Volume 135, Issue 5, Pages 1624-1635.e24 (November 2008) MicroRNAs Are Differentially Expressed in Ulcerative Colitis and Alter Expression of Macrophage Inflammatory Peptide-2α  Feng Wu, Michelle Zikusoka, Anil Trindade, Themistocles Dassopoulos, Mary L. Harris, Theodore M. Bayless, Steven R. Brant, Shukti Chakravarti, John H. Kwon  Gastroenterology  Volume 135, Issue 5, Pages 1624-1635.e24 (November 2008) DOI: 10.1053/j.gastro.2008.07.068 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 miRNA expression in human colon tissues. The expression of active UC-associated miRNAs was assessed in healthy control tissues as well as in active UC, inactive UC, IC, IBS, MC, and CD by qRT-PCR. The 6 most highly expressed, active UC-associated miRNAs are shown. Data are presented as box-whisker plots (box, 25%–75%; whisker, 5%–95%; line, median). *P < .05; **P < .005; ***P < .001. Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 miR-192 and MIP-2α localization in human colon tissues. Dual immunohistochemistry and in situ hybridization were performed on colon biopsy tissues from healthy controls and active UC for MIP-2α and miR-192, respectively. MIP-2α is not detected in epithelial cells of healthy control tissues but is detected in the epithelial cells and lamina propria cells of active UC tissues. miR-192 is localized to colonic epithelial cells of healthy control tissues, but not visible in epithelial layer of active UC tissues. Green, miR-192; red, MIP-2α; blue, DAPI nuclear staining. Pictures were imaged at ×40 magnification at 1024 × 1024 pixels resolution on a Zeiss LSM 510 Meta confocal microscope (20 μm scale). Controls for immunohistochemistry and in situ hybridization are shown in Supplementary Figure 2. Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 MIP-2α expression in human colon tissues and correlation with miR-192 expression. (A) MIP-2α mRNA expression in human colon biopsy tissues by qRT-PCR. Data are presented as MIP-2α expression relative to GAPDH (***P < .001). (B) Correlation of MIP-2α mRNA expression with miR-192 expression in individual biopsy samples from healthy control (open circles; n = 15), active UC (closed circles; n = 15), and all other tissues (open squares; n = 32). Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 MIP-2α and associated miRNA expression in TNF-α–stimulated HT29 colonic epithelial cells. MIP-2α mRNA expression (A) and protein secretion (B) in HT29 cells stimulated with TNF-α at various time points (*P < .05). (C) The expression of MIP-2α–associated miRNAs was assessed at 1 and 24 hours after TNF-α stimulation. The expression patterns of the 4 most highly expressed miRNAs are demonstrated. *P < .05; **P < .005, ***P < .001. Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 MIP-2α miRNA binding site mutation effects on reporter expression. (A) Schematic representation of MIP-2α mRNA with putative miRNA binding sites. (B) Sequence alignment and specific miRNA binding site mutations in the pMIR–MIP-2α 3′UTR reporter constructs. (C) Luciferase reporter activity in the pMIR–MIP-2α 3′UTR reporter construct and associated miRNA binding site mutations. Luciferase activity (normalized to Renilla luciferase activity) data is presented relative to the pMIR–MIP-2α 3′UTR reporter construct (*P < .05). Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 miR-192 inhibition of MIP-2α mRNA and protein expression. TNF-α–induced MIP-2α mRNA expression (A) and protein secretion (B) were significantly reduced in HT29 cells transfected with an miR-192 mimic. The control mimic had no effect. *P < .005; **P < .001. (C) TNF-α–induced RANTES expression was not inhibited by the miR-192 mimic. TNF-α–induced MIP-2α mRNA expression (D) and protein secretion (E) were also significantly reduced in HT29 cells transfected with a plasmid containing the genomic sequence of pre–miR-192. Transfection of a plasmid containing a scrambled miR-192 sequence had no effect (**P < .001). Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 Additional miRNA expression in human colon tissues. The expression of active UC-associated miRNAs was assessed in healthy control tissues as well as in active UC, inactive UC, IC, IBS, MC, and CD by qRT-PCR. The remaining 5 active UC-associated miRNAs, not included in Figure 1, are shown. Data is presented as box-whisker plots (box, 25%–75%; whisker, 5%–95%; line, median). *P < .05; **P < .005; ***P < .001). Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 MIP-2α immunohistochemistry and miR-192 in situ hybridization controls. Dual in situ hybridization (A) and immunohistochemistry (B) controls were performed on colon biopsy tissues from active UC. (A) In situ hybridization fluorescence (green) was seen in scattered lamina propria cells in the absence of probe; however, no fluorescence was seen in epithelial cells. (B) Immunohistochemical staining (red) was absent in all cells when using nonspecific goat serum as a control (blue, DAPI nuclear staining). Pictures were imaged at ×40 magnification at 1024 × 1024 pixels resolution on a Zeiss LSM 510 Meta confocal microscope. Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 3 MIP-2α–associated miRNA expression in TNF-α–stimulated HT29 colonic epithelial cells. The expression of MIP-2α–associated miRNAs was assessed at 1 and 24 hours after TNF-α stimulation. The expression patterns of the remaining 5 MIP-2α–associated miRNAs, not included in Figure 4, are demonstrated. *P < .05; **P < .005, ***P < .001. Gastroenterology 2008 135, 1624-1635.e24DOI: (10.1053/j.gastro.2008.07.068) Copyright © 2008 AGA Institute Terms and Conditions