Research On The Mechanism Of BPDE Inhibit Migration,Invasion And HR Repair Of Human Extravillous Trophoblastic Cells

Objective:Polycyclic aromatic compounds is the most common kind of persistent organic pollutants(POPs)environment,benzo[a]pyrene(BaP)as its representative,can cause a lot of bad women pregnancy outcome.The moderate invasion and migration in extravillous trophoblast were significantly reduced in many poor female reproductive outcomes,many studies have shown that BPDE,final metabolites of BaP,could negatively influence the ability of homologous recombination repair,but the underlying molecular mechanisms of both effacts are still unclear.In this paper,the influence of representative PAHs pollutant BaP and its metabolite BPDE on invasion and migration ability and DNA homologous recombination repair ability with underlying machanisms of extravillous trophoblast Swan 71 cells were studied,aiming to explore the causes of poor embryo development and pregnancy outcome in female reproductive toxicity studies for reference.Methods:1.The roles of BPDE in Swan 71 cell viability were measured by MTT assay.2.MTT assay was used to detect the effect of BPDE on cell proliferation.3.Wound assays and trans-well assays were performed to detect the influence of BPDE exposure,miRNA transfection,lncRNA transfection,and gene agonist treatment on invasion and migration of trophoblastic cells outside the chorionic membrane.4.The effects of BPDE exposure,miRNA transfection,lncRNA transfection,and gene agonist treatment on the migration ability of trophoblast cells outside the chorionic membrane were detected by scratch test.5.q-RT-PCR and western-blot experiments were used to detect the mRNA and protein expressions of invasion and migration pathway genes and homologous recombination repair pathway genes of the trophoblast cells outside the chorionic membrane after BPDE exposure,miRNA transfection,lncRNA transfection,and gene agonist treatment.6.The pseudopod function and nuclear damage of trophoblast cells after BPDE exposure were detected by transmission electron microscopy.7.The expression level and distribution of lncrna-HR in cells after BPDE exposure and BRCA1 overexpression were detected by RNA fluorescence in situ hybridization.8.Cellular immunofluorescence assays were used to detect the changes in the intracellular and nuclear expression levels of BRCA1,RAD51 and p-brca1 after BPDE exposure and after changes in the expression levels of lnc-HR.9.Direct interaction between homologous recombinant repair genes BRCA1,RAD51,p-brca1 and lnc-HR was detected by RIP assays.Results:Part I: influence and mechanism of BPDE on Swan 71 invasion and migration1.MTT results showed that BPDE exposure decreased cell viability of Swan 71 cells,and was dose-dependent with the concentration of BPDE(P*< 0.05).2.Wound assays and trans-well assays showed that BPDE could inhibit the migration and invasion ability of Swan 71 cells,and showed a dose-dependent relationship with BPDE(P**< 0.01).3.The results of transmission electron microscopy show that BPDE exposure can reduce the number and length of pseudopodia of Swan 71 cells.Moreover,the nucleus of Swan 71 will be significantly damaged,chromatin will be highly condensed and marginalized,and even cause nucleus disintegration,fragmentation and deformation.4.Western-blot and q-RT-PCR results showed that after the exposure of BPDE,the mRNA and protein levels of PI3K/AKT/CDC42/PAK1,the activation of this pathway activity could restore the invasion and migration function of BPDE on Swan 71 cells,indicating that BPDE could inhibit the invasion and migration function of Swan 71 cells by inhibiting the PI3K/AKT/CDC42/PAK1 pathway.5.High-throughput sequencing showed that BPDE exposure could induce significantly up-regulated expression of mir-194-3p in Swan 71 cells.Subsequent western-blot,q-RT-PCR,Wound assays and trans-well test results showed that BPDE-induced up-regulated mir-194-3p could reduce the mRNA and protein levels of PI3K/AKT/CDC42/PAK1,thus inhibiting the invasion and migration of Swan 71 cells.PartⅡ: the influence and mechanism of BPDE on Swan 71 homologous recombination repair6.Western-blot and cellular immunofluorescence results showed that lnc-HR could significantly up-regulate the protein expressions of the key genes BRCA1,p-brca1 and RAD51 in the homologous recombination repair pathway of Swan 71 cells,as well as the distribution of BRCA1 and RAD51 in the nucleus(p*<0.05,p**<0.01).7.High-throughput sequencing,q-RT-PCR and RNA FISH experiments have shown that BPDE exposure can reduce the expression of lnc-HR in Swan 71 cells and reduce its distribution in the nucleus(P**< 0.01).8.The results of comet electrophoresis experiment,transmission electron microscopy experiment and immunofluorescence experiment showed that: BPDE Swan 71 cells can be induced to double-stranded DNA fracture(P*<0.05,P**<0.01),and the protein expression of MRE11/RAD50/NBS1/CtIP/BRCA1/ were reduced,but total protein expression RAD51 increased with BPDE concentration increased,but the foci formation in the nucleus was significantly reduced.These results indicate that BPDE can significantly inhibit homologous recombination repair of Swan 71 cells.9.The results of cellular immunofluorescence and RIP assays showed that lnc-HR could directly bind to p-BRCA1 and RAD51,BPDE could weaken lnc-HR’s binding ability to them.Overexpression of lnc-HR can restore the expression of p-BRCA1 and RAD51 both in the cytoplasm and the nucleus,thus reduce the formation of DNA double-strand breakage caused by BPDE exposure.Conclusion:We found that BPDE can inhibit the invasion and migration ability of Swan 71 cells.The potential mechanism is as follows: BPDE induces the up-regulation of mir-194-3p expression in Swan 71 cells,then the up-regulated mir-194-3p inhibits the invasion and migration related pathway PI3K/AKT/CDC42/PAK1,thus reducing the invasion and migration ability of Swan 71 cells.In addition,BPDE can inhibit the repair ability of homologous recombination responsing to DSB.BPDE can reduce the mRNA expression level of lncRNA-lnc-HR.The reduced lnc-HR can influence the directly bingding between lnc-HR with the recombination genes BRCA1 and RAD51,thus weaken their stability and foci formation in the cell nucleus so that DSB accumulates obviously.In this study,the effect of BPDE on the invasion,migration and HR repair of trophoblast Swan 71 cells was observed,and the underlying molecular mechanisms were revealed,providing ideas for exploring the causes of poor embryo development and adverse pregnancy outcome in female reproductive toxicity studies.