Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer
Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer
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DOI：
10.1016/j.urolonc.2010.05.008
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发表时间：
2012-07-01
影响因子：
2.7
通讯作者：
Nakagawa, Masayuki
中科院分区：
文献类型：
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作者：
Chiyomaru, Takeshi;Enokida, Hideki;Nakagawa, Masayuki
目的：我们之前的研究表明，FSCN1在膀胱癌（BC）中可能具有致癌功能，其表达受特异性microRNAs （miRNAs）的调控。最近。LIM和SH3蛋白1 （LASP1）以及FSCN1已被报道为肌动蛋白丝束蛋白，它们位于附着于细胞膜内表面的相同复合物中。我们假设LASP1和FSCN1具有致癌功能，并受靶向LASP1 mRNA的mirna调控。方法：采用实时荧光定量pcr法检测86例临床标本中LASP1 mRNA的表达水平。用siRNA转染lasp1敲低的BC细胞系，通过XTT实验、伤口愈合实验和基质侵袭实验检测细胞活力。我们使用基于网络的软件来搜索靶向LASP1 mRNA的候选mirna，我们重点关注miR-1、miR-133a、miR-145和miR-218。荧光素酶报告基因检测用于确定mirna与LASP1 mRNA之间的实际结合位点。结果：Real-time RT-PCR结果显示，76例临床BC标本中LASP1 mRNA表达高于10例正常膀胱上皮（P < 0.05）。使用si- lasp1转染的BC细胞系进行的功能丧失研究表明，与对照组相比，转染物显著抑制了细胞活力（P < 0.0005），抑制了细胞迁移（P < 0.0001），并且入侵细胞数量减少（P < 0.005）。瞬时转染三种mirna （miR-1、miR-133a和miR-218）可抑制LASP1 mRNA的表达水平和蛋白水平，这三种mirna被预测为靶向LASP1 mRNA的mirna。荧光素酶报告基因实验显示，miR-1、miR-133a和miR-218转染体的发光强度显著降低（P < 0.05），表明这些mirna在LASP1 mRNA的3'非翻译区有实际的靶位。此外，miR-218、miR-1和miR-133a转染物显著抑制细胞活力（P < 0.001）。结论：我们的数据表明LASP1可能具有致癌功能，它可能受miR-1、miR-133a和miR-218的调控，这些mirna可能在BC中起肿瘤抑制作用。(C) 2012爱思唯尔公司版权所有。
Objective: Our previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently. LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA.Methods: The expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA.Results: Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P < 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P < 0.0005), cell migration inhibition (P < 0.0001), and a decrease in the number of invading cells (P < 0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P < 0.05), suggesting that these miRNAs have actual target sites in the 3' untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P < 0.001).Conclusion: Our data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC. (C) 2012 Elsevier Inc. All rights reserved.