Preliminary Study The Mechanism Of BPDE Induced Trophoblast Cell Cycle Arrest.

Objective:In order to understand the effect of BPDE on the cell cycle function in the trophoblast cell and its molecular mechanism,studying the mechanism of novelmir-105 regulating cell cycle.Methods:1.First we resuscitate the human trophoblast cell Swan 71 cell line and add BPDE culture in medium,then build the virus model.It was divided into five groups,one of which was the control group,and the other four groups were the concentration of 0.25μmol/L.0.5μmol/L,1.0μmol/L,and 1.5μmol/L.After 24h cultivation.MTT assay was used to detect the effect of BPDE on cell growth vitality,and flow cytometry was used to detect the cell cycle and the changes of apoptosis.The detection of comet assay with BPDE was used to treat double strand fracture of DNA.By using PCR and Western Blot assay to detect the difference expression of mRNA and protein in cell cycle which are correlation factor AKT.FoxO3a,P27,P21,CDK2 and ATM.chK1,cdc25A.Next we use high-throughput sequencing technology detect the difference expression miRNA of Swan71 cells and bioinfonnatics analysis was carried out by using DAVID software on the target genes of miRNA.The relationship between novel-mir-105 and the downstream target gene FoxO3a was detected by a double luciferase reporter.Data were analyzed with SPSS 17.0 and*P<0.05 was statistically significant.Results:1.The growth of trophoblast cells affected by BPDE.through using enzyme standard instrument detect the dosing group(0.25 umol/L,0.5 umolL,1.0 umol/L,1.5umol/L,2.0 umol/L,4.0 umoI/L)absorbance,compared with the control group,we observed that the absorbance significantly decreased and growth significantly inhibited.*P<0.05.2.BPDE can inhibit the cell cycle S phase of the extracellular trophoblastic cell cycle of the chorionic membrane,after 24 hours of infection,it will be fixed for 12 hours the flow cytometry will detect a decrease in G1 phase,increase in S phase,and no obvious changes in G2 phase.*P<0.05.3.Flow cytometry detection BPDE caused apoptosis of trophoblast cells,there was a significant change in apoptotic cells at the concentration of 1.0 μmol/L and 1.5μmol/L,*P<0.05.4.We used the comet assay to detect DNA damage,it turn out that BPDE increased DNA fragmentation in trophoblast cells.After the process of electrophoresis,the DNA was stained with PI and we found the DNA was towing.5.BPDE led to the down-regulation of cell protein AKT,ATM up-regulation,while it could increased expression of Foxo3a,P27 and P21 and decreased CDK2,BPDE inhibited the cell cycle of trophoblast cell Swan71 cells through AKT/FoxO3a/P27/P21/CDK2 pathway.6.The BPDE treatment induced the increase in 44 kinds of the differences of known miRNA differences in Swan 71 cells,and the reduction 55 kinds of differences of known miRNA differences;six kinds of novel-miRNA differences were up-regulated and 20 kinds of novel-miRNA differences were down-regulated.The novel-mir-105 was involved in the regulation of BPDE on Swan71 cell cycle.Transfecting the mimics or inhibitors could affect the cell cycle of trophoblast cells.7.The double-luciferase reporter gene experiment showed that the novelmir-105 could regulate the cell cycle by regulating the downstream gene FoxO3a.Conclusion:1.We successfully constructed the acute infection model through infecting trophoblast cell 24h by BPDE.2.BPDE has a toxic effect on trophoblast cells.BPDE can induce cell cycle arrest and apoptosis in the trophoblast cell Swan 71,block in S phase,and change the periodic protein.3.BPDE causes DNA damage in trophoblast cells,causing DNA breakage.4.At the same time,BPDE can induce the decline of novel-mir-105.transfection of novel-mir-105 mimics can reduce DNA damage,and transfecting of novel-mir-105 inhibitor aggravate DNA damage.5.Novel-mir-105 can affect cell cycle by regulating gene FoxO3a,the novelmir-105 mimics can reduce cycle arrest and DNA damage,which has certain guiding significance for the clinical treatment of cell damage caused by BPDE.