Volume 135, Issue 1, Pages (July 2008)

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Volume 135, Issue 1, Pages 257-269 (July 2008) MicroRNA-223 Is Commonly Repressed in Hepatocellular Carcinoma and Potentiates Expression of Stathmin1 Queenie W.–L. Wong, Raymond W.–M. Lung, Priscilla T.–Y. Law, Paul B.–S. Lai, Kathy Y.–Y. Chan, Ka–Fai To, Nathalie Wong Gastroenterology Volume 135, Issue 1, Pages 257-269 (July 2008) DOI: 10.1053/j.gastro.2008.04.003 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 (A) Seventy-five miRNAs displayed consistent differential expressions in 50% or more of cell lines examined. Error bar represents SEM. (B and C) A validating analysis by qPCR confirmed concurrent up-regulations of miR-222 and down-regulations of miR-223 in HCC cell lines studied. Gastroenterology 2008 135, 257-269DOI: (10.1053/j.gastro.2008.04.003) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 (A) Common up-regulations and down-regulations of miRNAs were suggested from profiling. Red depicts high expression levels, whereas green and grey correspond to low expression levels and nondetectable signals, respectively. (B and C) Hierarchical clustering of differential miRNAs revealed 2 distinct dendrograms that coincided with HCV- and HBV-related cell lines relative to nonviral-related cell lines. Gastroenterology 2008 135, 257-269DOI: (10.1053/j.gastro.2008.04.003) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Representative examples of Northern blot for miR-223 and miR-222 expressions in normal livers (nl), cell lines, HCC tumors, and paired adjacent nontumoral livers shown. (A) (1 and 2) Absent or negligible miR-223 expressions were suggested in tumors and cell lines. (3) qPCR analysis indicated consistent repressed miR-223 expressions in the 3 groups of tumors and cell lines (t) compared with adjacent nontumoral livers (tn) and NL. Box-plot lines represent median and interquartile ranges of the normalized threshold value (ct). Dotted lines denotes the median normalized Ct of normal livers. (B) (1 and 2) Distinct up-regulations of miR-222 were suggested in HCC tumors and cell lines. (3) High levels of miR-222 expressions were found in T relative to TN and NL from qPCR analysis in all 3 categories of samples studied. Gastroenterology 2008 135, 257-269DOI: (10.1053/j.gastro.2008.04.003) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 (A) Transfection with precursor pre-miR-223 and mature miR-223 induced a consistent decrease in cell viability in Hep3B, HKCI-10, and HKCI-C3. *P ≤ .01 relative to mock controls. (B) (1) Specific STMN1 knockdown resulted in reduced cell viability of Hep3B. (2) Western blot analysis for STMN1 protein expression in Hep3B, HKCI-10, and HKCI-C3 suggested a reduced STMN1 protein level in all 3 cell lines after mature miR-223 transfection. (C) Base pairing complement suggested the putative miR-223 binding position at 1–19 of the STMN1 3' UTR. The wild-type (wt), truncated (mut.1), and mutated (mut.2) constructs are shown with the seed region underlined and base substitutions in bold. The firefly luciferase activity was standardized to renilla luciferase control. *P = .02. Results from 3 independent experiments. Gastroenterology 2008 135, 257-269DOI: (10.1053/j.gastro.2008.04.003) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 (A) Over expressed genes indicated from mRNA microarray analysis have been ranked according to their relative expression levels. STMN1 ranked the most up-regulated gene. Box-plot lines represent the median and interquartile ranges. (B and C) An inverse relationship between miR-223 expressions with STMN1 mRNA and protein levels was suggested. (B) r* = -0.464 with a significant P value of .006. (C) r = -0.39. Gastroenterology 2008 135, 257-269DOI: (10.1053/j.gastro.2008.04.003) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Upper panel: histologic features of HCC (A, 100×; B, 400×) and (C and D) paired nontumoral liver from H&E stainings. Lower panel: (E and F) intense cytoplasmic staining of STMN1 is observed in HCC, whereas (G and H) the nontumoral hepatocytes are negative. Gastroenterology 2008 135, 257-269DOI: (10.1053/j.gastro.2008.04.003) Copyright © 2008 AGA Institute Terms and Conditions