| Objectives: Nonsyndromic cleft lip with palate(ns CLP)is a common congenital malformation with high neonatal morbidity and mortality.It is commonly caused by a failure of labial-palatal fusion in the early embryonic development.The occurrence of ns CLP varies with an average rate of 1 per 1,000 live births worldwide.Children with ns CLP may encounter numerous challenges adapting to social life,including feeding,language communication,hearing and dental development,and often require multiple surgeries and professional care.Current clinical strategies to diagnose orofacial clefts prenatally rely mainly on ultrasound,often performed around the middle-late of the pregnancy when the clefts have already occurred,but missing the optimal time for intervention and treatment options.An ultrasound examination is limited by several factors,including maternal obesity,fetal position,or operator skills.The etiology is complex and caused by genomic and environmental factors as well as their interactions.There are few studies on the pathogenesis of human ns CLP.Therefore,it is important to explore the early noninvasive biomarkers for prenatal diagnosis and the related pathogenic mechanisms.In this study,RNA sequencing analysis was performed on maternal plasma,white blood cells,plasma derived exosomes and lip tissues of foetuses in order to explore the potential biomarkers for ns CLP and identified a novel molecular mechanism of palatogenesis.Methods: 1.In the first part of this study,PIWI-interacting RNAs(pi RNAs)and micro RNAs(mi RNAs)were used as potential biomarkers for early noninvasive prenatal diagnosis in screening for ns CLP via RNA-sequencing on maternal plasma and plasma derived exosomes,combined with bioinformatics analysis and subsequent validations.1.1 The samples were collected from the ongoing Shengjing Birth Cohort study,bioinformatics analysis and real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)methods were used to detect the differentially expressed pi RNAs.1.2 The receiver operating characteristic(ROC)curve analyses were used to analyze the diagnostic ability of the hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 for ns CLP.1.3 The expression levels of hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 in the plasma derived exosomes of pregnant women with normal term delivery were detected by q RT-PCR analysis,and their spatial and temporal expression patterns were detected at eight consecutive time points of different gestation stages in the same pregnant women.1.4 q RT-PCR analysis was used to detect the expression levels of the above pi RNAs in the maternal plasma,umbilical cords,placentas,inner muscles of the embryonic calf and lip tissues.1.5 q RT-PCR analysis was used to detect the expression levels of hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 in other common birth defects(neural tube defects,NTDs,and congenital heart defects,CHDs).The ROC curve analyses were used to analyze the diagnostic ability of the above potential biomarkers for NTDs and CHDs.1.6 The expression levels of the hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 were detected in plasma derived exosomes at 15-19 weeks of pregnancy who carrying ns CLP foetuses by q RT-PCR analysis.1.7 The samples were collected from the ongoing Shengjing Birth Cohort study,bioinformatics analysis and q RT-PCR methods were used to detect the differentially expressed mi RNAs.1.8q RT-PCR analysis was used to detect these eight mi RNAs(hsa-let-7a-3p,hsa-let-7a-5p,hsa-let-7c-5p,hsa-let-7d-3p,hsa-let-7d-5p,hsa-let-7e-5p,hsa-let-7f-5p and hsa-mi R-98-5p)from Let-7 family in plasma and plasma derived exosomes.1.9The ROC curve analyses were used to analyze the diagnostic ability of the above potential mi RNAs biomarkers for ns CLP.2.RNA sequencing was performed on lip tissues of ns CLP foetuses and healthy controls to search for the related pathogenic genes(EN2,LIN28 A,hsa-let-7a-3p,HHIP and GLI2)and regulatory mechanisms.2.1q RT-PCR analysis and Western blot were used to detect the expression levels of EN2,LIN28 A,hsa-let-7a-3p,HHIP and GLI2 in embryonic lip tissue.2.2 We cultured HOK cells and human embryonic kidney 293 T cells.2.3 The dual luciferase reporter gene experiment was used to detect the binding effect and binding site of hsa-let-7a-3p and HHIP.2.4 The hsa-let-7a-3p expression silence/over expression HOK cell line was constructed by transient transfection,q RT-PCR and Western blot methods were used to detect the change levels of target genes HHIP and GLI2,and the CCK-8 experiment was used to detect the cell proliferation ability.2.5 Chromatin immunoprecipitation assay(Ch IP)and luciferase reporter gene experiment were used to clarify the binding effect and binding site of EN2 and LIN28 A promoter region,and then it was further confirmed that EN2 can transcriptionally activate LIN28 A expression in HOK cells.2.6 HOK cell lines with stable expression of EN2,LIN28 A or HHIP were constructed by cell transfection.The q RT-PCR analysis was used to detect the RNA expression levels of EN2,LIN28 A,hsa-let-7a-3p,HHIP and GLI2.Western blot analysis was used to detect the protein expression levels of EN2,LIN28 A,HHIP and GLI2.CCK-8 experiment was used to detect cell proliferation ability.Results: 1.The pi RNAs and mi RNAs in maternal plasma and plasma derived exosomes could be used as biomarkers for prenatal diagnosis of ns CLP.1.1hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 was significantly downregulated in plasma derived exosomes from pregnant women carrying ns CLP foetuses.1.2 At 24 weeks of pregnancy,hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 showed good diagnostic ability in prenatal diagnosis of ns CLP.1.3The above three pi RNAs showed a regular trend in different gestational ages during normal pregnancy.The expression levels increased at 4-5 weeks of gestation,reached the peak after 8-9 weeks of lip and palate development,and then began to decrease until after delivery.1.4 The expression of hsa-pi R-009228 and hsa-pi R-020496 showed no difference in the plasma,umbilical cord,placenta or inner calf muscle of ns CLP foetuses in pregnant women carrying ns CLP foetuses,and significantly downregulated in the lip tissues of ns CLP foetuses.hsa-pi R-016659 was also downregulated in plasma of pregnant women carrying ns CLP foetuses.1.5 The expression levels of hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496 were downregulated in plasma derived exosomes in pregnant women carrying NTDs foetuses,and had a good diagnostic efficiency in the prenatal diagnosis for NTDs.hsa-pi R-009228 was downregulated in exosomes of pregnant women carrying CHDs foetuses,while hsa-pi R-016659 was upregulated.The two pi RNAs can be used as biomarkers for prenatal diagnosis of CHDs.1.6 hsa-pi R-009228,hsa-pi R-016659,and hsa-pi R-020496 in maternal plasma derived exosomes had better diagnostic efficacy at the 15-19 weeks compared with at around 24 weeks.1.7 Eight mi RNAs from the Let-7 family(hsa-let-7a-3p,hsa-let-7a-5p,hsa-let-7c-5p,hsa-let-7d-3p,hsa-let-7d-5p,hsa-let-7e-5p,hsa-let-7f-5p and hsa-mi R-98-5p)were significantly down regulated in the whole plasma and plasma derived exosomes of pregnant women carrying ns CLP foetuses.1.8 The diagnostic efficiency of the eight mi RNAs differentially expressed is better in exosomes than that in plasma.In exosomes,the sensitivity of the eight mi RNAs panel is 100% and the specificity is 93.3%.2.In this second part of this study,the potential related genes were detected and the EN2/LIN28A/hsa-let-7a-3p/HHIP/GLI2 gene regulatory network was set.2.1 The expression levels of EN2,LIN28 A and HHIP genes were upregulated in the lip lesions;while hsa-let-7a-3p and GLI2 genes were downregulated in the lip lesions.2.2 HHIP is a potential target gene of hsa-let-7a-3p,and the interaction between the two is achieved through the binding site that exists in the 3’-end untranslated region of HHIP m RNA.2.3 In pre-hsa-let-7a-3p groups,the RNA and protein expression levels of HHIP was downregulated,GLI2 was upregulated and the proliferation of HOK cells was increased.In anti-hsa-let-7a-3p groups,the the RNA and protein expression levels of HHIP was upregulated,GLI2 was downregulated and the proliferation of HOK cells was decreased.2.4 EN2 targets and binds to the LIN28 A promoter region and plays a role in transcriptional activation.2.5 After silencing HHIP,it promotes the expression of GLI2 and promotes the proliferation of HOK cells.Silencing LIN28 A relieves the inhibitory effect on hsa-let-7a-3p.The expression of hsa-let-7a-3p increased,the expression level of HHIP decreased,and the expression level of GLI2 increased,which promoted HOK cell proliferation.After silencing EN2,the expression of LIN28 A and HHIP decreased,and the expression of hsa-let-7a-3p and GLI2 increased,which promoted the proliferation of HOK cells.Conclusions: 1.The three pi RNAs(hsa-pi R-009228,hsa-pi R-016659 and hsa-pi R-020496)were differentially expressed in exosomes and could be as biomarkers for prenatal diagnosis of ns CLP/NTDs.2.The differentially expressed hsa-pi R-009228 and hsa-pi R-016659 in exosomes could be as biomarkers for prenatal diagnosis of CHDs.3.Eight mi RNAs were differentially expressed in plasma and plasma derived exosomes(hsa-let-7a-3p,hsa-let-7a-5p,hsa-let-7c-5p,hsa-let-7d-3p,hsa-let-7d-5p,hsa-let-7e-5p,hsa-let-7f-5p and hsa-mi R-98-5p)and could be promising biomarkers for prenatal diagnosis of ns CLP.4.HHIP is a new target of hsa-let-7a-3p and interacts with hsa-let-7a-3p through a binding site located in the untranslated region of the 3’-end of HHIP m RNA.5.EN2 directly binds to the promoter region of LIN28 A to promote its transcription and play a role in inhibiting the proliferation of HOK cells.6.The EN2/LIN28A/hsa-let-7a-3p/HHIP/GLI2 regulatory pathway plays an important role in the development of cleft lip with palate.This study not only provide potential biomarkers with clinical application value for the prenatal diagnosis of ns CLP,but also explore a new potential molecular mechanism leading to the occurrence of nsCLP. |
PDF Full Text Request