| Congenial heart disease(CHD)is the top ranking of birth defect in China and has become the leading cause of non-infectious death for the infant.However,the etiology of CHD is complex and the pathogenesis remains to be elucidated.Genetic abnormalities(e.g.chromosomal abnormalities,single gene mutation,etc.)cannot be used to explain the occurrence of most of CHD,and genetic-environmental interaction has become the primary cause.Under the specific genetic background,environmental factors(such as drugs,poisons,etc.)can disturb the normal development of fetal heart during the embryonic period and result in cardiac structure malformation,especially the occurrence of non-syndromic CHD.At present,accumulating researches focused on the effect and mechanism of only one or several teratogen on cardiac development,which cannot fundamentally reduce the incidence of CHD.Only by decreasing the exposure to environmental teratogenic factors during pregnancy at the overall level,or decreasing and even blocking drugs or poisons transferred into the fetal side through the placenta,the incidence of CHD can be furthest declined at the first-level prevention.Hence,under the circumstance that mother is inevitably exposed to environmental teratogenic factors,to explore the transplacental transport of environmental teratogen from mother to fetus is very significant for decreasing fetal exposure on environmental teratogen,which may be an effective means to reduce the overall incidence of drugs-/toxins-induced cardiac malformation.In recent years,it has been found that there are a kind of membrane transporters located at the apical membrane and basement membrane of the “blood fetal barrier”,named ATP-binding cassette(ABC)transporters that play a significant role in controlling the transplacental transport rate of xenobiotics.Among them,P-glycoprotein(P-gp)encoded by ABCB1 gene in human has the most abundant expression and the most unambiguous function,which can exclude the xenobiotics from the fetal side into maternal side,and then decrease the fetal exposure to drugs or poisons.So,P-gp is a key component in regulating the transplacental transport of drugs or toxin.However,previous studies demonstrated that the expression of P-gp could be influenced to varying degree by different pathophysiological conditions and medication during pregnancy.Thereby,it could be important for reducing or blocking fetal teratogenic exposure and achieving a reduction in the incidence of birth defects,such as CHD,to clarify the regulatory mechanism of placental P-gp expression and search for the specific target for upregulating the expression and efflux function of placental P-gp.Long non-coding RNAs(lncRNAs)are classified as RNAs with a length of more than 200 nucleotides that can only be transcribed but do not produce a protein product.LncRNAs are widely present in eukaryotes with higher tissue and cellular specificity,which are involved in many important physiological and pathological processes and associated with the function of most organs(like placenta)and the development of many diseases(such as cancer,diabetes,preeclampsia and fetal growth restriction,etc.).Previous studies found that lncRNAs are linked to placental development and can affect its function,while the abnormal expressions of some lncRNAs are related to the occurrence and advancement of many pregnancy-associated diseases.Currently,oncology studies have shown that some lncRNAs can regulate the expression of tumor cell P-gp,which seems closely associated with the tumor drug resistance.But,little reports have been presented with respect to the regulation of lncRNA on placental P-gp.Given the similar biological features between placental trophoblast and tumor cell,the achievements from oncology studies indicate that investigating the regulation of lncRNA on placental P-gp appears to be of significance.Moreover,by reviewing related literature,we screened out the lncRNA urothelial carcinoma associated 1(UCA1)that might regulate the expression and efflux function of placental P-gp.Therefore,this study aimed to investigate the regulatory mechanism of lncRNA UCA1 on the expression and function of placental trophoblast P-gp,and expect to find a specific intervention target for safely and effectively upregulating placental P-gp expression and function to decrease the transplacental transport of drugs or poisons.This study can provide a new idea and basic theories to further reduce fetal exposure on drugs or poisons resulting in the abnormal development of fetal heart.Objectives1.To explore the effect of placental lncRNA UCA1 on the expression and function of P-gp in placental trophoblast.2.To clarify the regulatory mechanism of lncRNA UCA1 on the expression and function of P-gp in placental trophoblast.3.To establish the model of Bewo cell fusion induced by forskolin.4.To investigate the regulation of lncRNA UCA1 on the expression and function of P-gp in model of forskolin-induced fusion of Bewo cells.Material and Methods1.The placental Bewo cellls were cultured and divided into the experimental group and the control group.LncRNA UCA1 siRNAs(siRNA-1/-2)and negative control(NC)as well as the lncRNA UCA1 overexpression plasmid and NC plasmid were transfected into the Bewo cells using corresponding concentration,respectively.At 48 h after intervention,the expression of lncRNA UCA1 was detected by quantitative real time polymerase chain reaction(q RT-PCR),and then compared with the control group to confirm the successful transfection of lncRNA UCA1 siRNA-1/-2 and lncRNA UCA1 overexpression plasmid.Subsequently,the ABCB1 m RNA expression was detected by q RT-PCR;the P-gp protein expression was measured by cellular immunofluorescence(IF);and the efflux function of cellular P-gp was determined by multidrug resistance(MDR)direct dye efflux assay.The differences of both groups were analyzed.2.The placental Bewo cellls were cultured and RNA fluorescence In Situ hybridization(FISH)was used to ascertain the subcellular localization of lncRNA UCA1;miRNA microarray was used to detect the expression of miRNAs in Bewo cells intervened by lncRNA UCA1 siRNA and NC,and then select the miRNA with significantly different expression between the UCA1 siRNA group and the control group.Additionally,combined with the data obtained from the databases,the miRNA that probably involved in the regulation of lncRNA UCA1 on P-gp expression and function in placenta could be selected.Finally,the expression of selected miRNA in Bewo cell transfected by lncRNA UCA1 siRNA and overexpression plasmid was verified by q RT-PCR.3.The placental Bewo cellls were cultured and divided into the experimental group and the control group.The reagents of miRNA mimic and mimic control as well as miRNA inhibitor and inhibitor control were transfected into the Bewo cells using corresponding concentration for 48 h,respectively.The expression of the miRNA was detected by q RT-PCR and then compared with the control group to confirm the successful transfection of miRNA mimic and inhibitor.Subsequently,the ABCB1 m RNA expression was detected by q RT-PCR;the P-gp protein expression was measured by cellular IF;and the efflux function of cellular P-gp was MDR direct dye efflux assay.The differences of both groups were analyzed.4.Placental Bewo cells were cultured,and q RT-PCR,cellular IF and MDR direct dye efflux assay were applied to detect the ABCB1 m RNA expression,P-gp expression and its efflux function in Rescue assay,respectively.The direct relationship between lncRNA UCA1 and miRNA as well as between the miRNA and ABCB1 was determined by dual-luciferase reporter system assay.Besides,argonaute protein 2(AGO2)-RNA immunoprecipitation(RIP)-q PCR assay and RNA-Pull down-q PCR assay were performed to identify the interaction among lncRNA UCA1,miRNA and ABCB1 in placental Bewo cells.5.The placental Bewo cellls were cultured and divided into the intervention group and the vehicle control group.Forskolin(FSK)with the concentration of25 u M,50 u M and 100 u M was used to treat Bewo cells for 24 h,48h,72 h and 96 h in order to establish the Bewo fusion model.Dimethysulfoxide(DMSO)as the vehicle control was also applied to intervening Bewo cells using similar concentration and intervention time to FSK.After that,the expression levels of syncytotrophoblast makers of β-chain human chorionic gonadotropin(βh CG),Syncytin-2,Endoglin and Leptin gene in Bewo cells were detected by q RT-PCR;βh CG enzyme linked immune sorbent assay(ELISA)was conducted to measure the βh CG concentration in cell culture supernatant;The proliferation ability of Bewo cell was detected by WST-1 kit at different time points after 50 u M FSK treatment to screen out the optimal time point for this study;the expression and distribution of cellular E-cadherin was detected by cellular IF and the morphological changes of Bewo cells were observed using inverted phase contrast microscope at 24 h after 50 u M FSK intervention.6.After lncRNA UCA1 siRNA and NC,lncRNA UCA1 overexpression plasmid and NC plasmid,miRNA mimic and mimic control as well as miRNA inhibitor and inhibitor were applied to successfully transfect the model of FSK-induced fusion of Bewo cells using appropriated concentration,q RT-PCR,cellular IF and MDR direct dye efflux assay were applied to detect the ABCB1 m RNA expression,P-gp expression and its efflux function in the model,respectively.Results1.Compared with the control group,when successfully down-regulating the expression of placental lncRNA UCA1,cellular ABCB1 gene m RNA expression was significantly decreased(P<0.05),while the expression levels of placental ABCG2 gene and ABCC1 gene in placenta were obviously increased(P<0.05);also,cellular P-gp protein expression was significantly decreased and the efflux function of P-gp was accordingly reduced(P<0.05);meanwhile,the efflux activities of breast cancer resistance protein(BCRP)and multidrug resistance protein 1(MRP1)in Bewo cells were impacted by down-regulated lncRNA UCA1.When lncRNA UCA1 overexpressed in placental Bewo cells,the m RNA expression level of ABCB1 gene and the protein expression of P-gp were notably increased(P<0.05),and the efflux function of cellular P-gp was accordingly increased(P<0.05)in comparison with the control group.2.RNA-FISH identified that lncRNA UCA1 mainly located in the cytoplasm of placental Bewo cells.In terms of P value <0.05 and Fold Change ≥1.3,there were ten significantly different miRNAs determined by miRNA microarray between the lncRNA UCA1 siRNA group and the NC group,including eight up-regulated miRNAs and two down-regulated miRNAs.After comprehensively analyzing the ten significantly different miRNAs detected by miRNA microarray and the data obtained from databases,there were four miRNAs included at least in three aggregates but only miR-16-5p with notably upregulation(P<0.05)was screened out.Besides,using q RT-PCR to confirm that miR-16-5p expression in Bewo cells was significantly increased when down-regulating lncRNA UCA1 but miR-16-5p expression was obviously inhibited by overexpressed lncRNA UCA1(P<0.05),which was consistent with the miRNA microarray.3.Compared with the mimic control group,successfully increasing the expression of placental miR-16-5p expression could make cellular ABCB1 gene m RNA expression significantly decrease(P<0.05),while the expression levels of cellular ABCG2 gene and ABCC1 gene obviously increased(P<0.05);also,cellular P-gp protein expression was significantly decreased and its efflux function was accordingly reduced(P<0.05);meanwhile,the efflux activities of BCRP and MRP1 in Bewo cells were influenced by increased miR-16-5p.When inhibiting miR-16-5p expression in placental Bewo cells,the m RNA and protein expression of P-gp were notably increased(P<0.05),and the efflux function of cellular P-gp was accordingly increased(P<0.05)compared with the inhibitor control group.4.Rescue assay demonstrated that inhibited miR-16-5p could recover the effect of down-regulated lncRNA UCA1 on the m RNA and protein expression of placental P-gp as well as its efflux function in Bewo cells.Dual-luciferase reporter system confirmed in Bewo cells that lncRNA UCA1 could directly target the miR-16-5p,while miR-16-5p could also directly target ABCB1 coding DNA sequence(CDS)region.Besides,AGO2-RIP-q PCR assay detected the expressions of lncRNA UCA1,miR-16-5p and ABCB1 gene in Input group and in AGO2 group but without expressions in Ig G group to verify the interaction among lncRNA UCA1,miR-16-5p and ABCB1 gene.Further,RNA-Pull down-q PCR experiment demonstrated that the biotin probe targeting miR-16-5p pulled down more expression of lncRNA UCA1 in lncRNA UCA1 overexpression group than that in NC group to again prove the interaction between lncRNA UCA1 and miR-16-5p in Bewo cells.5.Compared with the DMSO groups,the expressions of βh CG,Syncytin-2,Endoglin and Leptin gene in Bewo cells were significantly increased in all FSK groups for every intervention concentration and at every time point(P<0.05).The concentration of βh CG in cell culture supernatant was notably increased in FKS group compared with the corresponding control group(P<0.05),especially the 25 u M and 50 u M plus the 24 h and 48 h.Combined with the related literature,the appropriated concentration of 50 u M was selected in following experiments.In addition,Bewo cell viability decreased significantly after 72 h in the blank group and the DMSO group,while in FSK group cell viability decreased rapidly after 48 h.Thus,considering the need of following experiments,24 h was selected as the time point to detect after intervention.Furthermore,using the inverted phase contrast microscope to observe the cellular morphology found that Bewo cells showed obvious morphological changes as “dendritic” processes and form multinucleate cells at 24 h after 50 u M FSK treatment;cellular IF demonstrated that the distribution and expression of cellular E-cadherin was decreased and multinucleate cells were observed,indicating that using 50 u M FSK to intervene Bewo cells for 24 h can establish the model of Bewo cell fusion.6.In model of FSK-induced fusion of Bewo cells,when successfully down-regulating the expression of lncRNA UCA1 or increased miR-16-5p expression,the expression and function of cellular P-gp were significantly decreased compared with the NC group or the mimic control group(P<0.05);besides,successfully overexpressing lncRNA UCA1 or inhibiting miR-16-5p expression,the expression and function of cellular P-gp were significantly increased in comparison the empty plasmid group or the inhibitor control group(P<0.05).However,either the up-regulation or the down-regulation of lncRNA UCA1 cannot affect the expression of miR-16-5p in the FSK-induced fusion model(P>0.05).Conclusions1.LncRNA UCA1 can positively regulate the expression and efflux function of P-gp in placental trophoblast.2.miR-16-5p can negatively regulate the expression and efflux function of P-gp in placental trophoblast by direct targeting the CDS regions of ABCB1.3.Down-regulation of lncRNA UCA1 and up-regulation of miR-16-5p can increase the expressions of ABCG2 and ABCC1 gene,and affect the function of BCRP and MRP1 in placental Bewo cells.4.LncRNA UCA1 can regulate the expression and efflux function of P-gp by directly targeting miR-16-5p in human placental Bewo cells.5.Using 50 u M FSK to intervene Bewo cells for 24 h can successfully establish the model of Bewo cell fusion and can morphologically and functionally differentiate into syncytiotrophoblast in vitro.6.In the model of FSK-induced fusion of Bewo cells,lncRNA UCA1 and miR-16-5p can similarly regulate the expression and efflux function of P-gp,but the upregulation or downregulation of lncRNA UCA1 expression has no impact on miR-16-5p expression,suggesting that the different mechanism could exist about the regulation of lncRNA UCA1 on the expression and efflux function of P-gp in placental syncytiotrophoblast. |
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