Study On The Role Of LncRNA TUG1 In The Pathogenesis Of Preeclampsia By Regulating The Proliferation Of Trophoblastic Cells

Preeclampsia(PE)is a special disease in pregnancy,with elevated blood pressure as the main clinical manifestation.PE is a placental disease,and its pathogenesis is unknown.Trophoblastic dysfunction caused by uterine spiral artery remodeling is one of the important pathogenesis.The biological behavior regulation of trophoblast involves various factors.In this paper,we studied the role of long non-coding RNA(Lnc RNA)in the process of uterine spiral artery recasting by regulating the biological behavior of trophoblastic cells.And we focused on the biological behavior patterns of trophoblasts regulated by long noncoding RNA TUG1 and its molecular mechanism.This paper is divided into two parts to explore the correlation between TUG1 and preeclampsia.The first part is to clarify the role of TUG1 in preeclampsia,leading to abnormalities in the biological behavior of trophoblast cells.Eventually cause spiral artery recasting.In this study,we first detected the expression of TUG1 in placenta of preeclampsia.The results showed that TUG1 in preeclampsia placenta was significantly lower than that in normal pregnancy.Then we study the biological behavior of trophoblastic cells by constructing high expression and low expression TUG1 cell models in vitro trophoblast cell lines.It is concluded that TUG1 can promote the proliferation,migration,invasion and angiogenesis of trophoblastic cells.In addition to clarifying its biological effects,we have carried out the second part of the study.That is,to explore the molecular mechanism of the role of TUG1 in trophoblasts.We have adopted nucleoplasmic separation RNAimmunoprecipitation technique,chromatin immunoprecipitation technique,combined with second-generation high-throughput sequencing,bioinformatics analysis,and reached a conclusion.We found that TUG1 can bind ezh2 to inhibit the expression of RND3 and maintain the proliferative activity of trophoblast in normal physiological process.But with the development of preeclampsia,the expression of TUG1 is down-regulated.The above mechanism was broken down,the inhibition of RND3 was removed,the cells were damaged,and the reconstruction of spiral artery was obstructed.These results confirm that the long noncoding RNA TUG1 is involved in the development of preeclampsia.And it’s by regulating the trophoblast.The results suggest that the long noncoding RNATUG1 may be a new target for preeclampsia therapy and provide a new idea for preeclampsia early pregnancy prediction and intervention.Objective: 1.To determine the expression of lnc RNA TUG1 in placenta of preeclampsia pregnant women as compared with normal pregnant women.2.To clarify the effect of long noncoding RNA TUG1 on the proliferation of trophoblast biological behavior.3.To clarify the effect of long noncoding RNA TUG1 on the biological behavior migration and invasion of trophoblastic cells.4.To determine the effect of long noncoding RNA TUG1 on the angiogenesis of trophoblast biological behavior.Methods: 1.The placental tissues of 52 pairs of pregnant women with preeclampsia and normal pregnancy were collected.The expression of TUG1 was detected by PCR,and the clinical data were analyzed.2.The low expression model of trophoblastic HTR-8 / v neo,JEG3 and human umbilical vein endothelial cell(HUVEC)transfected with small interfering RNA was used to verify the transfection efficiency.3.Trophoblast HTR-8 / v neo,JEG3 knockout TUG1,clone formation assay and edu proliferation assay to clarify the regulation of TUG1 on trophoblast proliferation.4.Trophoblast HTR-8 / v neo,JEG3 knockout low TUG1 after transwell experiment to clarify the regulation of TUG1 on trophoblastic invasion and migration5.Observe the number of reticular formation after knocking down the TUG1 in HUVEC 6.Trophoblast HTR-8 / vneo,JEG3 knockout TUG1 to clarify the regulation of TUG1 on trophoblastic life processes.Results: 1.The results of placental tissue showed that the expression of TUG1 in preeclampsia placenta was lower than that in normal pregnancy group.There were no statistical differences in smoking and age between the two groups,but there were significant differences in proteinuria,blood pressure and birth weight between the two groups.2.The expression of TUG1 was changed successfully in trophoblast and umbilical vein endothelial cells,and the transfection efficiency was improved.3.The results of clone formation assay,MTT and edu proliferation assay showed that the proliferative activity of trophoblastic cells decreased under the condition of low expression of TUG1.4.The results of transwell assay showed that the invasion and migration of trophoblastic cells decreased under the condition of low expression of TUG1.5.The results of umbilical vein endothelial cell formation experiment showed that under the condition of low expression of TUG1,the ability of angiogenesis decreased.6.The cycle detection of TUG1 showed that the cycle of trophoblastic cells stagnated under the condition of low expression of TUG1,and the apoptosis level of trophoblastic cells increased under the condition of low expression of TUG1,and the apoptosis level of trophoblastic cells was increased by flow cytometry.Conclusion:Long noncoding rna TUG1 can participate in the development of preeclampsia by regulating the biological behavior of trophoblast.The regulation of trophoblast is to promote trophoblast proliferation,trophoblast invasion and migration,angiogenesis and inhibition of apoptosis.Objective: 1.To determine the localization of long noncoding RNA TUG1 in cells.2.To identify the type of genes regulated by long chain non-coding rna TUG1 in trophoblastic cells 3.To identify the most prominent target genes regulated by long chain non coding rna TUG1 in trophoblastic cells 4.To clarify the pattern of long noncoding RNA TUG1 regulating genes in trophoblastic cells.5.Clarify the function of the target gene and verify Methods: 1.the dominant localization of TUG1 was observed by Nuclear and cytoplasmic separation experiment.2.generation high throughput RNA sequencing identified TUG1 downstream controlled gene class 3.analyze the sequencing data to find the most likely effect gene in the TUG1 downstream,that is,the target gene 4.predict how TUG1 regulates the target gene and rip to verify its binding protein 5.Predict how TUG1 regulates target genes and verify its mechanism by chip 6.Determine whether the expression of the target gene in the placental tissue of preeclampsia is consistent with the sequencing results 7.Verify the function of target gene in trophoblast is consistent with TUG1.8.the rescue experiment verifies the correctness of the target gene again.Results: 1.The results of nuclear and cytoplasmic separation experiments suggest that TUG1 is expressed in the nucleus preferentially.2.The TUG1 binding protein was predicted by the database.The rip showed that the binding kurtosis of Ezh2 was obvious.3.Analysis of the second generation of high-throughput sequencing results the analysis of TUG1 downstream controlled genes are mostly related to value-added migration 4.Analyzehigh throughput sequencing results by the second time,look for the most likely target gene: RND3,and verify it again 5.The results of chip experiment showed that ezh2 and RND3 promoter had binding peak and changed with the change of TUG1 expression.6.To verify the expression of RND3 in placenta of preeclampsia,it was found that RND3 was significantly overexpressed in placenta of preeclampsia.7.RND3 function test showed that it could promote cell apoptosis,arrest cell cycle and inhibit cell proliferation and migration.Conclusion: TUG1 inhibits RND3 expression by binding ezh2 to ensure RND3 promoter methylation level.This process was broken down during preeclampsia because of reduced expression of TUG1.The up-regulated expression of RND3 affects the invasion and migration of trophoblastic cells,resulting in excessive apoptosis of the cells,affecting the recasting of spiral arteries and promoting preeclampsia.