The Study On The Affection Of HDAC1 To The PRs' Expression And The Mechanism During The Onset Of Human Labor

Background and Purpose:Human pregnancy and parturition is an extremely complex physiological process.Pregnant human myometrium switch from a quiet state into a state of excitation-contraction and subsequent laboring.However,the precise mechanisms responsible for human parturition have yet to be resolved.Progesterone maintains the pregnant uterus in a state of rest and relaxation,and this antagonizing effect is withdrawn when a pregnancy comes to term.In most mammals,progesterone withdrawal is preceded by a drop in circulating maternal progesterone levels prior to the onset of labor.However,in humans,there is no detectable fall in maternal progesterone serum levels toward the end of pregnancy.Nonetheles,the administration of progesterone receptor(PR)antagonists,such as RU486,is known to increase myometrial contractility and induce labor.Therefore,th e concept of functional progesterone withdrawal,caused by reduced responsiveness of target tissues to progesterone in humans and higher primates,has been proposed as a clinical concept.Recent studies suggest that one important mechanism responsible for this functional withdrawal is changing levels of nuclear PR isoforms,which result in reduced responsiveness of progesterone upon uterine relaxation.PR gene is located in 11 q22-23,including 8 exons,length about 90 KB.PRA and PRB are the two major isoforms of the nuclear PR.PR-A is a truncated version of PR-B,lacking the first 164 N-terminal amino acids.The expression of PRA and PRB isoforms is controlled by 2 separate promoters but acts via single gene transcription of messenger RNAs(mRNAs).Progesterone receptor B,as the full-length functional receptor,mediates most progesterone effects,such as maintaining the uterus in a quiet state,and PRA,the truncated version,is thought to antagonize the effects of PRB.Related studies have revealed that t he expression of myometrial PRA relative to PRB becomes elevated at the onset of labor and have indicated that this differential PR isoform ratio suppresses myometrial responsiveness to progesterone,thereby triggering labor and delivery.These studies,the refore,highlight the importance of the precise mechanisms regulating PR isoform expression.However,the underlying mechanisms regulating differential expression of PR isoforms remain unclear.Recent studies have found that compared with PRB,PRA promoter region has a higher acetylation level,which provides a possibility to epigenetic regulation of "progesterone functional retreat".However,due to the fact that histone acetylation may be modified by several factors,for example: HATs or HDACs,the exact factors responsible still require further investigation.Meanwhile,studies of breast cancer have already shown that HDACs,especially HDAC1,can bind to the estrogen receptor(ER)promoter and efficiently regulate ER expression.HDACs members has different structures and biological functions,while human HDAC1 are divided to class I,and shown to share significant homology with the previously indentified Saccharomyce scerevisiae transcriptional regulator Rpd3/Sdi2 / Sds6.Our analysis of pregnant uterus found that accompanied by elevated PRA expression,HDAC1 dropped significantly.Therefore,combined with the recent studies,we hypothesized that HDAC1 could regulate PR epigenetically during the onset of labor.This study was designed to determine whether epigenetic HDAC1 is involved in the regulation of PR isoforms in the human pregnant myometrium and to investigate the underlying mechanisms involved.Results arising from this study will not only provide insight into the epigenetic-related mechanisms of human labor but also provide a potential interventional strategy with which to prevent preterm birth,overdue production and more labor disorders.Experimental methods:1.1 Comparing the HDAC1,PRA and PRB expression of the pregnant uterus,and PRA/PRB value in the absence of labor(NIL)and the active labor(IL)Myometrium tissue biopsies collected from singleton,term cesarean deliveries in the absence of labor(NIL)or in active labor(IL),and they were taken from the upper margin of the lower uterine segment.Extracting tissue mRNA and nucleoprotein,we tested mRNA level and protein expression levels in two groups using RT-PCR and western blot means.1.2 Isolation,Culture and identification of primary human myometrial cellsWe isolated and cultured primary human myometrial cells from pregnant human myometriumtissue.We then identified the primary cells of passage 4 by immunofluorescence with antibodies against the smooth muscle cell markers SM-MHC,a-SMA,calponin,and vimentin.The expression of these proteins was further verified by Western blot assays.1.3 ConstructingHDAC1 shRNA plasmid and an HDAC1-overexpressing plasmidHDAC1 sh RNA plasmid and an HDAC1-overexpressing plasmid were designed and constructed by Invitrogen,Inc.1.4 HDAC1 shRNA plasmid and an HDAC1-overexpressing plasmid transfected the human primary uterine smooth muscle cells(HUtSMCs)Using the P4 generation HUt SMCs,the plasmids were transfected by electroporation.Flow cytometrymeasureplasmid transfection efficiency.1.5 Testing HDAC1,PRA and PRB expression changes afterHDAC1 shRNA plasmid transfectionRT-PCR and western blot means analyze of HDAC1,PRA and PRB expression levels changes.1.6 Testing HDAC1,PRA and PRB expression changes after HDAC1-overexpressing plasmid transfectionRT-PCR and western blot means analyze of HDAC1,PRA and PRB expression levels changes.1.7 Progesterone receptor Gene Expression Is Regulated by HDAC1Using the ChIP�quantitative PCR(q PCR)method,we tested the Level of histone deacetylase 1(HDAC1)enrichment at the PR gene promoters in myometrial cells in which HDAC1 was knocked down(HDAC1-RNAi)or overexpressed(HDAC1 plasmid).1.8 Data AnalysisGraphPad Prism5 statistical analysis software was used.All data were doing normality test,and presented as mean � standard deviation.Double tail Student 't test was used to compare the differences between the two groups.Results:2.1 comparing the HDAC1,PRA and PRB expression of the pregnant uterus,and PRA/PRB value in the absence of labor(NIL)and the active labor(IL)We first detected the change in expression of these 2 genes at the mRNA and protein level in 10 samples of myometrium before and after labor.The quantitative reverse transcription-PCR assays showed that although PRB m RNA levels were not different between the IL and NIL tissues,the PRA mRNA levels and the PRA/PRB mRNA ratio were both markedly higher in the IL myometrium than in the NIL myometrium.However,HDAC1 m RNA levels were significantly lower in the IL tissues than in the NIL tissues.Using the PR antibody,which detects PRA and PRB isoforms simultaneously,Western blot analysis also indicated that the levels of PRA protein were markedly elevated and those of HDAC1 protein reduced in IL tissues,whereas no significant change was noted in these tissues in terms of PRB protein.2.2 Isolation,Culture and identification of Primary Human Myometrial CellsObserved under the microscope,typical spindle fibers could be seen in the organizations and long fusiform shape could be seen in the primary cells.Four smooth muscle cell markers SM-MHC,a-SMA,calponin,and vimentin were expressed in myometrial tissues and cultured primary cells.2.3 HDAC1 shRNA plasmid and an HDAC1-overexpressing plasmid transfected the HUtSMCsWith Immunofluorescence microscope,we can see a lot of GFP labels.Flow cytometry instrument measured plasmid transfection efficiency more than 60%.2.4 Testing HDAC1,PRA and PRB expression changes afterHDAC1-overexpressing plasmid transfectionBoth HDAC1 mRNA and protein were downregulated by the transfection of HDAC1 sh RNA plasmids.Silencing the HDAC1 gene in myometrial cells significantly enhanced PRA gene expression at both the mRNA and the protein level but did not alter the expression of the PRB gene,thus resulting in an increased PRA�PRB expression ratio.2.5 Testing HDAC1,PRA and PRB expression changes after HDAC1-overexpressing plasmid transfectionIn contrast,when primary myometrial cells were transfected with HDAC1-overexpression plasmids,the expression of the HDAC1 gene was increased and the expression of PRA was downregulated markedly at both the mRNA and the protein level,without any effect upon PRB gene expression but with a reduced PRA�PRB expression ratio.2.6 Progesterone receptor Gene Expression Is Regulated by HDAC1Using Ch IP�quantitative PCR(q PCR)method,we found that knocking down the HDAC1 gene reduced the level of PRA promoter enrichment with HDAC1 protein,whereas overexpression of the HDAC1 gene led to increased levels of PRA promoter enrichment.I n contrast,no significant changes were observed in PRB promoter enrichment with HDAC1 protein in cultured primary myometrial cells.Conclusion:1.The mechanism involving elevated expression of the PRA gene and a subsequent increase in PRA�PRB expression ratio in the pregnant human myometrium might contribute to labor.2.HDAC1 could directly bind to the promoter of PRA but not of the PRB gene,and regulate the expression of PRA gene.3.A decline in HDAC1 expression might initiate human labor by binding to the promoter of PRA,thus elevating the levels of histone acetylation and starting labor.