The Roles And Regulatory Mechanisms Of Novel Lnc-HZ04 In BPDE-induced Female Trophoblast Cell Apoptosis And The Occurrence Of Miscarriage

ObjectiveBenzopyrene（BaP）is widely present in environment,drinking water and food.The purpose of this study is to correlate the relationship between BPDE-induced trophoblast dysfunction and the occurrence of miscarriage,to identify new lncRNAs and microRNAs,and to find out differentially expressed signaling pathways,to reveal the regulatory effects of lncRNA and miRNA on signaling pathways,and to investigate their functional roles in BPDE-induced abortion,and to explore the molecular mechanisms how environmental harmful factors trigger miscarriage through trophoblast cell dysfunctionsMethods1.Novel lnc-HZ04 and novel miR-hz04 that are differentially expressed in BPDE-treated trophoblast cells were detected by high-throughput transcriptome sequencing.The relative expression levels of lnc-HZ04 and miR-hz04 were detected by RT-qPCR in BPDE-exposed trophoblast cells and in chorionic tissue of recurrent miscarriage（RM）patients.2.The effects of BPDE,lnc-HZ04 and miR-hz04 on trophoblast cell apoptosis were detected by flow cytometry.3.The effects of BPDE,lnc-HZ04 and miR-hz04 on IP₃R₁/p-CaMKII/SGCB signaling pathway were detected by RT-qPCR and Western blot4.The expression levels of IP₃R₁/p-CaMKII/SGCB signaling pathway in RM chorionic tissues were detected by RT-qPCR and Western blot.5.The effects of BPDE,lnc-HZ04 and miR-hz04 on intracellular free calcium ion levels were detected by fluorescence microscopy.6.RIP tests was used to detect the interaction between lnc-HZ04 and IP₃R₁,CaMKII,p-CaMKII and SGCB proteins.7.RNA stability assays were used to detect the effects of lnc-HZ04 and miR-hz04 on IP₃R₁ mRNA expression and the effects of miR-hz04 on lnc-HZ04 expression.8.The dual luciferase reporting assays,AGO2 RIP assays,MS2-RIP assays and RNA pull-down assays were used to detect the interactions between miR-hz04 and lnc-HZ04,as well as between miR-hz04 and IP₃R₁ mRNA.9.The BaP-exposed mouse model was established to detect the effect of BaP on the absorption rate of mouse embryos.The expression of miR-hz04 and the changes in IP₃R₁/p-CaMK2G/SGCB signaling pathway were detected by RT-qPCR and Western blot assays.Results1.The expression level of lnc-HZ04 was up-regulated（*p<0.05）and the expression level of miR-hz04 was down-regulated（***p<0.001）in both RM tissues and BPDE-exposed trophoblast cells.2.Flow cytometry showed that BPDE and lnc-HZ04 promoted cell apoptosis;while miR-hz04 inhibited cell apoptosis（*p<0.05）.3.RT-qPCR and Western blot results showed that IP₃R₁,CaMKII,p-CaMKII and SGCB were up-regulated in RM tissues.Both BPDE and lnc-HZ04 can promote the expression of IP₃R₁,CaMKII,p-CaMKII and SGCB.miR-hz04 inhibited the expression of IP₃R₁,CaMKII,p-CaMKII and SGCB.4.Fluorescence microscopy results showed that BPDE and lnc-HZ04 promoted intracellular calcium release;while miR-hz04 inhibited intracellular calcium release（**p<0.01）.5.RIP experiment results showed that IP₃R₁,CaMKII,p-CaMKII and SGCB had no interaction with lnc-HZ04.6.The RNA stability test results showed that miR-hz04 inhibited the stability of IP₃R₁ mRNA and lnc-HZ04（*p<0.05）.Lnc-HZ04 promoted the stability of IP₃R₁mRNA（*p<0.05）.miR-hz04 attenuated the lnc-HZ04-inhibited of IP₃R₁ mRNA degradation（*p<0.05）.7.The results of dual luciferase reporting assays,Ago2 RIP assays,MS2-RIP assays and RNA pull-down assays showed that miR-hz04 could directly bind to lnc-HZ04（***p<0.001）,and miR-hz04 could also directly bind to IP₃R₁ mRNA （***p<0.001）.Meanwhile,lnc-HZ04 and IP₃R₁ mRNA competitively bound to miR-hz04（***p<0.001）.8.The BaP-exposed mouse model showed that the absorption of mouse embryos increased after BaP exposure（**p<0.01）.RT-qPCR and Western blot results showed that the relative expression levels of IP₃R₁,Camk2g,p-Camk2g and Sgcb in placental tissues of BaP-exposed pregnant mic increased with BaP concentration.The relative expression of miR-hz04 was decreased in BaP-exposed mice tissues.ConclusionIn this study,a novel lnc-HZ04 and a novel miR-hz04 were identified.BPDE up-regulated the expression level of lnc-HZ04,which acted as a ce RNA of miR-hz04,competitively bound with miR-hz04,reduced the miR-hz04-inhibited stability and expression level of IP₃R₁ mRNA,and increased the expression level of IP₃R₁,increased the release of intracellular free calcium ions,activated CaMKII,increased CaMKII phosphorylation,and then activated SGCB to induce trophoblast apoptosis of trophoblast cells and the occurrence of miscarriage.