The Mechanism Of LncRNA-ATB Regulating The Biological Behavior Of Trophoblast Cells

ObjectivePreeclampsia(PE)is a multi-system disease characterized by new onset hypertension and other important organ damage after 20 weeks of pregnancy.It is one of the main reasons for the increased mortality of pregnant women and perinatal infants.Severe preeclampsia(s PE)can affect many important organs and systems,resulting in placental abruption,fetal growth restriction,premature delivery,HELLP syndrome and other adverse outcomes.At present,although the pathogenesis of PE is not understood completely,it is recognized that insufficient epithelial mesenchymal transition of trophoblast and the remolding of spiral artery are the starting points of PE.Long noncoding RNA(lnc RNA)is a kind of noncoding RNA whose length is more than 200 nucleotides.In recent years,the important role of lnc RNA in epigenetic and transcriptomic regulation has been increasingly recognized.Previous studies of our group found that the lnc RNA-ATB levels in placenta of patients with s PE were significantly decreased,and the low lnc RNA-ATB level could inhibit the biological behaviors of HTR8/Svneo trophoblast,such as proliferation,migration,invasion and tube-formation.This project aims to further explore the possible mechanism and key molecules of lnc RNA-ATB in regulating the biological behavior of trophoblast,so as to enrich the pathophysiology of s PE.Research objects and methods1.The first chapterA total of 48 pregnant women(28 cases with normal pregnancy and 20 cases with s PE)who were referred to the obstetrics unit of West China Second Hospital of Sichuan University were selected in the study.Placental samples of above patients were collected.In vitro cytological study,HTR8/Svneo trophoblast cell line was selected as the research object.(1)The lnc RNA-ATB specific binding proteins in HTR8/Svneo trophoblasts were identified by RNA-protein pull-down test and protein mass spectrometry analysis,and the binding proteins with higher relative expression levels were selected according to i BAQ.(2)The stable overexpression plasmid pc DNA3.1-ATB was constructed and transfected into HTR8/Svneo trophoblasts,and then,Western blotting was used to test the changes of the selected binding proteins’ level.(3)After that,RT-PCR and Western blot were used to detect the expressions of the selected binding proteins in the placenta samples.2.The second chapter(1)The specific enrichment of lnc RNA-ATB in HTR8/Svneo trophoblasts by PABPC1,which had the highest relative expression of lnc RNA-ATB binding proteins in the first chapter,was verified by RNA immunoprecipitation.(2)si RNAs that specifically interfered with the expression of PABPC1 and its interacting proteins p53 and MDM2 were constructed.Western blotting was used to test the changes of p53 and MDM2 levels after transfecting si RNA-PABPC1 into HTR8/Svneo trophoblasts,the changes of PABPC1 and MDM2 levels after transfecting si RNA-p53 and the changes of PABPC1 and p53 levels after transfecting si RNA-MDM2.(3)The p53 and MDM2 levels were detected by Western blotting after HTR8/Svneo trophoblasts were transfected with pc DNA3.1-ATB plasmid.(4)The p53 and MDM2 levels in placentas were detected by Western blotting.(5)After that,si RNA-MDM2 was transfected into HTR8/Svneo trophoblasts to identify the effects of si RNA-MDM2 on the proliferation,apoptosis,migration,invasion and tube-formation of HTR8/Svneo trophoblasts.3.The third chapterThe effects of si RNA-MDM2 on angiogenesis and inflammation related factors were detected by enzyme-linked immunosorbent assay(ELISA)to explore the possible mechanism of MDM2 regulating the biological behavior of trophoblast.The results were analyzed by SPSS 21.0 software(IBM,USA).The data with normal distribution were expressed by means ± standard deviation((?)±SD)and analyzed by independent sample t test.The data with non-normal distribution were expressed by median(interquartile interval)and analyzed by nonparametric Mann Whitney U test.When p < 0.05(bilateral),the results were statistically significant.Results1.The first chapter: To explore the target protein of lnc RNA-ATB1)lnc RNA-ATB can specifically bind PABPC1,PABPC4,EEF1 G,KRT18 and ANXA2.2)Overexpression of lnc RNA-ATB can promote the expression of the above selected binding proteins in HTR8/Svneo trophoblasts.3)The selected binding proteins’ levels were decreased in s PE group compared to those in normal pregnancy group.In the s PE group,the levels of the selected proteins were not different between term delivery and pre-delivery.The selected proteins’ levels in term delivery of s PE group were decreased compared to those in normal pregnancy group.2.The second chapter: Lnc RNA-ATB regulates trophoblast biological behavior through PABPC1/p53/MDM2 pathway1)PABPC1 can specifically enrich lnc RNA-ATB in HTR8/Svneo trophoblasts.2)Inhibition of PABPC1 has no effect on the p53 level in HTR8/Svneo trophoblasts but downregulates the expression of MDM2.Inhibition of p53 can downregulate the expression of MDM2 but has no effect on the PABPC1 level.Inhibition of MDM2 can downregulate the expression of p53 but has no effect on the PABPC1 level.3)Overexpression of lnc RNA-ATB can upregulate the expression of p53 and MDM2 in HTR8/Svneo trophoblasts.4)The p53 and MDM2 protein levels were decreased in s PE group compared to those in normal pregnancy group.In the s PE group,the levels of p53 and MDM2 were not different between term delivery and pre-delivery.The p53 and MDM2 levels in term delivery of s PE group were decreased compared to those in normal pregnancy group.5)Compared to that in normal pregnancy group,p53/MDM2 ratio was increased in s PE group.6)In placental samples of s PE group,there was a positive correlation between PABPC1 and MDM2.The same positive correlation existed between p53 and MDM2 in placental samples of s PE group.7)si MDM2 can decrease proliferation,migration,invasion and tube-formation of HTR8/Svneo trophoblasts,and promote the trophoblasts apoptosis.3.The third chapter: si MDM2 can regulate the expression of angiogenesis and inflammation-related factors1)si MDM2 significantly reduced the concentrations of PLGF,VEGF,Eng,IL-8 and IL-1β in the supernatant of HTR8/Svneo trophoblast medium.Conclusion1.Lnc RNA-ATB can regulate the expression of PABPC1,PABPC4,EEF1 G,KRT18and ANXA2.The levels of these selected binding proteins in placental samples of s PE patients were significantly decreased,and the expression difference was not related to gestational age of delivery.2.Lnc RNA-ATB/PABPC1/p53/MDM2 pathway existed in HTR8/Svneo trophoblasts.3.The levels of p53 and MDM2 in placental samples of s PE patients were significantly decreased,and not affected by gestational age of delivery.si MDM2 can decrease proliferation,migration,invasion and tube-formation of HTR8/Svneo trophoblasts,and promote the trophoblasts apoptosis.Therefore,we infer that lnc RNA-ATB can regulate the biological behavior of trophoblast through PABPC1/p53 /MDM2 pathway.4.si MDM2 can downregulate the expression of PLGF,VEGF,Eng,IL-8 and IL-1β.We infer that lnc RNA-ATB can regulate the expression of angiogenesis and inflammation-related factors through PABPC1/p53 /MDM2 pathway.In conclusion,lnc RNA-ATB may play an important role in the pathogenesis of s PE through PABPC1/p53/MDM2 pathway.