Bioinformatics prediction and experimental validation of microRNA-20a targeting Cyclin D1 in hepatocellular carcinoma
Bioinformatics prediction and experimental validation of microRNA-20a targeting Cyclin D1 in hepatocellular carcinoma
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DOI：
10.1177/1010428317698361
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发表时间：
2017-04-01
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影响因子：
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通讯作者：
Paryan, Mahdi
中科院分区：
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作者：
Karimkhanloo, Hamzeh;Mohammadi-Yeganeh, Samira;Paryan, Mahdi
肝细胞癌是原发性肝癌的主要形式，分别是男性和女性癌症相关死亡的第二大和第六大原因。大量研究表明，Wnt/β-catenin信号通路在肝细胞癌的生长、发展和分化过程中起着关键作用，是肝细胞癌中调控失调的主要信号通路之一。细胞周期蛋白D1是一种原癌基因，是Wnt信号通路的主要调节因子之一，在包括肝细胞癌在内的多种肿瘤中均有过表达。利用几个有效的生物信息学数据库，我们预测这些microRNAs能够靶向Cyclin D1信使RNA的3‘-非翻译区。根据结果，miR-20a被选为针对Cyclin D1信使RNA的排名最高的microRNA。用荧光素酶分析来证实生物信息学预测结果。用实时荧光定量聚合酶链式反应检测细胞周期蛋白D1的表达。然后，用含miR-20a的慢病毒转导HepG2细胞。检测miR-20a和Cyclin D1的表达。荧光素酶检测结果表明，miR-20a可以靶向Cyclin D1信使RNA的3‘非翻译区。此外，慢病毒诱导miR-20a后，HepG2细胞中Cyclin D1的表达下降了238倍。结果表明，miRNA-20a诱导对Cyclin D1基因的下调有显著作用。我们的结果表明miR-20a可以作为一种新的治疗候选基因和肝细胞癌诊断的生物标志物。
Hepatocellular carcinoma is the major form of primary liver cancer, which is the second and sixth leading cause of cancer-related death in men and women, respectively. Extensive research indicates that Wnt/beta-catenin signaling pathway, which plays a pivotal role in growth, development, and differentiation of hepatocellular carcinoma, is one of the major signaling pathways that is dysregulated in hepatocellular carcinoma. Cyclin D1 is a proto-oncogene and is one of the major regulators of Wnt signaling pathway, and its overexpression has been detected in various types of cancers including hepatocellular carcinoma. Using several validated bioinformatic databases, we predicted that the microRNAs are capable of targeting 3'-untranslated region of Cyclin D1 messenger RNA. According to the results, miR-20a was selected as the highest ranking microRNA targeting Cyclin D1 messenger RNA. Luciferase assay was recruited to confirm bioinformatic prediction results. Cyclin D1 expression was first assessed by quantitative real-time polymerase chain reaction in HepG2 cell line. Afterward, HepG2 cells were transduced by lentiviruses containing miR-20a. Then, the expression of miR-20a and Cyclin D1 was evaluated. The results of luciferase assay demonstrated targeting of 3'-untranslated region of Cyclin D1 messenger RNA by miR-20a. Furthermore, 238-fold decline in Cyclin D1 expression was observed after lentiviral induction of miR-20a in HepG2 cells. The results highlighted a considerable effect of miRNA-20a induction on the down-regulation of Cyclin D1 gene. Our results suggest that miR-20a can be used as a novel candidate for therapeutic purposes and a biomarker for hepatocellular carcinoma diagnosis.