The Relationship Of P53,MDM2 And Polymorphisms With Missed Abortion

Missed abortion refers to pregnancy at 20 weeks ago,in the absence of any outside interference factors,embryonic or fetal death in utero is not timely discharge. Missed abortion caused by many reasons such as the number of chromosomes, structural abnormalities,immune dysfunction,endocrine dysfunction,abnormal intrauterine environment,genetic tendency to thrombosis,systemic infection, environmental adverse factors,these factors are about the 50%incidence of missed abortion.The occurrence of missed abortion is probably more the result of factors, but the exact mechanism is not clear.Study found that a successful pregnancy depended on the necessary trophoblast apoptosis and appropriate sufficient angiogenesis.Apoptosis is the course under the control of gene activity of cell suicide,is a multi-cell organisms to control the body development,the maintenance of environmental stability,the initative procedure by the gene encoding the cell death process.Normal pregnancy has certain degree of trophoblast apoptosis,in embryonic development process,there is an appropriate degree of apoptosis within the lumen conducive to villi and chorionic villi branch formation and development.The success of pregnancy depends on enough villous angiogenesis,only with this can it supply adequate oxygen and nutrients.In early pregnancy,the early development of trophoblast cells(before 12 weeks of pregnancy) is a hypoxic environment,and the adaption of trophoblast cells to hypoxic environment is the key to successful pregnancy.And studies have proved that the process of angiogenesis was regulated by local oxygen concentration.However,in exceptional cases,villi dysplasia or degree of degenerative changes in both proliferation and degeneration,cytotrophoblast cells,syncytiotrophoblast cells significantly reduced or even disappear,villous trophoblast basement membrane and capillary basement membrane light degree of thickening,cytula cause developmental delay,failure of pregnancy,the occurrence of missed abortion.P53 protein as a transcription factor,by regulating the expression of multiple genes in cell cycle regulation and control,plays an important role in maintaining the integrity of cell genome,inducing cell differentiation and apoptosis.In normal cells, p53 gene remained at a relatively low level,so that does not interrupt the cell cycle or eventual cell death.When the cells were under DNA damage,p53 appropriate cell-mediated gene repair is activated;if too much to repair the damage,the p53-mediated apoptosis began to work,resulting in apoptosis,cell cycle arrest at G1 phase.Research had shown that the expression of trophoblast apoptosis-related factor p53 protein related to the occurrence of missed abortion.P53 is a short-lived protein in cells,the concentration of it is strict regulated and controlled by its negative regulator murine double minutes2(MDM2) gene.MDM2 is of the p53 regulation network and and as an important regulator of p53 involved in cell growth inhibition,apoptosis,cell cycle regulation and control processes.Through the study of the p53 and MDM2 negative feedback loop,they found that wild-type p53 protein can stimulate the high expression of MDM2 protein;however,the increase in the MDM2 protein can inhibit the function of wild-type p53 protein,thus increasing the role of the negative feedback cycle.Those can affect p53,MDM2 gene,or the factors of the p53-MDM2 interaction can have an important impact on cell fate (apoptosis,cell cycle arrest,etc.).When the change of the gene sequence was rare,it was called mutation;when it changed commonly,it was called gene polymorphism.Many gene single nucleotide change(SNP) will significant change the gene function,now,more and more eyelight are turning to the function of the polymorphism alleles,p53 exon 4 regulates the cell growth inhibition and apoptosis,and the 72 codons has the CCC/ CGC single nucleotide polymorphisms,then replaced arginine(Arg) by proline(Pro). Those included Arg and Pro p53 protein are all wild-type p53 protein,although in most cells the stability are same,but the ability of the two proteins of activating transcription,inhibiting cell growth and inducing apoptosis capacity are different.P ro-p53 seems to be more effective to activate transcription,so that upregulate the expression of the downstream gene;However the ability of Pro-p53 inhibiting cell growth and inducing apoptosis is weaker than Arg-p53.MDM2 gene promoter polymorphisms of the sequence can change the expression of MDM2 protein.Recently,inherent SNP309 in the MDM2 promoter polymorphism(first intron of the T→G change in nucleotide sites) was found.The research showed that the MDM2 protein carrying SNP309 G/G genotype of MDM2 protein also can enhance their effectiveness in stimulating the adoption of the combination of Sp1 protein,then enhancing the expression of MDM2 protein to reduce the stability of p53 than the MDM2 protein carrying the T/T genotype,thus changing the p53 mediated apoptosis in the response to DNA damage.Based on p53,MDM2 polymorphisms and p53,MDM2 play an important role in the regulation of apoptosis,cell cycle changes and angiogenesis,we divided our study into two parts,the first part mainly did the research of the polymorphism genotypes of p53 codon72 and MDM2 SNP309 in early pregnancy blood and villous samples and the relationship of the polymorphism of p53 codon72 and MDM2 SNP309 polymorphism with the expression of p53 and MDM2;The second part examined the expression of p53,MDM2,VEGF and HIF-1α,then transfected the recombined pcDNA3-MDM2 plasmid into trophoblast cell lines JEG-3,Bewo, investigated the mechanism of p53,MDM2 gene in the growing precess of villous trophoblast cells,further clarified the causes of missed abortion and to provide early diagnosis and treatment of the new strategy. PARTâ… THE RELATIONSHIP OF P53,MDM2 POLYMORPHISMS WITH MISSED ABORTIONObjective:(1) To investigate the polymorphisms of p53 codon72 and MDM2 SNP309 in the blood and villous samples of missed abortion and induced abortion patients, which attempt to discover the high risk genotype of missed abortion.(2) To investigate the relationship of p53 codon72 and MDM2 SNP309 polymorphisms with the mRNA and protein expression levels of p53 and MDM2, then to identify the mechanism of the effect of polymorphisms.Methods:(1) Extract the DNA of the blood and villous samples of missed abortion and induced abortion with DNA extract kit.(2) PCR with sequence specific primers(PCR-SSP) method to analysis the genotype of p53 codon72 in blood and villous samples of missed abortion and induced abortion groups.(3) PCR restriction fragment length polymorphism(PCR-RFLP) method to analysis the genotype of MDM2 SNP309 in blood and villous samples of missed abortion and induced abortion groups.(4)Extract mRNA of the villous samples of missed abortion and induced abortion, using qReal-time PCR method to examine the mRNA expression level of p53 and MDM2 in villous samples.(5) Immunohistochemistry method to examine the location and expression levels of p53,MDM2 protein in villous samples of missed abortion and induced abortion group.Results:(1) The genotypes of p53 codon72 in blood and villous samples:the distribution of p53 codon72 Pro/Pro genotype in missed abortion group was higher than induced abortion group(9.47%vs.3.66%;20.00%vs.12.50%),but in the comparison of the two group,the difference was not significant(P=0.094; P=0.330).The distribution of p53 codon72 Arg/Arg genotype in missed abortion group was lower than induced abortion group(34.74%vs.37.80%;20.00% vs.25.00%),but in the comparison of the two group,the difference was not significant(P=0.689;P=0.528).(2) The genotypes of MDM2 SNP309 in blood and villous samples:the distribution of MDM2 SNP309 G/G genotype in missed abortion group was higher than induced abortion group(32.63%vs.18.29%;28.33%vs.12.50%),and in the comparison of the two group,difference was significant(P=0.010;P=0.043). The distribution of MDM2 SNP309 T/T genotype in missed abortion group was lower than induced abortion group(21.05%vs.28.05%;31.67%vs.40.625%), and in the comparison oft he two group,difference was not significant(P=0.239; P=0.352).(3) The comparison of p53 mRNA expression in villous samples:p53 codon 72 Arg/Arg genotype induced higher p53 mRNA expression than Pro/Pro genotype, but in the comparison of the two genotypes,the difference was not significant (P=0.278);MDM2 SNP309 T/T genotype induced higher p53 mRNA expression than G/G genotype,but in the comparison of the two genotypes,the difference was not significant(P=0.367).(4) The location and expression of p53 protein in villous samples:p53 protein was mainly expressed in the nucleuses of villous cytotrophoblast,syneytiotrophoblast and extravillous trophoblastic cell column;p53 codon 72 Arg/Arg genotype can not induce higher expression of p53 protein with compared with other genotypes (P=0.563).MDM2 SNP309 T/T genotype can not induce higher expression of p53 protein with compared with other genotypes(P=0.289).(5) The comparison of MDM2 mRNA expression in villous samples:p53 codon 72 Pro/Pro genotype can induce higher expression of MDM2 mRNA than Arg/Arg genotype,and in the comparison of the two genotypes,the difference was significant(P=0.013);MDM2 SNP309 G/G genotype can induce higher expression of MDM2 mRNA than T/T genotype,and in the comparison of the two genotypes,the difference was significant(P=0.04).(6)The location and expression of MDM2 protein in villous tissues:MDM2 protein was mainly expressed in the nucleuses of villous cytotrophoblast,syneytiotrophoblast and extravillous trophoblastic cell column;p53 codon 72 Pro/Pro genotype can induce higher expression of MDM2 protein,and compared with other genotypes,the difference was significant(P=0.037),MDM2 SNP309 G/G genotype can induce higher expression of MDM2 protein,and compared with other genotypes,the difference was significant(P=0.001).Conclusions:(1)MDM2 SNP309 G/G genotype is the high risk genotype of missed abortion.(2) p53 codon72 Pro/Pro genotype and MDM2 SNP309 G/G genotype could induce high expression level of MDM2,then induced the occurrence of missed abortion.PARTâ…¡THE EFFECT OF P53,MDM2 ON MISSED ABORTIONObjective:(1)To explore the mRNA expression and protein expression and location of the related genes of p53,MDM2,vascular endothelial growth factor(VEGF) and hypoxia inducible transcription factors-1α(HIF-1α) in villous samples of missed abortion and induced abortion patients.(2)To explore the effect of p53,MDM2 on the growth of villous trophoblast cells.Methods:(1) qReal-time PCR method to detect the mRNA expression levels of p53,MDM2,VEGF and HIF-1αin villous samples of missed abortion and induced abortion groups.(2) Immunohistochemistry method to detect the protein expression levels and location of p53,MDM2,VEGF and HIF-1αin villous samples of missed abortion and induced abortion groups.(3) Extract the total mRNA of JEG-3 and Bewo,and amplying the MDM2 gene,to characterizate the expression of MDM2 mRNA in the two trophoblast cell lines.(4) The plasmid pcDNA3 and the recombinant plasmid pcDNA3-MDM2 were transfected into trophoblast cell lines JEG-3 and Bewo,separately;after the screening of G418,using reverse transcription-PCR(RT-PCR) method to detecte the expression of MDM2 mRNA,screening out the high expression cell.(5) With screening out high expression of MDM2 mRNA transfection cells given MDM2 antagonist(Nutlin3) treatment,under hypoxia(1%O₂;5%CO₂;94%N₂) conditions,the JEG-3 cell line was divided into five groups:A:JEG-3 group; B:JEG-3- pcDNA3-DMSO group;C,JEG-3-pcDNA3-Nutlin3 group;D, JEG-3-pcDNA3-MDM2-DMSO group;E,JEG-3-pcDNA3-MDM2-Nutlin3 group(the group of Bewo cell line was same to JEG-3 cell line).Above cells were collected after being cultured for 24 hours:1) Collecting 5×10⁵ cells added 1 ml 70%precooling alcohol-PBS stored at 4 degree,wind and percussion blending refrigerator overnight at before the treatment adopted by the PI flow cytometry to examing the changes of cell cycles in each group;2) The remaining cells were divided into two parts,the mRNA and protein expression levels of p53,MDM2,VEGF and HIF-1αwere detected by qReal-time PCR and Western blotting,separately.Results:(1) The relationship of the mRNA expression level of p53,MDM2,VEGF and HIF-1αin villous samples of missed abortion patients:In the villous smples of missed abortion patients,the expression of p53 was positive correlated with the expression of MDM2,HIF-1α(r=0.35;r=0.63),and the relationship was significant(P=0.01;P＜0.001);but negatively correlated to the expression of VEGF(r=-0.30),and the relationship was significant(P=0.03).The expression of MDM2 was positive correlated with the expression of HIF-1α(r=0.28),and the relationship was significant(P=0.04);and negatively correlated with the expression of VEGF(r=-0.08),but the relationship was not significant(P=0.57). The expression of HIF-1αwas negatively correlated with the expression of VEGF(r=-0.37),and the relationship was significant(P=0.007).(2) The relationship of the mRNA expression level of p53,MDM2,VEGF and HIF-1αin villous samples of induced abortion patients:In the villous samples of induced abortion patients,the expression of p53 was positive correlated with the expression of MDM2,VEGF and HIF-1α(r=0.31;r=0.48;r=0.67),and the relationship was significant(P=0.03;P=0.003;P＜0.001).The expression of MDM2 was positively correlated with the expression of VEGF(r=0.23),but the relationship was not significant(P=0.11);and negatively correlated with the expression of HIF-1α(r=-0.03),but the relationship was not significant (P=0.84).The expression of HIF-1αwas positively correlated with the expression of VEGF(r=0.35),and the relationship was significant(P=0.01).(3) The location of p53,MDM2,VEGF and HIF-1αprotein in early pregnancy villous samples:p53 and MDM2 protein was mainly expressed in the nucleuses of villous cytotrophoblast,syneytiotrophoblast and extravillous trophoblastic cell column. VEGF protein mainly expressed in the cytoplasm of syneytiotrophoblasts, cytotrophoblasts,extravillous trophoblastic cell columns of villouses and the villous mesenchymocyte of the two groups.HIF-1αprotein mainly expressed in the cytoplasm of syneytiotrophoblasts,cytotrophoblasts,extravillous trophoblastic cell columns of villouses and the villous mesenchymocyte of the two groups,occasionally expressed in the nucleuses of villous syneytiotrophoblast and extravillous trophoblastic cell column.(4) The comparison of the expression of p53,MDM2,VEGF and HIF-1αprotein in early pregnancy villous samples:Higher expression of p53,MDM2 and HIF-1αin villous samples of missed abortion group can be seen,and in the comparison of induced abortion group,the difference was significant(P＜0.001;P＜0.001;P=0.035);The expression of VEGF in villous samples of missed abortion group was less than induced abortion group,and in the comparison of the two group,the difference was significant(P=0.026).(5) The JEG-3 cell line had faint MDM2 mRNA expression level,Bewo cell line had little MDM2 mRNA expression.(6) Flow cytometry in examing the changes of cell cycle was found:After treatmented with Nutlin3,the cell cycles apparent stagnated at G1 phase,and the ratio of S phase decreased in all the groups of JEG-3 cell line.The cell cycles apparent stagnated at G1 phase,and the ratio of S phase decreased in the transfected of pcDNA3-MDM2 Bewo cell line.(7) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αmRNA expression levels of JEG-3 cells1) Comparison of p53 mRNA expression level:comparing A group and B group, p53 mRNA expression level was not different(P＞0.05);p53 mRNA expression in C group was significantly higher than B group,D group was significantly higher than the B group,E group was significantly higher than D group,and the difference was significant(P＜0.05).2) Comparison of MDM2 mRNA expression level:comparing A group and B group, MDM2 mRNA expression level was not different(P＞0.05);MDM2 mRNA expression in C group was significantly higher than B group,D group was significantly higher than C group,E group was significantly higher than D group, and the difference was significant(P＜0.05).3) Comparison of VEGF mRNA expression level:comparing A and B group,VEGF mRNA expression level was not different(P＞0.05);VEGF mRNA expression in C group was significantly lower than B group,D group was significant higher than B and E group,the difference was significant(P＜0.05).4) Comparison of HIF-1αmRNA expression level:comparing A and B group, HIF-1αmRNA expression level was not different(P＞0.05);HIF-1αmRNA expression in B group was significant higher than C group,D group was higher than E group,the difference was significant(P＜0.05).(8) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αmRNA expression levels of Bewo cells1) Comparison of p53 mRNA expression level:comparing A group and B group,B group and C group,p53 mRNA expression level was not different(P＞0.05);p53 mRNA expression in the D group was significantly higher than B group,E group was significantly higher than D group,the difference was significant(P＜0.05).2) Comparison of MDM2 mRNA expression level:the MDM2 mRNA expression was not detected in the A group,B group and C group;MDM2 mRNA expression level in the E group was significantly higher than D group,the difference was significant(P＜0.05).3) Comparison of VEGF mRNA expression level:comparing A,B and C group, VEGF mRNA expression level was not different(P＞0.05);VEGF mRNA expression in D group was significantly higher than B and E group,the differences were significant(P＜0.05).4) Comparison of HIF-1αmRNA expression level:comparing A group and B group, HIF-1αmRNA expression level was not different(P＞0.05);HIF-1αmRNA expression in D group was significantly higher than E group,the difference was significant(P＜0.05).(9) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αprotein expression levels of JEG-3 cells1) Comparison of p53 protein expression:B group was higher than A group,but lower than C group and D group,and D group was higher than C group.2) Comparison of MDM2 protein expression:B group was higher than A group,but lower than C group and D group,and D group was higher than C group.3) Comparison of VEGF protein expression:B group was lower than A group,and the expression of C group was significant high,D group was lower than C group. 4) Comparison of HIF-1αprotein expression:A group and B group had faint expression,C group increased the expression slightly,D group was lower than C group.(10) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αprotein expression levels of Bewo cells1) Comparison of p53 protein expression:Comparing A and B group,the expression of p53 protein was not different,C group was higher than A and B group,D group was higher than C group.2) Comparison of MDM2 protein expression:Comparing A and B group,the expression of MDM2 protein was not different,but lower than C group,D group was higher than C group.3) Comparison of VEGF protein expression:the expression in A group and B group was not different,D group was lower than C group.4) Comparison of HIF-1αprotein expression:the expression in A group and B group was not different,,D group was lower than C group.Conclusions;(1) Agiogenesis in villous of missed abortion patients decreased obviously.(2) p53,MDM2 participated in the angiogenesis of early pregnancy villous,but it is disparate that p53,MDM2 played a role of facilitating villous angiogenesis in normal early pregnancy,nevertheless when the DNA damaged or hypoxia intensified,p53,MDM2 would overexpress and could decrease the angiogenesis of early pregnancy villous.(3)p53,MDM2 gene regulated the growth of villous trophoblast cells through the regulated HIF-1αand VEGF,ultimately influenced the pregnancy outcome.