The Molecular Mechanism Study Of LPS Induced The UPA Promoter Elements

PARTâ… Vector construction and activity analysis of rat uPA gene regulation regionEXPERIMENTâ… Cloning and Analysis of the Promoter Region of Rat uPA GeneObjective:To clone and analyze the promoter sequence of rat urokinase plasminogenactivator protein gene.Methods:The genomic DNA was extracted from rat testicular tissue.According tourokinase plasminogen activator,the gene sense primer and antisense primer of uPA genewere designed and synthesized,then Touch-Down PCR were performed.After properpurification,the PCR product was sequenced,analyzed with the promoter predictionsoftware and compared with the DNA sequence of rattuas urokinase plasminogen activator.Results:The cloned uPA gene was about 1517bp in length,which contained a fullopen-reading frame with 21 bp in length exons,and the upper region of transcriptional startwas 1496 bp in length which was eucaryon transcriptional control area.The 5'UTR had apromoter region including a non-responsive TATA-box.Not only the GC-box bindingregion was found in this gene,but also active protein 1(AP1) and SP1were seen in otherregions.Conclusion:A 1517 bp uPA gene fragment (GenBank accession No.25916) was obtainedfrom rat genomic DNA library,containing eucaryon transcriptional control area with apromoter region,non-conspicuous TATA-box,GC-box and an extron.This non-responsiveTATA-box is located at the upper -30 region. EXPERIMENTâ…¡Verification of uPA gene core promoter in rat testisObjective:Analyzing the uPA gene promoter structure and activity of specifictranscriptional regulation region to examine the expression and regulation mechanism ofuPA,and to study the translation start uPA function of uPA gene5'and the non-coding region(NCR) in the Sertoli cells.Methods:Genomic DNA about 1.6 kb5' flanking area (flanking region) of uPA protein inthe rat genome sequences was isolated,and analyzed the length,structure,transcriptionstart site regulator frame work of rat uPA5' flanking fragment of the promoter.Five primerswere designed to amplify and to obtain rat uPA gene 5'flanking fragment of different length.After being purified and sequenced,the fragments of different length were inserted intoupstream of the plasmid PGL3-Enhancer which contained a luciferase reporter gene but didnot contain a promoter.Constructed vectors were verified by PCR and double-enzymedigestion.Recombinant vectors were transfected to Sertoli cells,and the lampyris luciferaseactivity in cell lysis products was detected by Lumat weak light detector 24h later tocompare the promoter activity among different fragments,so the core promoter of the uPAgene 5'flanking area could be determined.Results:Promoter transcriptional activity exists in these five fragments:P1-167/+40(211bp),P2-455 / +40(499bp),P3-758 / +40(802bp),P4-1156 / +40(1220bp),P5-1544 /+40(1588bp).In addition,P2-455 / +40(499bp) transcriptional activity is the strongest,followed by P1-167/+40 (211bp),P5-1544/+40(1588bp),whereas the transcriptionalactivity ofP3-758 / +40(802bp)and P4-1156 / +40(1220bp) fragments is very weak.Conclusion:The 1.6 kb upstream sequence of uPA gene transcription start site isindispensable for activation of rat uPA gene promoter,moreover P2-455/+40(499bp) sequence is the core of the uPA gene promoter.There is control of the promoter orenhancer and other cis-acting elements in -167 / -455,-1156 / -1544 region,and thetranscription of inhibitory factor and the negative regulatory factor exists in -455/-758,-758/-1156.Construction of the activity of core promoter containing sequences -455 /+40recombinant vector with the luciferase reporter gene is expected to detect specificDNA-binding protein related to uPA gene transcription,so this study lay a foundation forfurther clarification of the mechanism of uPA gene transcription regulation and the role ofuPA regulation during spermatogenesis.PARTâ…¡:Effect of LPS on the core transcription activity of uPA gene in rat Sertoli cellsEXPERIMENTâ… Effect of LPS on uPA gene transcription activityObjective:observing the regulation of uPA gene transcription induced by the LPS so as toexplore the specific uPA transcription regulatory mechanism.Methods:PGL₃ vector as control,and the verified recombinant vector PGL₃-uPA promoter499bp were transfected into Sertoli cells and induced with LPS individually,with inductionof time 0,12,24,48 hours separately.The luciferase activity changes of dual luciferasesystem were detected by Lumat weak-light detector before and after induction,at the sametime,luciferase activity differences between the two groups were observed.Results:LPS-induced luciferase activity changes were not very different in PGL₃ vectorcontrol group in 0,12,24,48hours,while there was difference in 0,12,24,48hours luciferaseactivity of LPS-induced recombinant vector PGL₃-499bp,especially the 24hours group. Compared with PGL₃-Control vector the recombinant vector PGL₃-uPA-499bp groupshowed larger changes in luciferase activity.Conclusion:The dual-luciferase detection results showed that the activity of core uPAgene transcription control region can be up-regulated by LPS.There was no significantdifference in PGL₃-Control vector with the CMV promoter induced by LPS.There weresignificant differences in the core region of uPA gene transcription after stimulation,but theLPS on the core uPA gene transcription control region did not increase as the stimulationtime increased,with the most obvious effect at the time point of 24 hours.A certain amountof LPS stimulation did not have time compliance,which suggested the increased activity ofuPA gene transcription did not show a time-depended manner.EXPERIMENTâ…¡uPA expression changes of rat Sertoli cells with LPS stimulationObjective:Based on LPS up-regulates the activity of uPA gene transcription control region,the effect of LPS on the uPA expression in Sertoli cells was further observed.Methods:Immunocytochemistry (IHC) was used to observe the secreted uPA protein afterLPS stimulated for 0,12,24,48 hours compared with control group,the statistical methodwas used to analyze the level of uPA protein expression.Results:The immunocytochemical results showed that uPA secretion in Sertoli cells wasmost significant at 12 hours by LPS stimulation.Conclusion:The results was consistent with preliminary studies that LPS could up-regulatethe activity of uPA gene transcription control region.It can be confirmed that LPSstimulated the secretion of uPA by increasing the level of gene transcription activity. Partâ…¢Study on rat uPA gene IFN stimulate-response mechanisms in male reproductivesystemEXPERIMENTâ… The levels of cytokine IL-1,IL-6 changed through LPS induced signaltransduction pathway.Objective:LPS stimulated Sertoli cell to increase secretion of uPA,but the relevance to thelevel of cytokines IL-1,IL-6 transcriptions was not understood yet.Studies had alreadyshown that the level of IL -1 and IL-6 could increased in supernatants of LPS stimulatedcells,including Sertoli cells,without dose and time compliance,to make a further study ofthe effect of IL-1,IL-6 on LPS-TLR4 signal transduction pathway and the relevance ofcytokine in signaling pathway.Methods:LPS and anti-IRF3 interference in the Sertoli cells for 0,12,24,48 hours.Levels ofIL-1 and IL-6 transcriptions were detected by real-time fluorescence quantitative PCR(QRT-PCR).The Results of comparative analysis of IL-1,IL-6 transcription level,mRNAchanges in a single-factor manner (LPS and anti -IRF3),underwent the statistical analysis.Results:The fluorescence quantitative PCR results showed that:(1) IL-1,IL-6 mRNA expression in LPS stimulation group was higher than the LPS +anti-IRF3 group which had statistical significance.(2) Compared with control group,no difference in IL-1,IL-6 expression level of mRNA inanti-IRF3 group was found,compared with the LPS group IL-1,IL-6 mRNA in these twogroups decreased with statistical significance.(3) Compared with blank control group and LPS + anti-IRF3 group the IL-1,IL-6 mRNAexpression level decreased in anti-IRF3 group,which had statistical significance.(4) The detection of IL-1,IL-6 mRNA level at 0,12,24,48hours points in each groupshowed LPS stimulation was no time compliance. Conclusion:IL-1 and IL-6 were the key cytokines in LPS-induced MyD88 independentsignal transduction pathway.Under LPS stimulation IL-1,IL-6 mRNA expression levelsdecreased with IRF3 antagonist—anti-IRF3,indicating that after IRF3antagonist—anti-IRF3 Sertoli cells had a significant resistance to LPS.Fluorescencequantitative results show that IL-1,IL-6 mRNA expression level did not change whensamples only be stutimulated by IRF3 antagonist anti-IRF3 in the absence of LPS,whichsuggested that exogenous stimulation factors was required for the induction of IRF3 intothe nucleus to play a role.Immune defense response of Sertoli cells was closely correlatedwith cytokine IL-1,IL-6 .Detection in the IL-1,IL-6 mRNA level in 0,12,24,48 hours ineach group was consistent with the results of preliminary studies,i.e.,the LPS stimulationreduced the time dependence.It is inferred that immune defense response of LPSstimulated Sertoli cells may be activated mainly through non-MyD88-dependent way ofsignal transduction pathway.The experiment laid a basis for further detailed analysis of themolecules in LPS-TRL4 signal transduction pathways in Sertoli cells.EXPERIMENTâ…¡Effect of uPA transcription region on LPS stimulated signaltransduction pathwayObjective:To determine whether the uPA gene mainly play a role in theMyD88-independent NF-κB/IRFs signal transduction pathway,the level of uPA mRNAand protein were measured in LPS,anti-IRF3,IFN-βin 0,12,24,48 hours interferencedSertoli cells,which can further understand the uPA gene in the signal transduction pathwayand provide a theoretical basis of the molecular mechanism.The experiment laid a basis forunderstanding important cis-regulatory elements and associated trans-acting factors of thepromoter region of genes,mechanism of nuclear gene-induced signal for the response,andthe regulation targets of uPA gene expression. Methods:In control,LPS,anti-IRF3,LPS + anti-IRF3,LPS + IFN-β,IFN-βsix groups,uPA mRNA and protein secreted by Sertoli cells were induced in vitro,with periods of time0,12,24,48 hours,respectively.Real-time PCR and Western-blot were used to detect uPAmRNA and protein level withβ-actin for the internal control at each induction time point,.Results:(1)The uPA mRNA levels in LPS + IFN-βgroup were higher than other groups,i.e.,therewas no statistical significance compared with the LPS group,but compared with IFN-βgroup there was statistical significance.(2)The uPA mRNA levels in control group were lower than LPS group,IFN-βgroup,LPS +IFN-βgroup,LPS+anti-IRF3 group,with statistical significance.(3)The uPA mRNA levels were higher in LPS + anti-IRF3 group than anti-IRF3 group withstatistical significance,but lower than the LPS group with statistically significance.(4) Compared with control group the uPA mRNA levels in anti-IRF3 group were notstatistically significant..(5) The uPA mRNA level in LPS + anti-IRF3 group was lower than the IFN-βgroup withobviously statistical significance,but uPA protein expression in IFN-βgroup was notsignificant compared with LPS + anti-IRF3 Group.(6)The uPA protein expression in LPS group with 12h stimulation had statisticalsignificance compared with 24,48 hours group in accordance with the previousimmunocytochemical results.(7)The level of uPA mRNA in 12 hours LPS stimulation group was not statisticallysignificant compared with 24,48 hours group.(8) The uPA mRNA and protein expression did not show time compliance based on0,12,24,48 hours observed in each group.Conclusion:The LPS and IFN-βhad the synergistic effect on LPS-TRL4 signals occurencein Sertoli cells.Increased expression of uPA can be induced by IFN-β.This indicated uPA may play a role in the cytokines secretory pathway stimulated by LPS-TRL4 MyD88independent NF-κB/IRFs signal transduction pathway.uPA also played a role inLPS-TRL4 signal transduction pathways,which may play a role in the defense mechanismof Sertoli cell to promote migration of extracellular matrix stimulated by LPS.In theeffect of IRF3 antagonistic-anti-IRF3,the uPA mRNA and protein level expressed loweronce again with LPS stimulation,which verified Sertoli cells were resistant to LPS after theantagonistic-anti-IRF3.Cytokines secreted by rat Sertoli cells promoted the expression ofuPA.In uPA gene transcription region there was uPA gene-specific DNA binding site-IRFstimulated responsive element -a new signal pathway,which demonstrated the regulation ofuPA gene expression targets.