Selective blockade of DCAMKL-1 results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism.
Selective blockade of DCAMKL-1 results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism.
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DOI：
10.1053/j.gastro.2009.05.004
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发表时间：
2009-08
期刊：
影响因子：
29.4
通讯作者：
Houchen CW
中科院分区：
文献类型：
--
作者：
Sureban SM;May R;Ramalingam S;Subramaniam D;Natarajan G;Anant S;Houchen CW
microRNA（miRNAs）是一类非蛋白质编码的RNA，可调控基因的表达。据报道，肿瘤抑制miRNA let-7a在胚胎干细胞和人类癌症中被转录后抑制。微管相关激酶DCAMKL-1是一种公认的肠干细胞标志物，在ApcMin/+腺瘤中表达。我们研究了DCAMKL-1对let-7a miRNA和癌基因c-Myc的表达以及在肿瘤发生中的作用。在人组织微阵列载玻片上进行DCAMKL-1的免疫染色。用DCAMKL-1小干扰（si）RNA转染HCT 116和SW 480细胞，并分析DCAMKL-1、c-Myc（使用免疫印迹和实时RT-PCR）和pri-let-7a miRNA（使用实时RT-PCR）水平。将DCAMKL-1 siRNA的脂质体制剂施用到裸鼠中的HCT 116异种移植物中，并测量肿瘤体积。使用在3' UTR处含有let-7a结合位点的质粒的荧光素酶报告基因测定来测量细胞系中的let-7a。使用DCAMKL-1和荧光激活细胞分选（FACS）从正常小鼠肠分离细胞，并进行pri-let-7a miRNA分析。DCAMKL-1在人结直肠癌中的表达增加。siRNA介导的DCAMKL-1阻断导致HCT 116肿瘤异种移植物生长停滞，pri-let-7a miRNA水平升高，荧光素酶活性相应降低，以及癌基因c-Myc表达降低。通过FACS分离的DCAMKL-1+细胞显示与更分化的细胞相比，pri-let-7a miRNA显著减少。DCAMKL-1是肠干细胞和结直肠癌细胞中let-7a miRNA生物合成的负调控因子;它可能代表基于抗癌干细胞策略的新靶点。
MicroRNAs (miRNAs) are non-protein coding RNAs that regulate gene expression. The tumor suppressor miRNA let-7a has been reported to be inhibited post-transcriptionally in embryonic stem cells and in human cancers. The microtubule-associated kinase DCAMKL-1 is a putative intestinal stem cell marker that is expressed in ApcMin/+ adenomas. We investigated the role of DCAMKL-1 on expression of let-7a miRNA and the oncogene c-Myc and in tumorigenesis. Immunostaining for DCAMKL-1 was performed on human tissue microarray slides. HCT116 and SW480 cells were transfected with DCAMKL-1 small interfering (si)RNA and analyzed for DCAMKL-1, c-Myc (using immunoblot and real-time RT-PCR) and pri-let-7a miRNA (using real-time RT-PCR) levels. A liposomal preparation of DCAMKL-1 siRNA was administered into HCT116 xenografts in nude mice and tumor volumes were measured. A luciferase reporter assay, with a plasmid containing a let-7a binding site at the 3’ UTR, was utilized to measure let-7a in cell lines. Cells were isolated from normal mouse intestine using DCAMKL-1 and fluorescence-activated cell sorting (FACS) and subjected to pri-let-7a miRNA analysis. Expression of DCAMKL-1 was increased in human colorectal cancers. siRNA-mediated blockade of DCAMKL-1 resulted in HCT116 tumor xenograft growth arrest, increased levels of pri-let-7a miRNA, a corresponding decrease in luciferase activity, as well as decreased expression of the oncogene c-Myc. DCAMKL-1+ cells isolated by FACS demonstrated a significant decrease in pri-let-7a miRNA, compared to more-differentiated cells. DCAMKL-1 is a negative regulator of let-7a miRNA biogenesis in intestinal stem and colorectal cancer cells; it could represent a novel target for anti-cancer stem cell-based strategies.
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