Identification Of New MiRNA-mRNA Networks In The Development Of Non-syndromic Cleft Lip With Or Without Cleft Palate

Objective:Non-syndromic cleft lip with or without cleft palate(NSCL/P)is one of the most common birth defects in human beings.MicroRNAs(miRNAs)are non-coding single-stranded RNAs,which are about 21-24 nucleotides in length.Increasing evidence suggests that numerous miRNAs may play critical roles in craniofacial development.This study aims to identify differentially expressed miRNAs(DEMs),differentially expressed genes(DEGs),as well as their interaction networks of NSCL/P by integrated bioinformatic analysis.Methods:Overlapping DEMs were selected from cleft palate patients(GSE47939)and murine embryonic orofacial tissues(GSE20880).Next,the target genes of DEMs were predicted and further filtered through differentially expressed genes(DEGs)from dental pulp stem cells(DPSCs)of NSCL/P patients and controls(GSE42589).Afterwards,two miRNA-mRNA networks(let-7c-5p-PIGA,miR-193a-3p-TGFB2)were identified after being investigated in MGI,MalaCards and DECIPHER.Finally,a series of functional experiments were carried out,including luciferase activity assay,cell apoptosis,cell proliferation,quantitative RT-PCR and western blot.In addition,association of genetic variants with the risk of NSCL/P were assessed in 504 cases and 455 controls.Results:Let-7c-5p,miR-193a-3p,and miR-423-3p were identified as DEMs in NSCL/P patients and their overexpression resulted in inhibited cell proliferation and promoted cell apoptosis in human embryonic palate mesenchyme(HEPM)and human oral epithelium cells(HOEC).PIGA and TGFB2 were target genes of let-7c-5p and miR-193a-3p,respectively.Their expression was detected in lip tissues from oral cleft patients and were negatively associated with the expression of let-7c-5p and miR-193a-3p.In addition,they were also associated with the occurrence of NSCL/P based on MGI,MalaCards and DECIPHER databases.TGFB2/rs77246858 was associated with the risk of NSCL/P.Conclusions:Let-7c-5p-PIGA and miR-193a-3p-TGFB2 networks may be involved in the development of NSCL/P.