Identification and characterization of microRNAs expressed in human breast cancer chemo-resistant MCF-7/Adr cells by Solexa deep-sequencing technology
Identification and characterization of microRNAs expressed in human breast cancer chemo-resistant MCF-7/Adr cells by Solexa deep-sequencing technology
复制标题
利用Solexa深度测序技术鉴定和表征人乳腺癌化疗耐药MCF-7/Adr细胞中表达的microRNA
DOI：
10.1016/j.biopha.2015.07.019
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发表时间：
2015
影响因子：
7.5
通讯作者：
Yin Hong
中科院分区：
文献类型：
--
作者：
Xu Pengfei;Wang Luyu;Huang Lei;Li Wenqu;Lv Shanshan;Lv Mingming;Ma Jingjing;Zhou Qian;Wu Xiaowei;Fu Ziyi;Lu Cheng;Yin Hong
背景/目的乳腺癌是女性最常见的肿瘤类型，化疗耐药一直是临床患者治疗的主要临床障碍。 miRNA 是被证明在乳腺癌化疗中发挥一定作用的因素之一。在本研究中，我们利用Solexa深度测序技术从体外样本中鉴定差异表达的miRNA，试图寻找miRNA与乳腺癌化疗耐药之间的新关系。方法用阿霉素脉冲选择人乳腺癌MCF-7细胞系（10个脉冲，每周一次，持续4小时，加入1μM阿霉素）以产生MCF-7/Adr细胞。从处理和未处理的 MCF-7 细胞中提取总 RNA，随后进行实时 PCR。建立MCF7NON和MCF7ADR两个小RNA文库，记录上述PCR产物的Solexa测序结果。所有测序结果均通过Stem-loop实时PCR验证。利用GO注释和KEGG分析程序富集差异表达的miRNA。结果结果显示，当与发夹结构比对时，MCF7ADR和MCF7NON文库中分别映射了214,822和378,597个reads。同时，当与成熟序列比对时，映射了 1323 和 520 个读数。此外，两个文库共表达了 310 个已知的成熟 miRNA。比较MCF7ADR组与MCF7NON组，18个miRNA的表达存在显着差异。 GO注释和KEGG分析显示靶基因在转录和发育调控以及Wnt信号通路、MAPK信号通路和TGF-β信号通路方面富集。结论结果证明Solexa深度测序是分析小RNA的强大而可靠的平台。对已确定的生物学过程和途径还需进一步研究，并加大对乳腺癌化疗耐药机制的研究力度。
Background/AimBreast cancer is the most common type of tumor in female and chemoresistance has been a major clinical obstacle to the treatment in clinical patients. miRNA was one of the factors demonstrated to play certain roles in chemoesistance in breast cancer. In this study, we exploited Solexa deep sequencing technology to identify differentially expressed miRNA from samples in vitro, trying to find novel relationship between miRNA and chemoresistance in breast cancer.MethodsThe human breast cancer MCF-7 cell line was pulse-selected with doxorubicin (10 pulses, once a week for 4 h, with 1 μM doxorubicin) to generate MCF-7/Adr cells. Total RNA was extracted from the treated and untreated MCF-7 cells and subsequently subjected to real time PCR. Two small RNA libraries of MCF7NON and MCF7ADR were established to record the Solexa sequencing results of the PCR products above. All the sequencing results were verified by Stem-loop real-time PCR. GO annotation and KEGG analysis program were exploited to enrich the differentially expressed miRNAs.ResultsThe results showed that 214,822 and 378,597 reads were mapped in the MCF7ADR and MCF7NON libraries when aligned to hairpin structure respectively. Meanwhile, 1323 and 520 reads were mapped when aligned to mature sequences. In addition, 310 known mature miRNAs were coexpressed in both libraries. Comparing the MCF7ADR group to the MCF7NON group, 18 miRNAs were significantly differentially expressed. GO annotation and KEGG analysis showed that the target genes were enriched in regulation of transcription and development as well as Wnt signaling pathway, MAPK signaling pathway and TGF-ß signaling pathway.ConclusionThe results proved that the Solexa deep sequencing was a powerful and reliable platform to analyze small RNAs. And further investigation should be conducted for the biological process and pathways that have been identified and more efforts should be made to research the mechanism of chemoresistance in breast cancer.