Mechanism Of HMGB1-TRL4 Enhancing Invasion And Proliferation Of Extravillous Trophoblast Cell Lines

Objective 1、 To investigate the effect of HMGB1 on the invasion and proliferation of human trophoblast cell line HTR8 / SVneo.2、 To study the possible molecular mechanism of the effect of HMGB1 on the invasion and proliferation of extracellular trophoblast cells.Methods: HTR8 / SVneo cells are immortalized human placental extrachorionic trophoblastic cells,whose functions and phenotypes are similar to those of in vivo extrophoblast cells.The normal proliferation and differentiation of trophoblast is an important guarantee for placental formation and its physiological function.Because of its infinite passage characteristics,trophoblast is often used to study the relationship between drug or poison and pregnancy.Therefore,many scholars have used the cell line to study the adverse pregnancy outcome and gestational disease related mechanisms in vitro.1、 To study the effect of HMGB1 on the invasiveness of extracellular trophoblast,HTR8/SVneo was randomly divided into normal control group and 50μg/L,100μg/L and 200μg/L HMGB1 stimulation groups.The cells were cultured for 6 h,12 h,24 h and 48 h respectively.The invasiveness of HTR8/SVneo cells was detected by Transwell invasion assay in vitro.2、 To study the effect of HMGB1 on cell proliferation,cells were randomly divided into normal control and 50μg/L,100μg/L and 200μg/L HMGB1 stimulation groups.At 6 h,12 h,24 h and 48 h respectively,the absorbance of the cells with different stimulation concentration at 450 nm was measured at different time points,and the proliferation of HTR8/SVneo cells was detected by CCK8 method.3、 RT-PCR was used to detect the expression of NF-κ B p65 and MMP-9 at the m RNA level.Si RNAs were designed and constructed for the HMGB1 gene,and the expression of HMGB1 in HTR8/SVneo was silenced.They were divided into three groups: specific interference group(si RNA HMGB1 group),plasmid negative control group and blank control group.First,three groups of cells were cultured and transfected.Then the relative expression of TLR4,NF-κ B p65 and MMP-9 m RNA was detected by real-time fluorescence quantitative PCRQ(RT-PCR).4、 The expression of TRL4,NF-κ B p65 and MMP-9 related proteins were detected by Western blot.4、 For TLR4 gene,si RNAs were designed and constructed.By silencing the expression of TLR4 in HTR8/SVneo,the expression of NF-κ B p65 and MMP-9 was detected by RT-PCR method.Western blotting was used to detect the expression of NF-κ B p65 and MMP-9 related proteins.Results 1.The effect of HMGB1 on cell invasion: collecting cells at different time points and different concentrations of stimulation,counting the number of cells at different time points and different concentrations of stimulation.There was statistical difference between different time groups(P < 0.001),there was interaction between time and stimulation concentration(P < 0.001),and there was no significant difference between time and stimulation concentration(P < 0.001).2、 The effect of HMGB1 on cell proliferation: there was significant difference between different time factors(P < 0.001),and there was no interaction between time and treatment factors(P > 0.05).3、 Compared with the control group(normal control group and plasmid negative control group),the m RNA expression of TLR4,NF-κ B p65 and MMP-9 in HTR8 / SVneo cells was decreased after HMGB1 was silenced.The expression of TLR4,NF-κB p65 and MMP-9 protein was decreased in HTR8 / SVneo cells.The expression of TLR4,NF-κ B p65,MMP-9 m RNA,TLR4,NF-κ B p65 and MMP-9 m RNA protein increased significantly after HMGB1 stimulation.4、Compared with the control group(normal control group and plasmid negative control group),the expression of TLR4,NF-κ B p65 and MMP-9 m RNA in HTR8 / SVneo cells was significantly decreased after TLR4 silencing.There was no significant difference in the expression of TLR4,NF-κ B p65 and MMP-9 protein in HTR8 / SVneo cell line,and no significant difference was found in the expression of TLR4,NF-κ B p65 and MMP-9 protein.The m RNA expression of TLR4,NF-κ B p65 and MMP-9 increased after HMGB1 stimulation.The expression of TLR4,NF-κ B p65 and MMP-9 protein increased.Conclusion: Exogenous HMGB1 enhances the invasion and proliferation of HTR8/SVneo.HMGB1-TLR4 may affect the invasion and proliferation of HTR8/Svneo by regulating the transcription level of NF-κ B and MMP-9.HMGB1 promotes the proliferation and invasion of HTR8/SVneo.When TLR4 recognized the ligand HMGB1,which is a molecular model of inflammation and damage associated molecular pattern,the signal pathway was turned on.The myeloid differentiation factor 88 was dimerized and aggregated,and NF-κ B was activated into the nucleus.The production of a large number of inflammatory factors such as cytokines,chemokines,interleukin,causing abnormal placental vascular recasting,leading to the occurrence and development of pre-eclampsia,leading to various complications of pre-eclampsia mother and child and adverse perinatal outcomes.