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Digestive Diseases and Sciences

, Volume 55, Issue 10, pp 2929–2939 | Cite as

Upregulated Expression of hITF in Crohn’s Disease and Screening of hITF Interactant by a Yeast Two-Hybrid System

  • Huaxiu Shi
  • Jianlin Ren
  • Hongzhi Xu
  • Jinshui Pan
  • Xiaoyan Hao
  • Lydia Leyland Barlow
  • Weiguo DongEmail author
Original Article

Abstract

Aims

To study the expression of human intestinal trefoil factor (hITF) mRNA in Crohn’s disease and to screen the cellular proteins that can interact with the hITF protein by a yeast two-hybrid system in order to explore the mechanism of hITF in protecting intestinal mucosa from injury.

Methods

Seventy-eight patients underwent double-balloon enteroscopy (DBE). Expression of hITF mRNA was detected by quantitative real-time polymerase chain reaction analysis (qRT-PCR). The hITF gene was amplified by PCR and cloned into vector pDEST32. The yeast cells cotransformed with pDEST32-hITF and the human jejunal cDNA library were plated in a selective SC-Leu-Trp-His-Ura medium. The subsequent screen was performed with χ-gal detection, and true-positive clones were sequenced and analyzed with bioinformatics. Co-immunoprecipitation (Co-IP) was performed to confirm the binding of putative proteins to the hITF protein.

Results

Thirty-nine patients were diagnosed with Crohn’s disease. We found that the expression of hITF mRNA is significantly increased in Crohn’s disease compared to normal controls. A total of ten colonies were selected and sequenced. Among these, six colonies were Homo sapiens zinc finger protein 193 (ZNF193), three colonies were Homo sapiens Aldo–keto reductase family 1C 1 (AKR1C1), and one colony was of an unknown gene. A reverse two-hybrid experiment and Co-IP indicated that ZNF193 and AKR1C1 might interact with hITF.

Conclusions

The expression of hITF mRNA is increased in Crohn’s disease. ZNF193 and AKR1C1 are proteins that can interact with the hITF protein by a yeast two-hybrid system and Co-IP, hITF may contribute to the mucosal repair through this interaction.

Keywords

Human intestinal trefoil factor Crohn’s disease Yeast two-hybrid system 

Notes

Acknowledgments

This work was supported in part by grant WQK05022005 from the Science and Technology Innovation Foundation of Fujian Province, China. The authors are grateful to the Department of Gastroenterology, Zhongshan Hospital, Xiamen University and the Department of Surgery, Renji Hospital of Shanghai Jiaotong Medical University. We thank Ms. Lydia Leyland Barlow and Dr. Wenyu Lin for their help in this manuscript edition.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Huaxiu Shi
    • 1
    • 2
    • 3
  • Jianlin Ren
    • 2
  • Hongzhi Xu
    • 2
  • Jinshui Pan
    • 2
  • Xiaoyan Hao
    • 2
  • Lydia Leyland Barlow
    • 3
  • Weiguo Dong
    • 1
    Email author
  1. 1.Department of GastroenterologyRenmin Hospital of Wuhan UniversityWuhanChina
  2. 2.Department of GastroenterologyZhongshan Hospital Xiamen UniversityXiamenChina
  3. 3.Gastrointestinal Unit, Center for the Study of Inflammatory Bowel DiseaseMassachusetts General Hospital and Harvard Medical SchoolBostonUSA

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