一种长非编码 RNA 和 microRNA 相互作用的新机制：CCAT2 通过抑制结肠癌细胞中 miR-145 的成熟过程来调节其表达。
A novel mechanism of lncRNA and miRNA interaction: CCAT2 regulates miR-145 expression by suppressing its maturation process in colon cancer cells.
机构信息
Department of Veterans Affairs Medical Center, 4646 John R, Detroit, MI, 48201, USA.
Departments of Internal Medicine, Wayne State University, Detroit, MI, 48201, USA.
出版信息
Mol Cancer. 2017 Sep 30;16(1):155. doi: 10.1186/s12943-017-0725-5.
BACKGROUND
Although both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. CCAT2 (Colon cancer-associated transcript-2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in CRC and is found to promote tumor growth. miRNAs, a class of naturally occurring short RNAs negatively control the expression of target genes by cleaving mRNA or through translation repression. Recently, we reported that miR-145 and miR-21 cooperate to regulate colon cancer stem cell (CSC) proliferation and differentiation. Considering that CCAT2 is mainly located in the nucleus and miRNA maturation process begins in the nucleus, we hypothesize that CCAT2 selectively blocks miR-145 maturation process, resulting in decreased mature miR-145 affecting colon CSC proliferation and differentiation.
METHODS
The levels of CCAT2 were manipulated by transfection of CCAT2 expression plasmid or knockdown by siRNA or by CRISPR/Cas9. Quantitative RT-PCR was performed to examine the expression of CCAT2 and pri-, pre- and mature miR-145/21. Fluorescence in situ hybridization (FISH) was used to visualize CCAT2 in the cells. In vitro processing of pri-miRNA-145 was performed using T7 RNA polymerase and recombinant human Dicer.
RESULTS
We have observed that modulated expression of CCAT2 regulates the expression of miR-145 in colon cancer HCT-116 and HT-29 cells. Knockout of CCAT2 increases miR-145 and negatively regulates miR-21 in HCT-116 cells, impairs proliferation and differentiation. In contrast, stable up-regulation of CCAT2 decreases mature miR-145 and increases the expression of several CSC markers in colon cancer cells. We have also observed that CCAT2 is enriched in the nucleus and correlates with the expression of pre-miR-145 but not pre-miR-21 in HCT-116 cells. These results indicate CCAT2 selectively blocks miR-145 maturation by inhibiting pre-miR-145 export to cytoplasm. Further, we revealed that CCAT2 blocks cleavage of pre-miR-145 by Dicer in vitro.
CONCLUSIONS
Our results identify CCAT2 as a negative regulator of miRNA-145 biogenesis, and expose a novel mechanism of lncRNA-miRNA crosstalk.
背景
尽管长链和 microRNAs 都被认为是结直肠癌（CRC）进展和转移的重要功能成分，但它们相互作用的机制仍知之甚少。CCAT2（结直肠癌相关转录物-2）是一种长链非编码 RNA（lncRNA），已在 CRC 中被报道过度表达，并被发现促进肿瘤生长。miRNAs 是一类天然存在的短 RNA，通过切割 mRNA 或通过翻译抑制来负调控靶基因的表达。最近，我们报道 miR-145 和 miR-21 合作调节结肠癌细胞干细胞（CSC）的增殖和分化。考虑到 CCAT2 主要位于细胞核中，并且 miRNA 成熟过程开始于细胞核中，我们假设 CCAT2 选择性地阻断 miR-145 的成熟过程，导致成熟 miR-145 减少，从而影响结肠 CSC 的增殖和分化。
方法
通过转染 CCAT2 表达质粒或 siRNA 或 CRISPR/Cas9 来操纵 CCAT2 的水平。通过定量 RT-PCR 检测 CCAT2 和 pri-、pre- 和成熟 miR-145/21 的表达。荧光原位杂交（FISH）用于观察细胞中的 CCAT2。使用 T7 RNA 聚合酶和重组人 Dicer 进行 pri-miRNA-145 的体外加工。
结果
我们观察到，CCAT2 的表达调节可调节结肠癌细胞 HCT-116 和 HT-29 中的 miR-145 表达。在 HCT-116 细胞中敲除 CCAT2 可增加 miR-145 并负调控 miR-21，从而损害增殖和分化。相反，稳定上调 CCAT2 可降低成熟 miR-145 并增加结肠癌细胞中几种 CSC 标志物的表达。我们还观察到，CCAT2 在细胞核中富集，并与 HCT-116 细胞中 pre-miR-145 的表达相关，但与 pre-miR-21 的表达无关。这些结果表明，CCAT2 通过抑制 pre-miR-145 向细胞质的输出来选择性地阻断 miR-145 的成熟。此外，我们揭示了 CCAT2 在体外阻断 Dicer 对 pre-miR-145 的切割。
结论
我们的结果将 CCAT2 鉴定为 miRNA-145 生物发生的负调节剂，并揭示了 lncRNA-miRNA 串扰的新机制。
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