Weiqi Tan1, Seng-Gee Lim1, Theresa M C Tan1. 1. Weiqi Tan, Theresa MC Tan, Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore S117597, Singapore.
Abstract
AIM: To determine the expression of microRNA-210 (miR-210) in hepatocellular carcinoma (HCC) and to examine its role using HCC cells. METHODS: The expression of miR-210 was determined in 21 pairs of HCC samples and the corresponding surrounding non-tumor tissues. The effects of miR-210 on proliferation and cell cycle progression were examined using HepG2 and HuH7 cells. Over-expression and inhibition of miR-210 was achieved by transfection of the cells with miR-210 mimic or inhibitor. Luciferase reporter constructs were used to identify the miR-210 interacting site on Yes1. Yes1 expression was examined after miR-210 transfection, as well as in the HCC samples. RESULTS: miR-210 was significantly up-regulated by 3.4 fold (P < 0.01) in the tumor samples. The over-expression of miR-210 significantly reduced cell proliferation compared to the mock-treated cells (68.9% ± 7.4% and 53.6% ± 5.0%, P < 0.05 for the HepG2 and HuH7 cells respectively). Analysis of the HuH7 cells transfected with miR-210 mimic by flow cytometry showed that the cells took a longer time to reach the G2/M phase. The interaction between miR-210 and the 3'UTR of the Yes1 transcript was confirmed using a luciferase reporter assay. Over-expression of miR-210 reduced the expression of Yes1 protein in both HuH7 and HepG2 cells. Tumors with a greater than four-fold increase in the expression of miR-210 showed consistently lower expressions of Yes1 in the tumors. In nocodazole-treated cells with a significant G2/M cell population, Yes1 protein was significantly reduced and pre-inhibition of miR-210 in HuH7 cells was able to prevent the reduction of Yes1 protein expression. Knock-down of Yes1 by siRNA also led to reduced cell proliferation (70.8% ± 7.5%, P < 0.05 in the HuH7 cells). CONCLUSION: Up-regulation of miR-210 inhibits cell proliferation. Yes1 is a target of miR-210 and affects cell proliferation in HCC.
AIM: To determine the expression of microRNA-210 (miR-210) in hepatocellular carcinoma (HCC) and to examine its role using HCC cells. METHODS: The expression of miR-210 was determined in 21 pairs of HCC samples and the corresponding surrounding non-tumor tissues. The effects of miR-210 on proliferation and cell cycle progression were examined using HepG2 and HuH7 cells. Over-expression and inhibition of miR-210 was achieved by transfection of the cells with miR-210 mimic or inhibitor. Luciferase reporter constructs were used to identify the miR-210 interacting site on Yes1. Yes1 expression was examined after miR-210 transfection, as well as in the HCC samples. RESULTS:miR-210 was significantly up-regulated by 3.4 fold (P < 0.01) in the tumor samples. The over-expression of miR-210 significantly reduced cell proliferation compared to the mock-treated cells (68.9% ± 7.4% and 53.6% ± 5.0%, P < 0.05 for the HepG2 and HuH7 cells respectively). Analysis of the HuH7 cells transfected with miR-210 mimic by flow cytometry showed that the cells took a longer time to reach the G2/M phase. The interaction between miR-210 and the 3'UTR of the Yes1 transcript was confirmed using a luciferase reporter assay. Over-expression of miR-210 reduced the expression of Yes1 protein in both HuH7 and HepG2 cells. Tumors with a greater than four-fold increase in the expression of miR-210 showed consistently lower expressions of Yes1 in the tumors. In nocodazole-treated cells with a significant G2/M cell population, Yes1 protein was significantly reduced and pre-inhibition of miR-210 in HuH7 cells was able to prevent the reduction of Yes1 protein expression. Knock-down of Yes1 by siRNA also led to reduced cell proliferation (70.8% ± 7.5%, P < 0.05 in the HuH7 cells). CONCLUSION: Up-regulation of miR-210 inhibits cell proliferation. Yes1 is a target of miR-210 and affects cell proliferation in HCC.
Authors: Zhan Zhang; Hong Sun; Hongyue Dai; Ryan M Walsh; Maki Imakura; Janell Schelter; Julja Burchard; Xudong Dai; Aaron N Chang; Robert L Diaz; Joseph R Marszalek; Steven R Bartz; Michael Carleton; Michele A Cleary; Peter S Linsley; Carla Grandori Journal: Cell Cycle Date: 2009-09-29 Impact factor: 4.534