;51(6):e12505. doi: 10.1111/cpr.12505. Epub 2018 Aug 24.
OBJECTIVES
MicroRNAs (miRNAs) as small noncoding RNA molecules function by regulating their target genes negatively. MiR-1258 was widely researched in multicancers, but its role remains unclear in colorectal cancer (CRC).
METHODS
The expression of miR-1258 and its specific target gene were detected in human CRC specimens and cell lines by miRNA RT-PCR, qRT-PCR and Western blot. The effects of miR-1258 on CRC proliferation were evaluated using CCK-8 assays, EdU incorporation, colony formation assays and cell-cycle assays; in vitro and the in vivo effects were investigated using a mouse tumorigenicity model. Luciferase reporter and RIP assays were employed to identify interactions between miR-1258 and its specific target gene.
RESULTS
MiR-1258 was downregulated in CRC tissues and CRC cell lines, and upregulated miR-1258 was proved to inhibit proliferation and arrest cell cycle at G0/G1 in vitro and vivo. Luciferase reporter, RIP and western blot assays revealed E2F8 to be a direct target of miR-1258. The effects of miR-1258 in proliferation and cell cycle regulation can be abolished by E2F8 through rescue experiments. By directly targeting E2F8, miR-1258 influenced the expression of several cell-cycle factors, including cyclin D1 (CCND1) and cyclin dependent kinase inhibitor 1A (p21).
CONCLUSION
MiR-1258 may function as a suppressive factor by negatively controlling E2F8, thus, highlighting the potential role of miR-1258 as a therapeutic target for human CRC.
目的
微小 RNA（miRNAs）作为小的非编码 RNA 分子，通过负向调控其靶基因发挥作用。miR-1258 在多种癌症中广泛研究，但在结直肠癌（CRC）中的作用尚不清楚。
方法
通过 miRNA RT-PCR、qRT-PCR 和 Western blot 检测人 CRC 标本和细胞系中 miR-1258 及其特异靶基因的表达。通过 CCK-8 测定、EdU 掺入、集落形成测定和细胞周期测定评估 miR-1258 对 CRC 增殖的影响；利用小鼠致瘤模型研究体外和体内的影响。利用荧光素酶报告和 RIP 测定来鉴定 miR-1258 与其特异靶基因之间的相互作用。
结果
miR-1258 在 CRC 组织和 CRC 细胞系中下调，上调 miR-1258 被证明可抑制体外和体内的增殖并使细胞周期停滞在 G0/G1 期。荧光素酶报告、RIP 和 Western blot 测定表明 E2F8 是 miR-1258 的直接靶基因。通过拯救实验可消除 miR-1258 在增殖和细胞周期调控中的作用。通过直接靶向 E2F8，miR-1258 影响了几个细胞周期因子的表达，包括细胞周期蛋白 D1（CCND1）和周期蛋白依赖性激酶抑制剂 1A（p21）。
结论
miR-1258 可能通过负向调控 E2F8 发挥抑制因子的作用，从而强调 miR-1258 作为人类 CRC 治疗靶点的潜在作用。
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