Ahsani Zeinab, Mohammadi-Yeganeh Samira, Kia Vahid, Karimkhanloo Hamzeh, Zarghami Nosratollah, Paryan Mahdi
Department of Medical Biotechnology, Faculty of Advanced Medical sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Appl Biochem Biotechnol. 2017 Mar;181(3):884-897. doi: 10.1007/s12010-016-2256-8. Epub 2016 Sep 29.
Hepatocellular carcinoma (HCC) is an invasive form of hepatic cancer arising from the accumulation of multiple genetic alterations. In this study, the causal role of disturbed canonical Wnt/β-catenin pathway was approved, and some of HCC-driven important gene candidates were determined. MicroRNAs (miRNAs), small non-coding RNAs, are the key regulators of important cancer genes, and their participation in tumorigenesis has been shown. By reviewing literature, WNT1 gene with functional significance was selected to approve miRNAs as new subjects for targeted therapy.For proper and fast miRNA detection and also confirmation of the role of bioinformatics in obtaining practical data, we benefited from different bioinformatics tools such as TargetScan, miRanda, and DIANA. In order to use an HCC model, we used HepG2 cell line. Luciferase assay was applied to assess the ability of the selected miRNAs in targeting WNT1 3'-UTR. To overexpress the selected miRNA in HepG2 cell line, viral construct was prepared. Quantitative real-time PCR was performed to evaluate selected miRNA and target gene expression levels. miR-122 was selected according to data concerning various bioinformatics tools.miR-122 was downregulated and WNT1 gene expression was upregulated in HepG2 cell line. After viral construct transduction, miR-122 expression was elevated and WNT1 expression was notably declined. Finally, we introduced WNT1 gene as one of the important genes in HCC, and also, we showed that miR-122 can regulate WNT1 gene expression.Moreover, our study determines the potential of bioinformatics analyses in providing accurate and reliable data for miRNA: messenger RNA (mRNA) prediction.
肝细胞癌(HCC)是一种侵袭性肝癌,由多种基因改变的积累引起。在本研究中,证实了经典Wnt/β-连环蛋白信号通路紊乱的因果作用,并确定了一些由HCC驱动的重要基因候选物。微小RNA(miRNA)是一类小的非编码RNA,是重要癌症基因的关键调节因子,其参与肿瘤发生已得到证实。通过文献综述,选择具有功能意义的WNT1基因来证实miRNA作为靶向治疗的新靶点。为了准确快速地检测miRNA,并确认生物信息学在获取实际数据中的作用,我们利用了不同的生物信息学工具,如TargetScan、miRanda和DIANA。为了使用HCC模型,我们使用了HepG2细胞系。应用荧光素酶报告基因检测来评估所选miRNA靶向WNT1 3'-UTR的能力。为了在HepG2细胞系中过表达所选miRNA,制备了病毒构建体。进行定量实时PCR以评估所选miRNA和靶基因的表达水平。根据各种生物信息学工具的数据选择了miR-122。在HepG2细胞系中,miR-122表达下调,WNT1基因表达上调。病毒构建体转导后,miR-122表达升高,WNT1表达显著下降。最后,我们将WNT1基因作为HCC中的重要基因之一进行了介绍,并且我们表明miR-122可以调节WNT1基因的表达。此外,我们的研究确定了生物信息学分析在为miRNA:信使RNA(mRNA)预测提供准确可靠数据方面的潜力。