The Roles And Mechanisms Of A Novel LncRNA-HZ09 In BPDE-inhibited Invasion And Migration Of Female Trophoblast Cells And The Occurrence Of Miscarriage

Objective: Benzo(a)pyrene(BaP)is a widespread environmental pollutant,which has been detected out from our environment,including food and drinking water.It shows carcinogenic,teratogenic and mutagenic effects.Its reproductive toxicity has gradually become a research hotspot.However,the studies on the effects of BaP and its metabolites on female trophoblast cells and the occurrence of miscarriage are still not enough.Therefore,the purpose of this thesis is to reveal the relationship between BPDE-induced dysfunctions of female trophoblast cells and the occurrence of miscarriage,to find out and identify new lnc RNAs and their regulatory signaling pathways,to explore the regulatory roles of lnc RNAs in the process of BPDE-induced miscarriage,to reveal the regulatory mechanism of miscarriage induced by polycyclic aromatic hydrocarbons,and also to provide scientific understanding in the occurrence of miscarriage.Method: Part 1: Identification of a New Lnc RNA(1)Using high-throughput sequencing technology,transcriptome in BPDE-exposed trophoblast cells was sequenced and the differentially expressed lnc RNAs were identified.(2)Using RACE assays,the full length of lnc-HZ09 was identified;the subcellular location of lnc-HZ09 was detected by FISH experiments.(3)lnc-HZ09,PLD1 or Hu R was knocked down or overexpressed in trophoblast cells.The invasion and migration were detected using transwell assays.Part 2: The regulation mechanisms of lnc-HZ09 in BPDE-inhibited invasion and migration of trophoblast cells(1)The expression of lnc-HZ09 and genes in this pathway was detected using RT-q PCR and Western blot assays.(2)The interactions among lnc-HZ09,PLD1 m RNA and Hu R were detected using RIP and RNA pull-down assays.M6 A RNA modification in lnc-HZ09 was detected using Me RIP experiments.(3)We used Ch IP experiments to detect the binding between PLD1 promoter region and transcription factor SP1,as well as lnc-HZ09 promoter region and transcription factor MSX1.(4)We used RT-q PCR and Western blot assays to detect lnc-HZ09 level and other gene levels in pathway in RM tissues.We used Ch IP experiment and Me RIP experiment to detect the binding of transcription factors to the promoter region and m6 A methylation modification,respectively;(5)The migration and invasion ability was detected in BPDE-treated trophoblast cells in transwell assays.The expression levels of lnc-HZ09 and other genes in this pathway were detected by RT-q PCR and Western blot experiments;we used Ch IP experiment and Me RIP experiment to detect the binding of transcription factors to the promoter region,and to detect the level of m6 A methylation modification.Part 3: Construction of an Animal Model A mouse miscarriage model was constructed by intragastric administration of 0,0.05,or 0.2 mg/kg BaP.RT-q PCR and Western blot experiments were used to detect the changes in genes in the related pathways in a random placenta of miscarried mice.Results Part 1: Identification of the novel lnc-HZ09 We have identified a novel lnc RNA(lnc-HZ09),which is differentially highly expressed after BPDE exposure.It is non-coding RNA and has a length of 366 nt.It is distributed to both the cytoplasm and the nucleus,and is mainly distributed to the cytoplasm.Lnc-HZ09 can obviously inhibit the invasion and migration of human trophoblast cells.Part 2: Regulation mechanisms of lnc-HZ09 in BPDE-inhibited invasion and migration of trophoblast cells In trophoblast cells,PLD1 promotes the expression of its downstream genes RAC1 and CDC42 and then promote cell invasion and migration.Compared with the HC group,the expression levels of PLD1,RAC1 and CDC42 in the RM tissues are relatively lower;whereas those of lnc-HZ09 are relatively higher.SP1,as a transcription factor of PLD1,can initiate the transcription of PLD1.RNA binding protein Hu R can bind to PLD1 m RNA and maintain its RNA stability.Lnc-HZ09 inhibits SP1 expression and competes with PLD1 m RNA to bind with Hu R.MSX1 acts as lnc-HZ09 transcription factor and promotes lnc-HZ09 transcription.Additionally,there are m6 A RNA methylation modification on lnc-HZ09.Methyltransferase METTL3 can promote the m6 A RNA methylation on lnc-HZ09 and increase its RNA stability.In BPDE-exposed human trophoblast cells,lnc-HZ09 shows positive regulation on PLD1 expression,same as that found in the untreated cells.BPDE up-regulates the expression of MSX1 and METTL3,increases lnc-HZ09 RNA stability,and reduces PLD1 m RNA stability.Compared with the HC group,the expression levels of SP1 in RM tissue are lower;whereas those of MSX1 and METTL3 are relatively higher.Moreover,the expression levels of lnc-HZ09 show a significant negative correlation with the expression levels of PLD1,RAC1 and CDC42 in the RM tissues.Part 3: Construction of Animal Model Lnc-HZ09 has no homology in mouse tissues.All of SP1,PLD1,RAC1 and CDC42 have homology in mice.Compared with the control group,the expression levels of SP1,PLD1,RAC1 and CDC42 in the miscarried mouse placental tissues after BaP exposure are obviously reduced.Conclusion In human trophoblast cells,we identified a new lnc RNA-HZ09.BPDE upregulates the expression levels of lnc-HZ09 by promoting the expression of MSX1 and m6 A RNA methylation modification in lnc-HZ09.After overexpression of lnc-HZ09,the expression of SP1 and the stability of PLD1 m RNA are significantly reduced to reduce the level of PLD1,which ultimately weakens the invasion and migration of human trophoblast cells.This alteration may be closely related with the occurrence of unexplained miscarriage.