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Human trefoil factor 3 (hTFF3) is a small peptide of potential therapeutic value. Themechanisms underlying the transcriptional regulation of hTFF3 remain unclear. Thepurpose of this study was to identify the core functional elements for theself-induction action of hTFF3 and transcription factors. First, truncated promoterswere constructed to identify the functional regions of the hTFF3 promoter.
Next,point mutation, chromatin immunoprecipitation, RNA interference, and geneoverexpression experiments were performed to analyze the transcriptional bindingsites responsible for the self-induced transcription of hTFF3. Our results revealedthe −1450 bp to −1400 bp fragment ofthe hTFF3 promoter was the functional region for the self-induction action of hTFF3.Bioinformatics analysis confirmed that a STAT3 binding site is present in the−1417 bp to −1409 bp region.Subsequently, site-directed mutagenesis analysis determined that this STAT3 bindingsite was critical for the self-induction effect of hTFF3. ChIP experiments confirmedthat STAT3 binds to the hTFF3 promoter. STAT3 overexpression and knockdownexperiments revealed that STAT3 enhanced the self-induction effect and theexpression of hTFF3.
This study confirmed that hTFF3 exhibits self-induction action,and that STAT3 is the key transcription factor to maintain the function ofself-induction. Human trefoil factor 3 (hTFF3) is a small polypeptide secreted by intestinal gobletcells. There are six cysteine residues in the amino acid sequence of hTFF3, sequentiallyconnected in pairs by disulfide bonds to form three ring structures.This structure confers stability, and thus provides resistance against acidic and basicconditions, as well as protease hydrolysis. The structural stability protects hTFF3 fromdamages in the gastrointestinal tract, which is a complex environment. HTFF3 has beenunder intensive research by numerous scholars since its discovery.
A large number ofstudies have shown that hTFF3 plays imperative roles in the maintenance and repair ofthe intestinal mucosa. The regulation of hTFF3 is complex andprecise, and many kinds of substances are involved in the regulation of hTFF3expression. Some studies have found that proteins of the trefoil factor family exhibitthe phenomenon of “self-induction” to enhance their ownexpression. However, the exact regulatory mechanisms remain unclear.The present study successfully amplified hTFF3 promoter fragments of varying lengths,and identified the −1450 to −1400 bp region as thefunctional region for its self-induction.
Bioinformatics and site-directed mutagenesisanalyses revealed that the STAT3 binding site, located in the region of−1417 to −1409 bp, is necessary for theself-induction of hTFF3. We further proved that STAT3 binds to hTFF3 promoter toregulate its transcription.
Our study lays a foundation for elucidating the regulatorymechanisms of hTFF3. HTFF3 enhances the transcription of its promoterPlasmids expressing the full-length hTFF3 promoter or control DNA weretransfected into HEK293 cells and LS174T cells, and different concentrations ofhTFF3 was added at 24 h post-transfection. The relative luciferaseactivity was measured using a dual-luciferase reporter system at48 h. The results showed that the luciferase activity of cellsexpressing the hTFF3 promoter was 15-fold higher than that of the negativecontrol expressing pGL3-basic, and the enhanced luciferase activity showed adose-dependent effect with increasing concentrations of hTFF3 treatment.
Theluciferase activity of cells expressing the hTFF3 promoter was 45-fold higherthan that of the negative control expressing pGL3-basic when treated with50 μg/mL hTFF3. This difference was significant(P. The full-length promoter of hTFF3 was transfected into HEK 293 cells and LS174T cells, and the transfected cells were stimulated with different concentrations of hTFF3 at 24 h post-transfection. The relative fluorescence intensity was calculated as the ratio of the firefly fluorescence intensity to Renilla fluorescence intensity. At least three independent experiments were performed under each condition for this experiment. Data are presented mean ± S.D. Effects of hTFF3 on the transcription of its promoter fragments of varyinglengthsWe transfected fragments of the hTFF3 promoter into LS174T 293 cells and HEKcells, and treated with exogenous hTFF3.
It showed that the luciferase activityin LS174T cells was significantly higher than that in HEK293 cells, regardlessof the length of the transfected promoter. The luciferase activities of cellsexpressing pGL3−1826 and pGL3−1519 were relativelyhigher, and the luciferase activities of cells expressing pGL3−1070and shorter length fragments were significantly lower. Therefore, to further narrow down on the functional region, weconstructed eight truncated fragments between −1519 bpand −1070 bp. As shown in,cells expressing pGL3−1450 had a luciferase activity 41-fold morethan that of cells expressing pGL3-basic, and cells expressingpGL3−1400 and GL3−1100 exhibited relatively lowluciferase activities, which were only 10–15 fold higher than thatof the cells expressing pGL3-basic.
HTFF3 promoter fragments of varying lengths were transfected into HEK293 cells and LS174T cells, and the relative fluorescence intensity was calculated as the ratio of the firefly fluorescence intensity to Renilla fluorescence intensity. At least three independent experiments were performed under each condition. ( A) Represents the promoter length of −1826 bp to −100 bp; ( B) represents −1450 bp to −1100 bp.
Data are presented mean ± S.D. Mutations of −1417 bp to−1409 bp reduced transcription of the hTFF3promoterWe entered the DNA sequence of the −1450 to−1400 bp region of hTFF3 promoter into TFSEARCHdatabase. Interestingly, a STAT3 binding site (TTCCTGGAA) was found in theregion of −1417 bp to −1409 bp,with a score of 94.2.
Therefore, we mutated the core region of the STAT3 bindingsite from CTG to ACT. As shown in, the luciferaseactivity of cells expressing pGL3−1826 was 45-fold higher than thatof cells expressing pGL3-basic, while the activity of cells expressing themutant reporter decreased to only 17-fold higher than that of cells expressingpGL3-basic. Verification of the binding activity of STAT3 to the hTFF3promoterIn order to confirm the interaction between STAT3 and the hTFF3 promoter, ChIPassay was performed.
After fixation, sonication, immunoprecipitation, reversalof link, PCR, and other steps, PCR products were subjected to DNA gelelectrophoresis. Our results showed that intense DNA bands were detected in theSTAT3 ChIP sample and the input sample, while no DNA band was detected in thenegative control, IgG ChIP. AG490 is a specificinhibitor of the transcription factor STAT3, which can specifically block thetranscription activity of STAT3 protein. Luciferase activityassay demonstrated that AG490 could potently inhibit the self-induction of hTFF3on its own promoter. ( A) LS174Tcells were fixed with formaldehyde following ultrasonic fragmentation. Next, STAT3 antibody, positive control Pol RNA II antibody, or negative control IgG antibody were added to the fragmented mixtures, respectively. The region containing the STAT3 binding site was amplified by PCR with specific primers.
( B) HEK293 or LS174T cells were co-transfected with pGL3−1826 and pRL-TK, and different concentrations of AG490 were added into the culture medium 1 h before hTFF3 addition. The relative fluorescence intensity was detected after 24 h.
Data are presented mean ± S.D. Transcriptional activation of the hTFF3 promoter by STAT3To determine whether the transcription of hTFF3 promoter was affected by STAT3upregulation, pGL3−1826 or mutant pGL3−1826 and STAT3eukaryotic expression vectors were co-transfected. We found that luciferaseactivity of cells expressing the hTFF3 promoter gradually increased withincreasing amounts of STAT3 plasmid in a dose-dependent manner. While theluciferase activities of cells expressing mutant pGL3−1826 did notshow the increase. Real-time RT-PCR analysisshowed that mRNA expression of hTFF3 was significantly elevated upon STAT3overexpression.
Western blot results alsodemonstrated upregulation of hTFF3 at the protein level. Conversely, RNAi knockdown was used to downregulate STAT3expression, and the promoter activity and expression of hTFF3 were determined.The results showed that transcription of the hTFF3 promoter significantlydecreased upon STAT3 RNAi knockdown. While co-transfection of mutantpGL3−1826 with STAT3 knockdown plasmids did not decrease theluciferase activities. Real-time RT-PCR analysisshowed that both STAT3 and hTFF3 mRNA levels were decreased in the STAT3knockdown cells. Western blot results alsoshowed that protein expression of hTFF3 was significantly decreased upon STAT3RNAi knockdown. Intestinal mucosal injury underlies the pathogenesis of many diseases. Therefore, it is of great importance to maintain the integrity of the intestinalmucosa.
HTFF3 is a small polypeptide of potential therapeutic value, and its mainpharmacological action is to ameliorate gastrointestinal mucosal injuries caused byvarious factors and to promote repair of the damaged mucosa.There are six cysteine residues in the amino acid sequence of hTFF3, connected inpairs by disulfide bonds to form three ring structures. This structure makes itstable, thus resistant to acidic and basic conditions, and protease hydrolysis. Thestructural stability protects hTFF3 from damages in the gastrointestinal tract,which is a complex environment, and also enables physiological functions of hTFF3. Avariety of substances has been found to regulate hTFF3 expression by thecorresponding response elements, such as, upstream stimulatory factor (USF),interleukin, and hypoxia inducible factor 1 (HIF-1).However, its basic regulatory mechanisms remained unclear. This study validated theself-induction effect of hTFF3 on its own promoter, and identified the functionalregion for the action of self-induction. We also identified the transcriptionfactors binding to the hTFF3 promoter, which will help us elucidate the regulatorymechanisms of hTFF3 expression.
This study confirmed the transcriptionally enhancingeffect of hTFF3 on its promoter. The results showed that with increasing amounts ofhTFF3, the transcription of the hTFF3 promoter was gradually enhanced in adose-dependent manner, indicating that hTFF3 has a strong self-induction effect onits own promoter.
When hTFF3 concentration was more than50 μg/mL, transcription of the hTFF3 promoter entered into aplateau stage, thus the concentration of hTFF3 used in the subsequent experimentswas 50 μg/mL. To search forthe functional region of the hTFF3 promoter important for its self-induction,truncated fragments of the hTFF3 promoter were tested by luciferase assay. Ourresults demonstrated that the transcriptional activities in cells expressingpromoter fragments of −1826 bp and−1519 bp were restively high; while the activities of cellsexpressing fragments downstream of −1070 bp decreasedsignificantly, suggesting that the functional region of hTFF3 was located between−1519 bp to −1070 bp. To identifythe core region of the hTFF3 promoter, eight different truncated vectors between−1519 bp and −1070 bp wereconstructed. The results showed that cells expressing the−1450 bp promoter fragment exhibited higher activity, andcells expressing the promoter fragment with the region of−1400 bp to −1100 bp exhibitedsignificantly decreased activity. The results show that the core functional regionof the hTFF3 promoter could be narrowed down to −1450 bp to−1400 bp. Subsequently, we performed bioinformatics analysisof the hTFF3 promoter and searched for transcription factors in the TFSEARCHdatabase.
The search revealed that a STAT3 binding site (TTCCTGGAA) was discoveredin the −1417 bp to −1409 bp region,with a score of 94.2. Therefore, the core region of the STAT3 binding site wasmutated (CTG to ACT).
As shown in, the luciferaseactivity of cells expressing pGL3−1826 was over 45-fold higher than thatof cells expressing pGL3-basic, while the activity of cells expressing the mutantreporter decreased to only 17-fold of cells that express pGL3-basic. Therefore, wespeculate that −1417 bp to −1409 bpis the core region of hTFF3 for its self-induction mechanisms. STAT3 is a shuttleprotein, which is present in the cytoplasm in the absence of stimulus, and cantranslocate into the nucleus to bind specific DNA sequences upon activation. STAT3 has dual functions in signal transduction and transcriptionalregulation. The STAT3 protein is widely expressed in differenttypes of human tissues and cells, and is involved in cell proliferation,differentiation, apoptosis, and a variety of physiological functions.
It is also associated with the physiological and pathological functions ofinflammation, tumor, and immune response. To test whetherthe self-induction effect of hTFF3 was affected by the STAT3 binding site at−1417 bp to −1409 bp, we firstperformed ChIP on the hTFF3 promoter (pGL3−1826), which is the best wayto study the binding activity in vivo between transcription factors andpromoters.
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The results showed that STAT3 can bind to the hTFF3 promoter invivo. Next, we used a specific STAT3 inhibitor, AG490, to block the bindingactivity of STAT3 to the hTFF3 promoter. Results of the luciferase reporter assayshowed that AG490 could significantly reverse the self-induction effect of hTFF3 onits promoter activity in cells treated with different concentrations of AG490 for24 h. All of the above results confirmed that STAT3 can bind to thehTFF3 promoter.
Subsequently, the regulatory effect of STAT3 on hTFF3 self-inductionwas determined by STAT3 overexpression and knockdown assays. A eukaryotic expressionvector of STAT3 was constructed and co-transfected with pGL3−1826 intoLS174 cells.
We found that transcription of the hTFF3 promoter and expression levelsof hTFF3 were enhanced by co-transfection of STAT3 in a dose-dependent manner. Incontrast, when STAT3 expression was decreased by RNAi knockdown, we found that thetranscription of the hTFF3 promoter decreased by 40% in STAT3 knockdown cells. Realtime RT-PCR analysis showed that the STAT3 mRNA expression level decreased toapproximately 40% of the level before knockdown and that the hTFF3 mRNA levels weredownregulated to approximately 50% of the level before knockdown. Western blotanalysis showed that upon RNAi knockdown, hTFF3 protein expression also decreasedsignificantly. In summary, this study first confirmed the self-induction effect ofhTFF3.
By amplifying the hTFF3 promoter to generate truncated mutants of varyinglengths, the functional region (−1450 bp to−1400 bp) of the hTFF3 promoter was determined.Bioinformatics analysis confirmed a STAT3 binding site located in the region of−1417 bp to −1409 bp, andsite-directed mutagenesis analysis revealed that this binding site was essential forthe self-induction effect of hTFF3. ChIP experiments proved that in the presence ofhTFF3, STAT3 binds to the hTFF3 promoter. Furthermore, STAT3 overexpression andknockdown assays demonstrated that STAT3 enhanced the self-induction effect of hTFF3on its own promoter and the expression of hTFF3. Cell cultureHuman embryonic kidney (HEK) cell line HEK293 and colon cancer cell line LS174Twere purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEMsupplemented with 10% fetal bovine serum, and penicillin and streptomycin(100 U/mL) at 37 °C in 5% CO2.
Culturemedium was replaced every other day and cells were passaged every3–4 days at a ratio of 1:3. Properly shaped cells were used forexperiments. Generation of the full-length hTFF3 promoter construct and truncatedmutant constructsConsidering the full-length of the hTFF3 promoter (−1826 to+19 bp) as a template, primers targeting the 5′-untranslated region (5′ -UTR) sequence (AB038162) of hTFF3 inGenBank were designed, and KpnI and HindIII enzyme cutting sites wereintroduced in the upstream and downstream primers, respectively. HTFF3promoter fragments of different lengths (truncation mutants) were amplifiedand subsequently subjected to double enzyme digestion with KpnI and HindIII.The purified DNA fragments were ligated with pGL3-basic vector, and positiveclones were selected for enzyme digestion with KpnI and HindIII. Finally,these constructs were subjected to sequence confirmation. AliBaba 2.1software was used to analyze the transcription factor binding sites toensure that novel binding sites were not introduced into the constructs. Construction of STAT3 expression vectorTotal RNA extracted from HEK293 cells was reverse-transcribed into cDNA.
TheSTAT3 gene sequence (NM139276) was obtained from GenBank, and Primer 5.0software was used to design the following primers: forward,5′-CCCAAGCTTATGGCCCAATGGAATCAGCT-3′; and reverse,5′-CCGCTCGAGTCACATGGGGGAGGTAGCGC-3′.PfuUltra™ DNA polymerase (Stratagene, USA) was used to amplify theSTAT3 gene. PCR products were double-digested with HindIII and XhoI, and clonedinto the pCDNA3.1(+) vector. The ligated clones were subjected to doubledigestion and sequencing.
Double-stranded small interfering RNA (siRNA)RNA interference was used to silence endogenous STAT3 expression. Control siRNAand human STAT3-specific siRNA were purchased from Santa Cruz (Santa Cruz, CA,USA), and the experiments were conducted according to themanufacturer’s instructions. Cell transfection and luciferase assayHEK293 and LS174T cells undergoing logarithmic growth were seeded in 96-wellplates, and were subjected to transfection with jetPEI reagent kit(Polyplus-Transfection, France) at 80% confluence. The ratio of jetPEI toplasmid DNA was 2:1. Control pGL3, pGL3-hTFF3, and pGL3 basic plasmids weretransfected at 100 ng/well. Transfection of each plasmid wasperformed in triplicate wells, and 3 ng of pRL-TK plasmid wasco-transfected in each well as a loading control.
Transfection medium wasreplaced with fresh medium six hours after transfection. At 48 hpost-transfection, different concentrations of hTFF3 protein were added, andcells were collected for analysis after 48 h. AG490, a smallmolecule inhibitor of STAT3-DNA binding, was added to the medium prior to hTFF3treatment. Pudhupettai full movie free download mp4 songs.
In STAT3 overexpression and knockdown experiments, 100 ngluciferase reporter plasmid, 3 ng pRL-TK plasmid, and STAT3overexpression or knockdown plasmids were transfected into cells, and differentconcentrations of hTFF3 were added into the culture medium at24 h-post transfection. Cells were collected for analysis at48 h post-transfection. Luciferase activity was measured using thedual reporter assay system (Promega, Madison, Wisconsin, USA), and relativefluorescence intensity was defined as the ratio of the firefly fluorescenceintensity to Renilla fluorescence intensity. Three independentexperiments were performed for each experimental condition. Chromatin immunoprecipitation (ChIP) AssayChIP assays were performed using the ChIP-IT kit (Active Motif, USA) according tothe manufacturer’s instructions. HEK293 cells or LS174T cells wereroutinely cultured to 70–80% confluence, and subsequently fixed bymedium containing 1% formaldehyde. After rinsing with pre-chilled PBS, glycinewas added to the cells to stop fixation, the cells were rinsed again withpre-chilled PBS.
Next, the fixed cells were collected for cell lysis followed bycentrifugation to collect the cell nuclei. The centrifuged nuclei werere-suspended and sonicated by ultrasound to shear the chromatin into500 bp fragments. After treatment with RNase A andproteinase K, the effect of chromatin shearing was determined by agarose gelelectrophoresis on a 1% agarose gel. Protein G beads were used to clearunspecific antibody binding in the chromatin lipid, and STAT3 antibody wassubsequently added to the cleared chromatin.
RNA Pol II and IgG antibodies wereused as positive and negative controls, respectively. The mixture was incubatedat 4 °C overnight, followed by the addition of Protein Gbeads. After washing, the formaldehyde crosslinks in the elutedantibody-chromatin complexes were reversed, and DNA was purified. The purifiedDNA was then amplified by PCR. The PCR primers were: forward,5′-CAGAGGCTCCTGGAAGGG-3′; and reverse,5′-CAACCTCCTGCAGTGGAC-3′ (143 bpproduct). Quantitative real-time RT-PCRTotal RNA was extracted from cells using TRIzol reagent (Invitrogen, New York,CA, USA) according to the manufacturer’s instructions.
Total RNA wasreverse-transcribed into cDNA using the PrimeScript RT Master Mix (Perfect RealTime) Kit (TaKaRa, Dalian, China). Using cDNA as a template, two pairs ofprimers were used for real-time PCR analysis. HTFF3 primers were forward,5′-CCAAGGACAGGGTGGACTG-3′; and reverse,5′-AAGGTGCATTCTGCTTCCTG-3′. STAT3 primers were forward,5′-ATCACGCCTTCTACAGACTGC-3′; and reverse,5′-CATCCTGGAGATTCTCTACCACT-3′. PCR reaction system with20 μL volume: 0.5 μL cDNAtemplate, 0.25 μL forward primer,0.25 μL reverse primer, 10 μLRNase-free ddH2O, 8 μL2.5 × Real Master Mix (SYBR Green I).Reaction conditions: pre-denaturation: 95 °C for 10 s, 1cycle; PCR reaction: 95 °C for 15 s,60 °C for 60 s, 40 cycles. Statistical analysis wasperformed following data collection.
Western blotCells were collected and lysed, and total protein was quantified using BCAProtein Quantification Kit (Pierce, USA). Forty micrograms of total protein wasloaded in each lane, and a 15% SDS-PAGE gel was run to separate the proteins.After electrophoresis, proteins were transferred onto a cellulose nitratemembrane, and incubated with anti-hTFF3 or anti-STAT3 polyclonal antibodies, oranti-β-actin monoclonal antibody (Abcam, Cambridge, MA, USA),respectively, overnight at 4 °C. Subsequently, themembrane was incubated with horseradish peroxidase-labeled anti-mouse IgGsecondary antibody at room temperature for 2 h.
Chemiluminescencesignals were quantified using an ECL imager, and analyzed using Quantity Onesoftware (Bio-Rad, Hercules, CA, USA). Statistical AnalysisStatistical analysis was performed using SPSS Software (version 16.0). Data arepresented as mean ± S.D. Results wereanalyzed using unpaired t tests, andP. 1.Suemori, S., Lynch-Devaney, K.
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Autoinduction of the trefoil factor 2 (TFF2) promoter requires an upstream cis-acting element. Biochemical and biophysical research communications 293, 366–374, 10.1016/S0006-291X(02)00199-7 (2002). 5.Joung, Y. Combination of AG490, a Jak2 inhibitor, and methylsulfonylmethane synergistically suppresses bladder tumor growth via the Jak2/STAT3 pathway. International journal of oncology 44, 883–895, 10.3892/ijo.2014.2250 (2014). 6.Huang, C.
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