Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells
Role of miR-19b and its target mRNAs in 5-fluorouracil resistance in colon cancer cells
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DOI：
10.1007/s00535-012-0547-6
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发表时间：
2012-08-01
影响因子：
6.3
通讯作者：
Rokutan, Kazuhito
中科院分区：
文献类型：
--
作者：
Kurokawa, Ken;Tanahashi, Toshihito;Rokutan, Kazuhito
结肠直肠癌的耐药性被认为是由microRNA表达的变化介导的，但microRNA的具体身份和作用在很大程度上尚不清楚。我们检测了5-氟尿嘧啶（5-FU）耐药对microRNA表达的影响。两种类型的5-FU耐药结肠癌细胞来源于DLD-1和KM 12 C细胞系。用含有723个microRNA的微阵列分析microRNA的表达，并通过定量实时聚合酶链反应（qRT-PCR）进行验证。为了研究microRNA的下游介导因子，我们使用microRNA：mRNA免疫沉淀（RIP）-芯片和途径分析工具来鉴定microRNA潜在的直接靶点，在5-FU耐药细胞中，miR-19 b和miR-21过表达。值得注意的是，miR-19 b在DLD-1耐药细胞中上调3.47倍，尽管暴露于5-FU，但细胞周期谱没有改变。在转染miR-19 b后，将特异性mRNA募集到用Ago 2抗体分离的microRNA：mRNA复合物中，并进行全基因组转录分析。在该分析中，66种靶mRNA在来自DLD-1抗性细胞的microRNA：mRNA复合物中富集至少5.0倍。mRNA靶点的抗肿瘤通路分析显著（P < 0.05）表明，“细胞周期”类别可能是与5-FU耐药相关的分子和细胞功能领域。在候选mRNA靶点中，SFPQ和MYBL 2已与细胞周期功能相关。我们发现miR-19 b响应5-FU而上调，并且在5-FU处理下，miR-19 b介导细胞周期的潜在靶点。我们的研究为深入了解结直肠癌5-FU耐药的机制提供了重要线索。
Drug resistance in colorectal cancers is assumed to be mediated by changes in the expression of microRNAs, but the specific identities and roles of microRNAs are largely unclear. We examined the effect of 5-fluorouracil (5-FU) resistance on microRNA expression.Two types of 5-FU-resistant colon cancer cells were derived from the DLD-1 and KM12C cell lines. The expressions of microRNAs were profiled with a microarray containing 723 microRNAs and validated by quantitative real-time polymerase chain reaction (qRT-PCR). To survey the downstream mediators of microRNA, we used a microRNA:mRNA immunoprecipitation (RIP)-Chip and pathway analysis tool to identify potential direct targets of microRNA.In response to 5-FU, miR-19b and miR-21 were over-expressed in 5-FU-resistant cells. Of note, miR-19b was up-regulated 3.47-fold in the DLD-1 resistant cells, which exhibited no alteration in cell cycle profiles despite exposure to 5-FU. After transfection of miR-19b, specific mRNAs were recruited to microRNA:mRNA complexes isolated with Ago2 antibody and subjected to whole-genome transcriptional analysis. In this analysis, 66 target mRNAs were enriched by at least 5.0-fold in the microRNA:mRNA complexes from DLD-1 resistant cells. Ingenuity pathway analysis of mRNA targets significantly (P < 0.05) indicated the category "Cell Cycle" as a probable area of the molecular and cellular function related with 5-FU resistance. Among candidate mRNA targets, SFPQ and MYBL2 have been linked to cell cycle functions.We revealed up-regulation of miR-19b in response to 5-FU and potential targets of miR-19b mediating the cell cycle under treatment with 5-FU. Our study provides an important insight into the mechanism of 5-FU resistance in colorectal cancers.