TP53, CDKN1A And BAX Expression In The Placental Villi From Recurrent Miscarriage

Objective: Recurrent miscarriage(RM) is a disease with complex causesand seriously jeopardizes family happiness. The etiology of RM iscomplicated, which includes Genetic defects, anatomical anomalies, endocrinedisorders, immune factors, infection, thrombosis, and environmental factors, etal. At present more than50%of the patients can’t find the pathogenesis. TP53gene may induce cell growth retardation, apoptosis, cell differentiation andDNA repair, etc. However, apoptosis is the basis of spontaneous abortion. Thevarious function of TP53gene mainly through the activation or inhibit a largenumber of downstream genes to finished. TP53gene mediated its downstreamgene including BAX and CDKN1A gene, and both of them are importantsignaling pathways of signaling pathway. TP53protein may mediateexpression of the downstream CDKN1A gene, then the cells stagnation in theG1phase, and induce cell apoptosis finally. BAX gene has the function ofpromoting apoptosis, and start the related mitochondrial apoptosis pathway bythe TP53gene activation directly. Therefore we choose chorionic villus ofnormal early pregnancy and unexplained spontaneous abortion as researchobjects. Then detection villi tissue cell apoptosis and regulatory gene TP53gene and its downstream gene CDKN1A, BAX gene expression. To discussthe mechanism of action that apoptosis related protein TP53gene and itsdownstream gene CDKN1A, BAX gene in RM.Methods: Thirty women with recurring miscarriages were enrolled asexperimental group, and thirty women with normal reproduction served ascontrol group. All patients were positive for urinary hCG. All chorionic villusspecimens were obtained by vacuum aspiration. Each specimen was dividedinto two parts. One part was flash-frozen in liquid nitrogen and used for PCRas described below. Another part was fixed with10%formalin overnight at room temperature and paraffin embedded. Slices of5μm were prepared,mounted on glass slides with APES, and used for immunohistochemicalstaining.Detection of TP53geneDewaxed tissue slice by conventional immunohistochemistry methods.TP53antibody purchased combined with the associated antigens within anorganization,4℃overnight. Horseradish peroxidase combine2hours at roomtemperature, DAB color development for10minutes. Neutral balsammounted after hematoxylin staining the nuclei. Double blind method analysisof positive expression rate of TP53gene. TP53in the nuclei are claybankparticle as positive. Select3field(×200) of each sample to take pictures,select villi tissue area, using gray scale Image j software measurement, eachwith3slices of the average gray scale values for statistical processing.Detection of CDKN1A, BAX mRNAUsing real time quantitative PCR method for CDKN1A and BAX mRNAexpression detection. Villi tissue total RNA extraction is as follows: ground byvilli of Cryopreserved in liquid nitrogen by liquid nitrogen,100mgorganization1mL Trizol, take500μl suspension add200μl mix chloroformcentrifugal15min back12000rpm.Supernatant and mix equal volumeisopropanol12000rpm after centrifugation15min,75%washing with ethanolprecipitation, dry precipitation in water dissolved in50μl RNase-free. Firstchains of DNA synthesis by reverse transcription Kit (Fast Quant RT Kit)instructions for reverse transcriptase. SYBR Premix Ex Taq used for thepreparation of RT-PCR reaction system. Response procedures: predegenerated:95℃,30sec;PCR amplification:95℃,30sec;60℃,30sec；84℃,1sec；40cycles,84℃fluorescence is read.2-△Ctmethod to calculate CDKN1A andrelative expression of BAX mRNA in villi tissue.Detection of apoptosisUsing TUNEL(terminal-deoxynucleotidyl transferase mediated nick endlabeling) to detect the Villi tissue cells and experimental process according tothe DeadEnd Fluorometric TUNEL System kit instructions. Finally using SlowFade Gold Antifade Reagent with DAPI to mounting the sample.Under fluorescence microscopy, DAPI recognize all the nucleus, greenfluorescent particles within the nucleus is positive cells, apoptotic cells.Eachsample individually at520nm (green TUNEL) and430nm (blue DAPI)wavelength pictures, and merged using Photoshop CS5. Analysis offluorescence ratio was done using Image-Pro Plus6.0.Results:1Expression of TP53gene in Villi tissueImmunohistochemistry results show that the expression of TP53gene inthe cell nucleus mainly and that is brown or tan particles. TP53proteinexpressed in villi tissue in patients with recurrent miscarriage of significantlyhigher than normal villi tissue. Expressive situation has been evaluatedpositively from the TP53protein. Between the control group and theexperimental group’s villus tissue specimens.The average gray level of thecontrol group were significantly higher than the experimental group in villitissue, after comparison P=0.000, with statistical significance(P<0.05).2Expression of CDKN1A and BAX mRNA in villi tissueThe results showed that compared with the normal group, the mRNAlevel of CDKN1A gene in the experimental group were significantly higherthan the control group in villi tissue, and P=0.015, the results were deemedstatistically significant (P<0.05). BAX mRNA level in the experimental groupwere significantly higher than the control group in villi tissue, and P=0.019,the results were deemed statistically significant (P<0.05).3Apoptosis of chorionic villusTUNEL detection showed that apoptotic cells Appeard in trophoblastcells of chorionic villus tissues in both of the control group and theexperimental group, but there were a smaller number of apoptotic cells in thecontrol group and a greater number of apoptotic cells in the experimentalgroup, and P=0.024, the results were deemed statistically significant (P<0.05).Conclusions: TP53induced its downstream genes CDKN1A and BAXabnormal expression may play an important role in the course of RM. Thus, the apoptosis pathways controlled by TP53may be involved in thepathogenesis of RM.