The Relationship Between The Expression Of PAPD4 And The Invasiveness Of Trophoblasts In Placental Accreta Spectrum

During pregnancy,a portion of placental cells called villous trophoblast cells(CTBs)differentiate and gain the ability to invade the uterus and its vasculature [In contrast,CTB opens up the ends of the uterine veins,which passively expand to accommodate increasing blood flow back into the maternal circulation.In most chorionic villi of early pregnancy,CTB forms a polarized monolayer that attaches to the base of the trophoblastic basement membrane and to the apex of the fusion trophoblastic layer that is in direct contact with maternal blood.However,in many locations,usually near the tip of the chorionic membrane,the rejection mechanism promotes the attachment of the CTB from the placenta to the cell lines on the surface of the uterine cavity.These cell lines guide the passage of the CTB subpopulation,which usually restricts the advancing CTB to the medial third of the myometrium.In 2018,the International Federation of Obstetrics and Gynaecology collectively referred to different types of Placenta implantation disorders as Placenta accrete spectrum disorders PAS.PAS was first described in 1937,when cesarean sections were becoming more common.Epidemiological studies have identified prior uterine surgery,of which C-section is the most common type,as the greatest risk factor for developing PAS.In this case,invasive CTB follows the scar and can enter deeper parts of the uterus that would normally not be removed.As C-section rates have risen,the number of PAS cases has increased simultaneously,from1:30,000 pregnancies in 1950 to 1:71 recently.Since the mechanism of PAS is unclear,it is interesting to explore the mechanism of this very harmful disease.Polyadenylate RNA polymerase D4(poly(A)RNA polymerase D4,PAPD4)(also known as TENT2)is an RNA polyadenylate enzyme that regulates the length of the terminal poly(A)of RNA by co-forming RNA posttranscriptional modified complexes with other proteins.And then control the stability and translation activity of RNA.The mechanism of PAPD4 regulating cell invasion is very complex.Maxwell Burroughs et al.showed that Ataxin-2 formed a complex with PAPD4 and PABPC1 to induce polyadenylation of its target m RNA.Therefore,they investigated whether DDX6 was involved in the functional Ataxin-2 polyadenylate complex.When 5 Flag-DDX6 and 5 MycPAPD4 HEK293 T cell lysates were expressed by immunoprecipitation with anti-Flag antibody,Five Flag-DDX6 were co-precipitated with five Myc PAPD4 and endogenous PABPC1 and Ataxin-2.As PAPD4 decreases,many mi RNA genes are also affected,and possibly m RNA transcripts themselves.Some experiments have observed that m RNA target expression has a significant decrease trend after PAPD4 knockdown.Previous experiments have shown that m RNA target expression of mi R-122 is increased after PAPD4 knockout in mouse liver.This may indicate that PAPD4 is active against the precursor mi RNA 39 terminal in addition to acting on mi RNA.The present study only found up-regulation of PAPD4 in PAS.Therefore,we thought:Can the up-regulation of PAPD4 regulate the invasion of placental trophoblast? This study was to investigate whether up-regulation of PAPD4 can regulate the invasion of placental trophoblast.research method1 Groups are divided into Control group and PAPD4 group.Control group: cells were not treated;PAPD4 group: Cells were transfected with PAPD4.The rest of the operations are the same for both groups.2.HTR8/SVneo cells were transfected with sh RNA and transfected with transfection reagent.HTR8/SVneo cells were incubated at 37℃ and cultured in complete medium for 24 h.The invasion test was conducted by Transwell coating the upper compartment membrane with diluted matrix glue.The cells were counted by images obtained through an inverted microscope.Five fields were randomly selected for counting and the average number of cell invasion was calculated.3.In cell scratch experiment,HTR8/SVneo cells were used to plate and transfect 6-well plates,and 3 replicates were performed in each group.When the cell confluence reached 80%～ 90% at 37 ℃ and 50 m L /LCO2,scratches were performed in the well plates with 200 u L gun head in the vertical direction,and the superneant was removed.The gaps between cells were washed gently with PBS,and then complete culture medium was added,and the culture was continued at 37 ℃.Photos were taken of the gaps between cells in each group at 0h and24 h,respectively.4.The tissue blocks of placenta samples were washed with pre-cooled PBS for 2-3 times by Western blot.The protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to cellulose nitrate membrane,which was then exposed with PAPD4 antibody.Finally,ECL developer was used for exposure and the film was scanned and archival.Photo Shop removes the color,and the Alpha software processing system analyzes the optical density value of the target strip.5.Trizol method was used to extract tissue blocks of placenta samples and RNA from trophoblast cells cultured with DMEM/F12.The purity and concentration of RNA were measured.c DNA was obtained by reverse transcription and real-time fluorescence quantitative PCR was performed.GAPDH was used as internal reference,and △△Ct method was used for data analysis.6.Statistical analysis was performed using SPSS 26.0 and Graphp Prism 8 software for statistical analysis of experimental results,and measurement data were expressed as x ± s.t test was used to analyze the significance of measurement data,and P < 0.05 was considered statistically significant.ResultScratch test results showed that the scratch healing rate of PAPD4 group[(80.57±9.21)%],the scratch healing rate of Control group [(22.34±4.12)%],the Control group was significantly lower(P < 0.01).There were 181.06 ± 5.32 cells invaded in PAPD4 group and 72.04 ± 3.38 cells invaded in Control group,which was significantly less in Control group(P < 0.01).Due to limited experimental time,there were only 4 cases in each group,which could not be processed statistically.However,PAPD4 protein in placenta showed an increasing trend.ConclusionIt was preliminarily proved that PAPD4 can regulate the invasion force of placental trophoblast through scratch experiment and cell invasion experiment.It is possible to reduce the incidence of PAS by regulating PAPD4.Of course,regulating PAPD4 is not easy to achieve,because it is not clear how much PAPD4 is optimal.This needs further study.