| Objective: The differentially expressed mi RNAs in non-syndromic cleft lip and palate patients was screened by mi RNA sequencing,and the lnc RNAs and m RNAs with potential binding sites were predicted and screened by bioinformatics database.To analyze the regulatory axis of lnc RNA-mi RNA genes that may be involved in NSCL/P,laying a foundation for further research on the potential regulatory mechanism of nc RNAs and m RNAs in NSCL/P.Methods: The peripheral blood of patients in CPO group,CL/P group and normal children were collected from 15 cases each,altogether 45 cases.The total RNA was extracted after plasma separation.3 samples from each group were analyzed by mi RNA sequencing to screen the differentially expressed mi RNAs.Using bioinformatics database and lnc RNA GSE183527,m RNA GSE42589 data set in GEO database to predict and screen lnc RNAs and m RNAs with potential binding sites with differential mi RNAs,enrichment analysis of GO and KEGG pathway and PPI network analysis by m RNAs.Cytoscape software was used to preliminarily construct the ce RNA network and screen lnc RNA-mi RNA gene regulatory axis that may be involved in NSCL/P,the selected mi RNA,lnc RNA and m RNA were used to verify the expression of RT-q PCR and analyze the diagnostic efficiency of ROC in 12 samples of each group.Results:(1)In the plasma samples of NSCL/P,47 differentially expressed mi RNAs were identified in CPO group,including hsa-mi R-212-3p,hsa-mi R-204-3p/5p,etc.36 differentially expressed mi RNAs were identified in CL/P group,including hsa-mi R-1249-3p,hsa-mi R-579-3p,etc.24 differentially expressed mi RNAs were identified in CPO group compared with CL/P group,including hsa-mi R-212-3p,hsa-mi R-6511b-3p,etc.48 differentially expressed mi RNAs among CPO group,CL/P group and control group,and the common up/down-regulated mi RNAs included hsa-mi R-200b-3p,hsa-mi R-130b-3p,etc.(2)Bioinformatics combined with GEO dataset predicted 2337 co-expressed m RNAs and 11 co-expressed lnc RNAs.Through PPI analysis,a m RNAs network with 165 nodes and 293 edges was identified,and the top 20 key genes were screened,including BRCA1,CDK1,SMAD3,POLA1,MCM5,MCM6,BLM,POLD3,CDC6,POLA2,RFC5,BRCA2,MCM8,SMAD2,TIPIN,RAD51,CDC7,FANCM,FANCL,MYC.The 20 key genes are mainly concentrated in DNA replication,cell cycle,cell response to DNA damage stimulation and cell cycle,DNA replication,TGF-βsignal pathway,Hippo signal pathway,mismatch repair,cancer and other signal pathways.(3)After comprehensive screening of lnc RNA-mi RNA-m RNA,a ce RNA network with 13 nodes and 19 edges is constructed,and the potential ce RNA regulation axis was verified by RT-q PCR.The results showed that the lnc RNA NEAT1 was down-regulation in CPO group(P<0.05),but it was no statistical significance in CL/P group(P>0.05);The expression of SMAD2 was down-regulated in CPO group and CL/P group with statistical significance(P<0.05);The expression of hsa-mi R-200b-3p was up-regulated in CPO group(P<0.05),but it was no statistical significance in CL/P group(P>0.05).The expression trend of RT-q PCR of hsa-mi R-200b-3p was consistent with that of mi RNA sequencing.ROC curve analysis showed that the AUC of mi R-200b-3p in diagnosing CPO was 0.819,the AUC in diagnosing CL/P was 0.833,and the AUC in diagnosing NSCL/P was 0.826.Conclusion:(1)There are different mi RNA expression profiles in CPO and CL/P patients,suggesting that the etiology of CPO and CL/P may have different regulatory mechanisms.(2)Differential mi RNAs represented by mi R-200b-3p,may participate in the mechanism of NSCL/P by regulating the related target genes of cell cycle,TGF-βsignaling pathway and DNA damage repair response.(3)The lnc RNA NEAT1-mi R-200b-3p-SMAD2 regulatory axis may be involved in the formation of cleft palate,which is worthy of further study. |
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