Gastroenterology

Gastroenterology

Volume 138, Issue 1, January 2010, Pages 231-240.e5
Gastroenterology

Basic—Alimentary Tract
RNA-Binding Protein Quaking, a Critical Regulator of Colon Epithelial Differentiation and a Suppressor of Colon Cancer

https://doi.org/10.1053/j.gastro.2009.08.001Get rights and content

Background & Aims

Colon cancer is one of the best understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death. Post-transcriptional regulation mediated by RNA-binding proteins or microRNAs coordinately targets multiple genes, holding promise involved in colon cancer initiation and development. Here we studied the role of RNA-binding protein quaking (QKI) in colon cancer.

Methods

We observed the expression pattern of QKI in normal colon and colon cancers through reverse-transcription polymerase chain reaction and Western blot. Bisulfite sequencing and methylation-specific PCR were applied for QKI promoter methylation analysis. We used enterocyte differentiation markers and soft agar assay to test the role of QKI in colon differentiation and colon cancer development. 3′ Untranslated region (UTR) reporter assay and RNA-immunoprecipitation were used to confirm the interaction between QKI and β-catenin or p27.

Results

QKI is significantly down-regulated and even absent in some colon cancers, which is at least partially because of the promoter hypermethylation. Forced expression of QKI in the colon cancer cells increased the expression of enterocyte differentiation marker intestinal alkaline phosphatase and lactase, together with the enhancement of p27Kip1 protein level, and membrane localized β-catenin. Finally, QKI overexpression reduced the proliferation and tumorigenesis ability.

Conclusions

Our study establishes that QKI functions as a principal regulator in the differentiation of colon epithelium and a suppressor of carcinogenesis through coordinately targeting multiple genes associated with cell growth and differentiation, whose deregulation by methylation is involved in colon cancer onset and progress.

Section snippets

Tissue Sample Collection

Frozen tumor and normal colon mucosa were collected from colectomy specimens from Tangdu Hospital, the University of the Fourth Military Medical University. Tissue was frozen in liquid nitrogen within 30 minutes of resection. Nonneoplastic mucosa from colon was dissected free of muscle and histologically confirmed to be tumor free by frozen section. For cancer tissue, tumor purity of over 70% was confirmed by frozen section for each case before submission for DNA and protein extraction.

Expression Pattern of QKI in Normal Colon and Colon Cancers

To establish the role of QKI in the differentiation of colon epithelium, we first analyzed the expression of QKI in normal colon and colon cancers. Expression levels of QKI were compared among normal gastrointenstinal epithelial cell lines (GES-1 and IEC-6) and cancerous cell lines (HCT116, HT29, SW480, SW620, and Colo205 cells). High expression of QKI in normal GES-1 and IEC-6 cells; median expression in HT29 cells; and low expression in HCT116, SW480, and SW620 cells were observed both at RNA

Discussion

Our study investigated the mechanism how QKI regulates colon epithelial cell differentiation and the role of its aberrant expression in colon cancer evolution. The data demonstrate for the first time that QKI expression, especially QKI6, is widely distributed in normal colon epithelium while absent or very lowly expressed in colon cancer cells because of high methylation of the promoter region. QKI expression in differentiating HT29 cells resulted in sustained accumulation of p27kip1, decreased

Acknowledgments

G.Y., H.F., and J.Z. contributed equally to this work and should be considered as co-first authors.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by Foundation for Excellent Doctoral Level Students of Fourth Military Medical University (200706), National Science Foundation of China (NSF: 30470387; NSF: 30570396; NSF: 30628025), and National Key Basic Research and Development Program 2009CB521704.

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