The expression of trefoil factors in colorectal cancer with clinic pathology procession and overexpression of hTFF2 in E. coli and its invitro activity
Cancer is one of leading causes of disease death in developed countries and many developing countries, and new cases and deaths of colorectal cancer have ranked third in the various types of cancer. Statistics show that the incidence of colorectal cancer rapidly rises and has been ranked second in all types of cancer in Beijing and Shanghai, China. The development and progression of colorectal cancer involve in a series of cell and molecular changes, including the abnormal gene structure and gene expression abnormalities. Trefoil factor (TFF) is small protein peptide found with the special domain of the protein trefoil factor by different research groups in the late 80s to early 90. The domain is characterized by a peptide containing 38 -40 amino acid and including six conserved cysteine residues to form disulfide bonds in 1-5,2-4,3-6 way to create a close three-leafed structure . Present study has found that there are three trefoil factors in mammals, which are synthesized by different cells within the mucosal tissues and are secreted into the mucosal surface to protect the mucosal. When the mucosal injury, TFF involve in the repair and reconstruction of injury mucosal by promoting epithelial cell migration, inhibiting apoptosis and promoting angiogenesis through various channels. Trefoil factors may promote the development of cancer when they express in tumor tissue. Studies have shown that trefoil factors abnormal expression in tumor may be related to the occurrence and development process of many cancers. We detected TFF1 and TFF3 gene exons sequence in colorectal cancer tissues through DNA sequencing to definite whether mutations. With QRT-PCR and immunohistochemistry we also definited TFF1 and TFF3 mRNA and protein expression levels in colorectal cancer tissues and analyzed the relationship between their expression and colorectal cancer clinical and pathological features. Meanwhile, we used ELISA to detected serum levels of TFF1 and TFF3 in colorectal cancer patients, and analyzed their clinical relations, and gradually investigated whether the two trefoil factors may be useful as serum markers of colorectal cancer. And we got the following results: ①We found a high frequency (C →T) mutation locating upstream of-2bp of the start codon in the TFF1 gene 5 - untranslated region. Its Mutation frequency was 40%. In other non-coding regions were also found several mutations with lower frequency. However, TFF3 gene mutation was not found; ②Our results indicated that the mRNA levels of TFF1 and TFF3 in colorectal cancer tissues varied greatly in different patients. And its relation to Clinical Pathology was without a reasonable statistics because of the less sample cases. TFF1 and TFF3 protein expression positive rate were 90% and 94% in colorectal cancer tissues respectively. We also found there was no statistical significance between TFF1 expression and clinical and pathological types of colorectal cancer, and up regulation of TFF3 expression was related to lymph node metastasis; ③Our results also displayed that patients with colorectal cancer demonstrated an approximately four-fold increase in plasma levels of TFF1 compared
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with healthy people, which was the first report. The serum level of TFF3 in colorectal cancer patients was three times higher than that of the normal population. Whether TFF1 and TFF3 can be used as serum markers of colorectal cancer still need to improve the related information and for further study.
TFF1 and TFF3 include one trefoil domain respectively, containing a free cysteine thiol near the C-terminal. TFF1 and TFF3 homodimer formation through the disulfide bonds is the main form of its activity. TFF2 contains two trefoil domains, and in the extraterritorial trefoil structure near the N-terminal and its C-terminal have a cysteine respectively. The two cysteines linked by disulfide bonds to form a compact structure. We used PET system to clone and express human TFF2 (hTFF2), as well as extra-territorial structure of TFF2 trefoil unlock disulfide mutant TFF2 (MhTFF2), and detected cell migration activity. The results were highly expressed in hTFF2 and MhTFF2, cytoplasmic total protein accounted for more than 40%. Purified sample was obtained by affinity chromatography and the sample purity was more than 95%. Scratch test on the HCT116 cell line experiment showed that hTFF2 and MhTFF2 had migrating effect on HCT116 cells, the cell migration number was about 1.5 times as the control group of BSA.
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