Wang Lumin, Yao Jiayi, Shi Xin, Hu Lili, Li Zongfang, Song Tusheng, Huang Chen
Department of Genetics and Molecular Biology, Xi'an Jiaotong University Health Science Center, No,76 West Yanta Road, Xi'an, Shaanxi 710061, P,R, China.
BMC Cancer. 2013 Oct 2;13:448. doi: 10.1186/1471-2407-13-448.
MicroRNAs are regulators that can play an essential role in tumorigenesis. Although miR-302 families have been suggested to be tumor repressors in human cancer, the mechanism by which they suppress tumor development remains to be defined. In this study, we discover that miR302b suppresses tumor proliferation may due to directly targeting EGFR in human hepatocellular carcinoma (HCC).
QRT-PCR was used to assess miR-302b and EGFR expression in 27 pairs of clinical hepatocellular carcinoma tissues and their corresponding adjacent nontumorous liver tissues. MTT, colony formation, immunofluorescence staining, and cell cycle assays were used to examine the tumor suppressor role of miR302b in cell proliferation. Luciferase assays were performed to assess the EGFR was a novel target of miR-302b. Western blot assay was used to validate the protein expression level.
We demonstrated that miR-302b was frequently down-regulated, whereas EGFR was up-regulated in 27 pairs of clinical HCC and non-tumorous counterparts. The dual-luciferase reporter assays revealed that EGFR was a novel target of miR-302b. Re-expression of miR-302b resulted in the inhibition of proliferation in hepatocellular carcinoma SMMC-7721 cells. The silencing of EGFR by miR-302b or siEGFR led to down-regulation of proliferation-related proteins, such as AKT2, CCND1, and CDK2.
miR-302b suppresses HCC growth may due to targeting the EGFR/AKT2/CCND1 pathway.
微小RNA是在肿瘤发生过程中发挥重要作用的调节因子。尽管miR - 302家族在人类癌症中被认为是肿瘤抑制因子，但其抑制肿瘤发展的机制仍有待明确。在本研究中，我们发现miR - 302b抑制肿瘤增殖可能是由于其在人类肝细胞癌（HCC）中直接靶向表皮生长因子受体（EGFR）。
采用定量逆转录聚合酶链反应（QRT - PCR）检测27对临床肝细胞癌组织及其相应的癌旁非肿瘤肝组织中miR - 302b和EGFR的表达。使用MTT法、集落形成实验、免疫荧光染色和细胞周期分析检测miR - 302b在细胞增殖中的肿瘤抑制作用。进行荧光素酶报告基因实验以评估EGFR是否为miR - 302b的新靶点。采用蛋白质免疫印迹法验证蛋白表达水平。
我们发现，在27对临床肝癌及癌旁组织中，miR - 302b经常下调，而EGFR上调。双荧光素酶报告基因实验表明EGFR是miR - 302b的新靶点。miR - 302b的重新表达导致肝癌SMMC - 7721细胞增殖受到抑制。miR - 302b或小干扰RNA（siEGFR）沉默EGFR导致增殖相关蛋白如AKT2、细胞周期蛋白D1（CCND1）和细胞周期蛋白依赖性激酶2（CDK2）表达下调。
miR - 302b可能通过靶向EGFR/AKT2/CCND1信号通路抑制肝癌生长。
