Volume 138, Issue 1, Pages 231-240.e5 (January 2010) RNA-Binding Protein Quaking, a Critical Regulator of Colon Epithelial Differentiation and a Suppressor of Colon Cancer  Guodong Yang, Haiyan Fu, Jie Zhang, Xiaozhao Lu, Fang Yu, Liang Jin, Liyuan Bai, Bo Huang, Lan Shen, Yue Feng, Libo Yao, Zifan Lu  Gastroenterology  Volume 138, Issue 1, Pages 231-240.e5 (January 2010) DOI: 10.1053/j.gastro.2009.08.001 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Expression pattern of QKI in normal and neoplastic colonic epithelium. (A) RNA levels of QKI in multiple colon cancer cell lines, normal small intestinal epithelial IEC-6 cell line, and normal gastric epithelium GES-1 were quantified by comparative RT-PCR. Adherent cells were cultured to reach confluence and harvested for examination. β-actin served as an internal control to ensure equal loading. (B) Protein levels of QKI in the above cell lines were detected by Western blot, and GAPDH served as an internal control to ensure equal loading. (C) Immunostaining for QKI in normal and cancerous colon epithelium in 10 human colon cancer specimens and paired adjacent normal tissue collected at surgical resection. QKI expression was much lower in most cancerous tissues than that in the adjacent normal counterparts. Representative data are provided. (D) Protein levels of QKI in the above patients were detected by Western blot, and β-actin served as a loading control. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Methylation status of QKI promoter region. (A) Methylation status of QKI promoter region in multiple human cell lines. Methylated promoter and the total level (served as a loading control) of each cell line were amplified with specific primers. (B) Methylation status of QKI promoter region in normal and cancerous colon tissues. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Differentiation promoting role of QKI. (A) QKI expression under postconfluence growth induced differentiation. Cells were seeded at 80% confluence at day 0 or continued to grow for an additional 1 or 2 days. QKI expression was significantly up-regulated in colon cancer cell HT29 after 2-day culture, which is correlated with the increased expression of p27. α-Tubulin serves as a control to ensure equal loading. (B) Analysis of QKI overexpression. Cells were infected with the indicated adenovirus, and QKI expression was analyzed by Western blot. (C) QKI overexpression efficiently induced the expression of lactase and E-cadherin. Compared with the control, infection of Ad-QKI5 or Ad-QKI6 in HT29 cells for 4 days significantly up-regulated the expression of enterocyte marker lactase and E-cadherin. QKI overexpression does not change the β-catenin mRNA level. Representative data of 3 different experiments are shown. (D) Effects of QKI on the expression of intestinal alkaline phosphatase (IAP). HT29 cells were infected with Ad-QKI5 or Ad-QKI6 or the control for 3 days before harvest for IAP detection. Data are expressed as means ± SD (n = 3). *P < .05. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 QKI enhances the mRNA stability of p27 at high confluence condition. (A) p27 mRNA stability in HT29 cells at low density, high density, and high density with QKI knockdown. Cells cultured in the above conditions were further treated with Actinomycin D at time 0, and RNA was extracted at indicated time to examine the RNA stability. Increased mRNA stability was detected in high-density culture, which is partially blocked by QKI RNAi. Data presented here are representative of 3 independent experiments. (B) Quantification of the data from Figure 4A. Intensity of the bands was analyzed by ImageJ software (NIH, Bethesda, MD) and expressed as mean ± SD. *P < .05. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 QKI alters the subcellular distribution of β-catenin. (A) QKI overexpression decreased the cytoplasmic and nuclear β-catenin while increased the membrane bound β-catenin. α-Tubulin and Lamin B served as internal controls. (B) QKI expression reduced the Topflash activity. Topflash or Fopflash reporter plasmid and 50-ng internal control vector pRL-TK were cotransfected with the indicated plasmids; and, 36 hours later, cells were lysed, and luciferase activity was examined. Fold induction by QKI (Topflash/Fopflash) was calculated and expressed as means ± SD (n = 3). *P < .05. (C) QKI regulates β-catenin expression through 3′UTR. Two hundred-nanograms β-catenin 3′UTR, Δβ-catenin 3′UTR1, or the Δβ-catenin 3′UTR2 reporter and 50 ng internal control vector pRL-TK were cotransfected with the indicated plasmids for 24 hours before luciferase activity examination. β-catenin 3′UTR and the Δβ-catenin 3′UTR1 responded similarly to QKI overexpression, whereas Δβ-catenin 3′UTR2 did not respond to QKI overexpression. Fold induction was calculated and expressed as means ± SD (n = 3). *P < .05. (D) In vivo interaction between β-catenin and QKI. pcDNA3.1-3 × flag-QKI5 transfected HT29 cells were immunoprecipitated with anti-flag antibody or the negative control IgG. The presence of β-catenin mRNA in the immunoprecipitation was detected by RT-PCR and visualized by ethidium bromide staining. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Tumor suppressive role of QKI. (A) Growth curves of HCT116 and HT29 cells infected with indicated adenovirus (n = 3). QKI, especially QKI6, significantly blocked the growth of the tumor cells. (B) Colony formation of HCT116 cells infected with indicated adenovirus in semisolid medium (n = 3). Cells on soft agar plates were grown for 2 weeks before colonies were visualized microscopically. A representative view of them is shown. (C) Quantification of colony formation data from B. Colonies were counted in a blinded fashion. Those with a diameter >50 μm are defined as large and those <50 μm as small colonies. *P < .05. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Endogenous expression of QKI isoforms in HT29 cells. Confluent HT29 cells were harvested for RNA extraction and RT-PCR. Specific primers for total QKI, QKI5, and QKI6 were applied for specific amplification of different isoforms of QKI. β-actin served as an internal control. Both QKI5 and QKI6 were endogenously expressed in HT29 cells. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 5-aza-dC treatment rescuing the expression of QKI and promoting the intestinal epithelial differentiation in Colo205 cells. (A) Colo205 cells were treated with 1 mmol/L 5-aza-dC for 7 days before harvest for RT-PCR analysis. 5-aza-dC treatment rescued the QKI expression and increased the lactase level. (B) Seven-day treatment of 5-aza-dC increased the intestinal alkaline phosphatase (IAP) activity in Colo205 cells. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Inhibition of confluence-induced epithelial differentiation in HT29 cells by knockdown of endogenous QKI. (A) Knockdown efficiency of the 2 different small interfering RNAs targeting QKI. (B) Cells transfected with control siRNA or siRNAs targeting QKI were cultured at high confluence for 2 days before harvest for IAP analysis. Si-QKI inhibited the intestinal alkaline phosphatase (IAP) activity. Si-QKI inhibited the expression of lactase. (C) Cells were treated the same as above and harvested for RT-PCR. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 Interaction between QKI5 and p27. (A) Overexpression of QKI5 increased the expression of p27 in a high density-dependent manner. HT29 cells infected with control or QKI expression adenovirus were cultured at high or low density for an additional 24 hours, and p27 expression was analyzed by Western blot. (B) Interaction between QKI and p27. pcDNA3.1-3 flag-QKI5 transfected HT29 cells were immunoprecipitated with anti-flag antibody or the negative control IgG. The presence of p27 mRNA in the immunoprecipitation was detected by RT-PCR. Input represents the existence of p27 mRNA in the cell. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 Interaction between QKI6 and β-catenin. pcDNA3.1-flag-QKI6 transfected HT29 cells were immunoprecipitated with anti-flag antibody or the negative control IgG. The presence of β-catenin mRNA in the immunoprecipitation was detected by RT-PCR and visualized by ethidium bromide staining. Input represents the existence of β-catenin in the cell. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 6 Redistribution of β-catenin from cytosol and nuclear to membrane in cells cultured at different densities. Cells seeded on the coverslip at low density or high density were fixed for immunofluorescence analysis. Strong green fluorescence (signal for β-catenin) was seen in cytosol and nuclear in cells grown at low density, whereas strong green fluorescence was seen on the membrane when cells were grown at high density. Nuclear was visualized as blue fluorescence by DAPI staining. Scale bar, 50 μm. Gastroenterology 2010 138, 231-240.e5DOI: (10.1053/j.gastro.2009.08.001) Copyright © 2010 AGA Institute Terms and Conditions