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Association between High miR-211 microRNA Expression and the Poor Prognosis of Oral Carcinoma

¹ Institute of Oral Biology, School of Dentistry, National Yang-Ming University, No. 155, Li-Nong St., Sec.2, Taipei, Taiwan 112;
² Department of Oral and Maxillofacial Surgery, Taipei MacKay Memorial Hospital, Taipei, Taiwan;
³ MacKay Medicine, Nursing and Management College, Taipei, Taiwan; and
⁴ Biosettia, San Diego, CA, USA

View larger version (18K): [in this window] [in a new window]   Figure 1. miR-211 expression in microdissected cells. (a) We performed stem-loop Q-RT-PCR analysis to detect expression of miR-211 in microdissected cells. The scattered dot plots of miR-211 expression in OPL and OSCC cells appear in the left columns. Each dot represents the mean value of triplicate analyses. The miR-211 expression of N0 plus N1 tumors and N2 tumors are shown as box- and whisker-plots. miR-211 expression is higher in N2 tumors than in the other tumors (p = 0.011). (b) miR-211 expression is significantly higher in tumors with vascular invasion than in the other tumors (p = 0.002). Box- and whisker-plot. In (a,b), horizontal lines, median; +, mean. (c) Kaplan-Meier analysis. The tumors were divided into two groups with the median or 0 of Ct as cut-offs. Tumors with higher miR-211 expression ( Ct > median) and tumors with miR-211 higher than matched non-cancerous mucosa ( Ct > 0) were significantly associated with worse survival. (d) Scattered dot plot of miR-211 expression and miR-204 expression in all OSCC samples, showing a significant correlation between the expression of miR-211 and miR-204. Dot lines indicate 95% confidence interval. **p < 0.01; unpaired t test.

View larger version (51K): [in this window] [in a new window]   Figure 2. Enforced miR-211 expression in cells. (a) Green fluorescence in SAS-GFP and SAS-miR-211 cells (upper), OECM-1-GFP and OECM-1-miR-211 cells (middle), and NHOK1-GFP and NHOK1-miR-211 cells (lower). Bars, 100 µm. (b) Stem-loop Q-RT-PCR analysis detected up-regulation of miR-211 in SAS-miR-211, OECM-1-miR-211, NHOK1-miR-211, and NHOK2-miR-211 cells compared with controls. SAS-GFP is the baseline. (c) Reporter assay in SAS cells. Each miR-31 or miR-211 reporter plasmid was co-transfected with pCMV-Luc to SAS-GFP and SAS-miR-211 cells. The luciferase activity was used to normalize transfection efficiency. The β-galactosidase activity in cells transfected with empty pCMV-LacZ vector was used to normalize β-galactosidase activity. Compared with controls, SAS-miR-211 cells having specific miR-211 targeting on the antisense sequences of miR-211 significantly repressed β-galactosidase activity (p = 0.025). The antisense sequences of miR-31 did not affect β-galactosidase activity in SAS-miR-211 cells. (d) Reporter assay in OECM-1 cells showing significant repression of β-galactosidase activity in OECM-1-miR-211 cells in relation to controls (p = 0.005). Data shown are mean ± SE from triplicate analyses (in b) and two distinctive triplicate analyses (in c,d). *p < 0.05, **p < 0.01; Mann-Whitney test.

View larger version (14K): [in this window] [in a new window]   Figure 3. Phenotypic changes of SAS cells with enforced miR-211 expression. (a-c) Cell proliferation, migration, and anchorage-independent colony formation, respectively. Significant increase in proliferation (at day 6 to day 8), migration, and anchorage-independent colony formation was noted in SAS-miR-211 cells relative to SAS-GFP cells. SAS-miR-211 cells grew to a plateau phase after day 8. Data shown are mean ± SE from two distinctive triplicate analyses. (d) Tumorigenesis assay showed greater tumorigenicity in SAS-miR-211 xenografts than in SAS-GFP xenografts following the injection of 1 x 105 cells (solid lines) or 2.5 x 105 cells (dotted lines). Data shown are mean ± SE from 5 or 6 nude mice. *p < 0.05; **p < 0.01; ***p < 0.001; Mann-Whitney test.

Journal of Dental Research, Vol. 87, No. 11, 1063-1068 (2008)
DOI: 10.1177/154405910808701116


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