Comprehensive Analysis Of Long Noncoding RNA Regulates And CeRNA Regulatory Networks In Non-syndromic Orofacial Clefts

Object:Orofacial Clefts（OFCs）is is a congenital,hereditary craniofacial developmental defect and accounts for the highest proportion of craniofacial malformations.OFCs are generally classified as non-syndromic orofacial clefts（NSOFC）,or syndromic orofacial clefts（SOFCs）.The etiology and pathogenesis of non-syndromic orofacial clefts（NSOFC）which account for the majority of OFCs are largely unknown.In recent years,long non-coding RNAs（lnc RNAs）are thought to play important roles in non-syndromic orofacial clefts（NSOFC）,but there is no detailed report on this.Thus,we will explore the role of lnc RNAs in NSOFC,and we will establish competing endogenous RNA networks（ce RNA networks）associated with the lnc RNAs.These findings will also provide new sights for the pathogenesis of NSOFC.Methods:In this study,the children with NSOFC but no other disease were selected from October 2019 to September 2021 in the department of Maxillofacial Surgery of Gansu Provincial People’s Hospital.Clinically,NSOFC can be categorized as either cleft palate only（NSCPO）or cleft lip with or without cleft palate（NSCL/P）.Healthy people were considered as blank control.A total of 92 patients were included in the study.The general data and epidemiologically investigated were collected before operation in patients.Tissues excised from the trimmed wound edge were reserved as experimental samples;adjacent normal tissue was used as a positive control,and tissue from healthy individuals was used as a blank control.Target lnc RNAs in the collected tissues were identified using Arraystar Human lnc RNA microarray V5.0 combined with bioinformatics methods and quantitative reverse transcription PCR（RT-q PCR）.Gene ontology（GO）enrichment,and Target Scan predictions were employed to construct competing endogenous RNA networks（ce RNA networks）and explore their potential functions.Immunohistochemical（IHC）staining and RT-q PCR were used to verify the target m RNAs.Results:RNA-Seq revealed 24 upregulated and 43 downregulated lnc RNAs;MALAT1 and NEAT1 were screened and validated using RT-q PCR.Common NSOFC risk factors were positively correlated with MALAT1 and NEAT1 expression.Common risk factors of NSOFC were positive correlation associated with relative expression of lnc RNA MALAT1 and NEAT1(OR_MALAT1=28.111,95%CI:4.054-194.923,OR_NEAT1=30.556,95%CI:4.422-211.142).Bioinformatics predicted 4 ce RNA networks:MALAT1-hsa-mi R-1224-3p-SP1,MALAT1-hsa-mi R-6734-5p/hsa-mi R-1224-3p-WNT10A,NEAT1-hsa-mi R-140-3p.1-CXCR4,and NEAT1-hsa-mi R-199a-3p/hsa-mi R-199b-3p/hsa-mi R-3129-5p-ZEB1.GO enrichment focused on the potential functions of ce RNA networks,such as organic cyclic compound biosynthetic process,organic substance metabolic process,membrane-enclosed lumen,organelle lumen,Wnt-protein binding,metal ion binding,and so on.RT-q PCR and IHC data were consistent with respect to expression levels of proteins and the m RNAs that encode them.SP1 was significantly down-regulated,while WNT10A,CXCR4,and ZEB1 were up-regulated in NSOFC tissues compared to two control group（P<0.01）.Conclusion:MALAT1 and NEAT1,up-regulated lnc RNAs in NSOFC,are associated with the disease severity of NSOFC,and four ce RNA networks were constructed.Our research provided new insights for the pathogenesis and therapy of NSOFC and suggested that MALAT1and NEAT1 might act as potential therapeutic targets and prognostic biomarkers for NSOFC.