The Roles And Regulatory Mechanism Of A Novel LncRNA-31515 In BPDE-induced Recurrent Miscarriage By Inhibition Of Trophoblast Cell Invasion And Migration

Objective: Polycyclic aromatic hydrocarbons are persistent organic pollutants in our widespread environment.Previous studies have shown that polycyclic aromatic hydrocarbons may cause miscarriage,but epidemiological studies of risk factors for miscarriage have limitations due to small sample size or single factor.A representative of polycyclic aromatic hydrocarbons(PAHs),benzo[a]pyrene(Ba P)and its metabolite BPDE,cause abortion by inhibiting invasion and migration of trophoblast cells.However,the underlying mechanism remains largely unknown.This paper conducts a meta-analysis of the risk factors of abortion,expecting to comprehensively and systematically investigate the main risk factors of abortion.It was found that exposure to polycyclic aromatic hydrocarbons is one of the most important risk factors that lead to abortion.According to the meta-analysis results,the typical representative of polycyclic aromatic hydrocarbons,benzo[a]pyrene,and its metabolite BPDE,were further studied at the levels of cell,human tissue,and animal model.We want to know(1)the harmful effects of benzo[a]pyrene and its metabolite BPDE on abortion;(2)the regulatory mechanism of lnc RNA 31515 in Ba P/BPDE-induced miscarriage by inhibiting invasion and migration of trophoblast cells.It is expected to discover biomarkers of recurrent abortion,and to provide theoretical basis for early diagnosis and clinical treatment of abortion Part 1: Meta analysis of risk factors for abortionMethods: The research on the risk factors of miscarriage published in China How Net,Wanfang Medical Net,Weipu Journal,Pub Med,Springer and other databases was searched from construction to October 2019.Meta-analysis was performed on the data using Revman5.3 and Stata12.0 software.Results: After clear criteria for inclusion and exclusion,the final number of included articles was 18,and the meta-analysis results showed that the combined OR value of each influencing factor and their 95% CI value were finally: smoking(OR = 2.86,95% CI: 2.40-3.40),polycyclic aromatic hydrocarbons Exposure(OR = 1.33,95% CI: 0.97-1.83),living environment(OR = 2.05,95% CI: 1.56-2.70),radiation(OR = 2.94,95% CI: 2.21-3.90),drinking(OR = 1.99,95% CI: 1.45-2.72).Conclusion: The results suggest that smoking,PAH exposure,living in nearby cities or traffic roads,radiation,and drinking are all risk factors for miscarriage.Among them,high-risk smoking and nearby living environment contain PAH exposure,suggesting that there are many factors that cause miscarriage,and PAHs account for a large part of these risk factors.This Meta analysis laid a statistical foundation for studies on the toxic effects of PAHs on miscarriage.Avoiding exposure to PAHs could reduce the risk of miscarriage.Part 2: Mechanism of a novel lnc RNA-31515 in BPDE-induced abortion by Inhibiting Invasion and Migration of Trophoblast CellsMethods:(1)High-throughput sequencing was used to detect differentially expressed m RNA and lnc RNA in the tissues of aborted people and trophoblast cells after BPDE treatment.(2)MTS cell viability test,flow cytometry,and Trans-well test were used to determine the effects of lnc RNA regulation on trophoblast cell viability,apoptosis,and invasion and migration.(3)Q-RT-PCR and Western-Blot test methods were used to determine the m RNA and protein levels of invasion and migration-related genes in BPDE exposure,transfected si RNA / plasmid,unexplained abortion population,voluntary abortion population,and Ba P exposed mouse tissues.(4)The position and expression of lnc31515,EGR1 and NF-κB in cells before and after BPDE exposure were detected by immunofluorescence technology.(5)Co-immunoprecipitation test was used to detect the interaction between EGR1 and NF-κB genes.(6)RNA immunocoprecipitation experiment was used to detect the interaction between lnc31515 and EGR1 gene.Results:(1)Through cell and animal experiments,it was found that Ba P and BPDE inhibited invasion,migration and proliferation of trophoblast cells,promoted apoptotic function,and increased the abortion rate of C57 BL /6 mice.(2)Trans-well,q-RT-PCR,Western-Blot and other methods were found in cells and human tissues,and the new lnc RNA-lnc31515 played an important role in the process of abortion caused by BPDE.(3)Trans-well from the human tissue also found that the invasion and migration of villous trophoblast cells in tissues of patients with recurrent abortion was significantly reduced.(4)Through Western-Blot,RNA immunoprecipitation,co-immunoprecipitation and other cell experiments,it was found that lnc31515 inhibits the interaction between EGR1 and NF-κB by combining with EGR1,and further reduces the invasion and migration ability by reducing the activation capacity of CXCL12.The final outcome is a miscarriage occurred.Conclusion: The research on human trophoblast cells,human abortion tissue,and mice model with Ba P-induced abortion shows that:(1)Ba P and BPDE have harmful effects on abortion,leading to an increase in abortion rate;(2)a new lnc RNA-lnc31515 inhibits the binding of EGR1 to NF-κB,and then reduce the activation of CXCL12,which further reduces the invasion and migration of trophoblast cells and triggers abortion;(3)lnc31515 might be used as a biomarker for miscarriage,providing theoretical basis for early diagnosis and clinical treatment.Full text conclusion In this paper,meta-statistical analysis and experiments with cell,human tissue,and mice model have shown that Ba P and its metabolite BPDE can cause miscarriage and have toxic effects on female reproduction.Mechanismtically,lnc RNA-31515 reduces the activation of CXCL12 by inhibiting the binding of EGR1 to NF-κB.This inhibited EGR1/NF-κB/CXCL12 pathway further weakens the invasion and migration of trophoblast cells and induces abortion after exposure of Ba P or BPDE.This work provides theoretical basis for early prevention,early diagnosis,and clinical treatment of abortion.