Molecular Cloning And Transcriptional Activity Analysis Of Human Intestinal Trefoil Factor Gene Promoter

Background: Intestinal trefoil factor (ITF) is a small polypeptide, secreted abundantly onto mucosal surface by the goblet cells of gastrointestinal tract[1]. ITF has one structurally characteristic'trefoil'domain which is defined as a sequence of 38-39 amino acid residues[2], in which six cysteines are disulphide-link in a 1-5, 2-4, and 3-6 configuration. The amino acid sequence, together with the disulphide bonds, forms a characteristic three-leaved structure which has given the peptide its name. ITF appears to play an important role in protection, repair and healing of the gastrointestinal mucosa after injury[3]. Up to now, the research of ITF is extensive and profound, including tissue distribution, gene localization, recombinant expression, amino acid sequence and spatial structure analysis as well as mass of function research[4, 5]. But the regulation mechanism of hITF transcription is slow-moving. The promoter region of hITF gene was cloned by Seib T at first[6], and several classical transcription factor response elements such as AP1 and Sp1 binding consensus sequence were predicted. Now some new members like hypoxia inducible factor 1 response element(HRE) and butyrate response element (BRE) were confirmed[7]. Evidence showed that some of them can facilitate the transcription when encountering stimulation, hence the expression of hITF was enhanced. The new progress in the field of rodent ITF promoter was found GCRE (goblet cell response element) and GCSI(goblet cell silencer inhibitor) positive regulatory element[8, 9], which could facilitate hITF gene transcription by alone or company with other regulatory element. No similar elements were reported in hITF promoter region until now, therefore some further studies needed to be done.Objective: To explore the transcriptional regulatory mechanisms of hITF by cloning hITF promoter and transcriptional activity analysis Methods:1. The region spanning -2331/+79 of hITF gene was amplified from genomic DNA by PCR, then subcloned into the pGL-3 basic luciferase reporter plasmid (pGL-3 hITF pro-2500)and transiently transfected into LS-174T or HEK-293 cells. The cells were lysed and measured luciferase activity after transfection 48 hours.2. Subsequently, some lopping plasmids such as pGL-3 hITF proVx2.5(-2331/+53), pGL-3 hITF-2000, pGL-3 hITF pro-1500, pGL-3 hITF pro-1000, pGL-3 hITF pro-500, pGL-3 hITF pro-400, pGL-3 hITF pro-300, pGL-3 hITF pro-280, pGL-3 hITF pro-260, pGL-3 hITF pro-240, pGL-3 hITF pro-220, pGL-3 hITF pro-200, pGL-3 hITF pro-100, pGL-3 hITF pro-50, and some mutational plasmids: mut1(+54/+59), mut2(+60/+65), mut3(+66/+71), mut4(+72/+77),mut MIX1(-278/-270) and mut Sp1②(-266/-256) were made and validated by the same way.3. Through measured luciferase activity and using Patch, Alibaba2 software analysis roughly decide the approximate location of the positive and negative regulatory regions, and finally confirm the enhancer and repressor elements by using mutation assays.4. In computations, SPSS (version 13.0) statistical package program was used. The relative transcriptional activity was presented with mean±S.D., the difference between fragments was analysised by One-way ANOVA(Dunnett T3). A difference of P< 0.05 was considered significant.Results:1. Restriction enzyme analysis and sequencing result showed that the sequence of cloning fragment was identical with hITF promoter gene sequence reported in genebank.2. The hITF pro-pGL3-basic expression vector was confirmed by restriction enzyme digestion and sequencing, and named pGL-3 hITF pro-2500(-2331/+79). Transcriptional activity analysis showed that there were low activity of cloning hITF promoter transfected into both LS-174T and HEK-293 cells.3. Through gradually lopping and mutation analysis, finally confirm there are repressor element in+53/+79 and enhancer element in -280/-251 regions of hITF promoter. The -280/-251 transcription enhancer regions exist two Sp1 binding domain, situated in -278/-270 and -266/-256 respectively.Conclusion:1. There is repressor element in +53/+79 regions of hITF promoter gene order, it maybe inhibit the hITF isoform transcription and expression.2. Two specific Sp1 binding site in -278/-270 and -266/-256 regions of hITF promoter gene order, it maybe dominantly contribute to positive transcription regulatory of hITF.