Volume 145, Issue 6, Pages 1300-1311 (December 2013) Bile Acids Increase Levels of MicroRNAs 221 and 222, Leading to Degradation of CDX2 During Esophageal Carcinogenesis  Juntaro Matsuzaki, Hidekazu Suzuki, Hitoshi Tsugawa, Mitsuhiro Watanabe, Sharif Hossain, Eri Arai, Yoshimasa Saito, Shigeki Sekine, Toshihiro Akaike, Yae Kanai, Ken–Ichi Mukaisho, Johan Auwerx, Toshifumi Hibi  Gastroenterology  Volume 145, Issue 6, Pages 1300-1311 (December 2013) DOI: 10.1053/j.gastro.2013.08.008 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 Differences of MIRs 221 and 222, CDX2, and p27Kip1 expression between human Barrett's esophagus and esophageal adenocarcinoma. (A) The levels of MIRs 221 and 222 by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The levels of MIRs 221 and 222 were higher in EAC than in BE (n = 11). (B) The levels of MIR 221 and MIR 222 were correlated. (C) The levels of MIRs 221 and 222 were associated with the depth of invasion using Pearson' correlation analysis. (D) H&E stain and fluorescent staining for CDX2, p27Kip1, and Ki-67 were performed on serial sections of BE and EAC. Expression of CDX2 and p27Kip1 was observed especially the surface lesion of Barrett's metaplasia, but gradually decreased through the metaplasia-dysplasia-adenocarcinoma sequence. Ki-67 staining was much greater in EAC than in Barrett's metaplasia. Scale bars = 40 μm. Data by quantitative reverse transcription polymerase chain reaction were repeated 3 times. Data represent mean ± SEM (∗P < .05; ∗∗P < .01). Ad, adentitia; MP, muscularis propria; SM, submucosa. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 MIRs 221 and 222 suppressed CDX2 with down-regulation of p27Kip1. (A) Western blotting of CDX2 and p27Kip1 after the transfection of pre-MIR 221 and 222 precursors with or without p27Kip1 expression vector (pCMV5-p27) in OE33, OE19, or BAR-T. (B) Western blotting and densitometry measures of CDX2 and p27Kip1 after transfection with the inhibitors of MIRs 221 and 222 in OE33 or OE19. (C) The mRNA expression of MUC2 by quantitative reverse transcription polymerase chain reaction after transfection with the inhibitors of MIRs 221 and 222 in OE33 or OE19. (D) Cell proliferation activity by bromodeoxyuridine enzyme-linked immunosorbent assay after the transfection with the inhibitors of MIRs 221 and 222 in OE33 or OE19. Data were repeated 3 times in duplicate. Data represent mean ± SEM. ∗P < .05; ∗∗P < .01. Ctrl, negative control; NS, not significant. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 MIRs 221 and 222 were enhanced during exposure to bile acids by activation of FXR. (A) The levels of MIRs 221 and 222 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in OE33. The levels of MIRs 221 and 222 were enhanced during exposure to 200 μM CA or 10 μM CDCA. (B) The levels of MIRs 221 and 222 by qRT-PCR in OE33. MIRs 221 and 222 were enhanced by exposure to the FXR agonist in OE33. The TGR5 agonist did not affect the levels of MIRs 221 and 222. (C) The levels of MIRs 221 and 222 by qRT-PCR in OE33. The FXR antagonist (guggulsterone) antagonized the CA-dependent increase in the levels of MIRs 221 and 222. (D) Western blotting and densitometry measures of CDX2 and p27Kip1 during exposure to the FXR agonist or the TGR5 agonist. (E) The mRNA expression of MUC2 by qRT-PCR during exposure to the FXR agonist or the TGR5 agonist. Data were repeated three times in duplicate. Data represent mean ± SEM. ∗P < .05; ∗∗P < .01. ago., agonist; NS, not significant. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 Exposure to bile acid enhanced CDX2 degradation through the ubiquitin-proteasome system. (A) Western blotting and densitometry measures of CDX2 and p27Kip1 during exposure to 25 μg/mL cycloheximide in combination with 20 μM lactacystin or 5 μM MG132 in OE33 or OE19. (B) Western blotting and densitometry measures of CDX2 and p27Kip1 during exposure to the FXR agonist in HET1A+Cdx2. (C) The levels of MIRs 221 and 222 by qRT-PCR. MIRs 221 and 222 were enhanced by exposure to the FXR agonist in HET1A+Cdx2, but not in HET1A+LacZ. The TGR5 agonist did not affect the levels of MIRs 221 and 222. (D) Cell proliferation activity by bromodeoxyuridine enzyme-linked immunosorbent assay during exposure to CA in HET1A+Cdx2 or HET1A+LacZ. (E) Western blotting and densitometry measures of CDX2 in HET1A+Cdx2 during exposure to CA in combination with 20 μM lactacystin or 5 μM MG132 in HET1A+Cdx2. (F) Western blotting of CDX2 after the isolation of polyubiquitylated CDX2 using His-tagged tandem ubiquitin binding entities (TUBE). Under conditions of lactacystin treatment, polyubiquitylated CDX2 (Ub-CDX2) was increased during exposure to CA, and nonubiquitylated CDX2 (non-Ub-CDX2) was decreased. Data were repeated 3 times in duplicate. Data represent mean ± SEM. ∗P < .05; ∗∗P < .01. ago., agonist. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 Transfection of CDX2 enhanced the expression of FXR. (A) Fluorescent immunocytochemistry of FXR. The immunostaining of FXR was increased in HET1A+Cdx2 as compared with HET1A+LacZ. Scale bars = 20 μm. (B) Fluorescent immunocytochemistry of FXR. The immunostaining of FXR was increased in Cdx2-transfected BAR-T as compared with LacZ-transfected BAR-T. Scale bars = 20 μm. (C) The mRNA expression of FXR by quantitative reverse transcription polymerase chain reaction with the transfection of CDX2 or LacZ in HET1A, EPC1-hTERT, BAR-T, OE33, and OE19. (D) Transcriptional activity of FXR by luciferase reporter assay. Although the activities of FXR were not altered by the FXR agonist in HET1A+LacZ, these were significantly increased in HET1A+Cdx2. Data were repeated 3 times in duplicate. Data represent mean ± SEM. NS, not significant; ∗∗P < .01. ago., agonist; FXRE, farnesoid X receptor responsive element. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 The injection of the inhibitors of MIRs 221 and 222 reduced tumor growth and enhanced CDX2 expression. (A) The experimental design is illustrated; 3 × 106 cells of OE33 were subcutaneously injected into both flanks of 7-week-old male immunodeficient NOD/SCID/IL-2Rγnull (NOG) mice (n = 5). Seven days after the injection of cells, 10 pmoL of either negative control (Ctrl.) or with the inhibitors of MIRs 221 and 222 were injected around OE33-derived tumors for 7 days. (B) A representative finding of OE33-derived tumors on the flank of a NOG mouse is shown. A tumor treated with the inhibitors of MIRs 221 and 222 was smaller than negative control treated one. (C) Tumor growth curves depicting the average volumes of tumors are shown. (D) The levels of MIRs 221 and 222 by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Suppression of MIRs 221 and 222 was confirmed by the injection of the inhibitors of MIRs 221 and 222. (E) The mRNA expression of MUC2 by qRT-PCR. The expression of MUC2 was significantly up-regulated by the inhibitors of MIRs 221 and 222. (F) Representative H&E stain and fluorescent staining for CDX2. In the negative control-treated tumor, CDX2 staining was scarcely observed. On the other hand, in the MIR inhibitor-treated tumor, CDX2 staining was clearly observed in tumor cells. Scale bars = 40 μm. Data by qRT-PCR were repeated 3 times. Data represent mean ± SEM.∗P < .05; ∗∗P < .01. Ctrl, negative control. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 Schematic representation how bile acids promote the degradation of CDX2. Gastroenterology 2013 145, 1300-1311DOI: (10.1053/j.gastro.2013.08.008) Copyright © 2013 AGA Institute Terms and Conditions