Variants In MiRNA Regulome And Their Association With The Risk Of Non-syndromic Orofacial Clefts

Background:Non-syndromic orofacial clefts（NSOCs）are influenced by genetic and environmental factors,including non-syndromic cleft lip only（NSCLO）,non-syndromic cleft lip with palate（NSCLP）and non-syndromic cleft palate only（NSCPO）.The non-syndromic cleft lip only and non-syndromic cleft lip with palate are also considered as non-syndromic cleft lip with or without cleft palate（NSCL/P）.Micro RNAs（miRNAs）are 20 to 24 nucleotide long small non-coding RNAs discovered to be closely associated with craniofacial development.In particular,single-nucleotide polymorphisms（SNPs）in the miRNA regulome can influence the miRNA transcription and the regulation of targeted genes.Objective:We aimed to explore if SNPs in miRNA regulome were associated with risks of non-syndromic orofacial clefts（NSOCs）and its subgroups.Materials and methods:Firstly,we integrated the miRBase v22,the UCSC common SNPs（150）and the GEO database（GSE97752）to identify SNPs in miRNA regulome（promoter and enhancer）.Second,a two-stage case-control study with 2,004 NSOC cases and 1,891 healthy controls were genotyped to identify SNPs and the risk of specific orofacial cleft subgroups.Stage I consisted of NSCL/P Genome-Wide CHB1and CHB2 arrays data with 504 cases and 455 controls,while Stage II comprised another independent cohort with 1,500 cases and 1,386 controls.At last,bioinformatics analysis and cell and molecular biology techniques were applied to explore the possible biological mechanism of the susceptible SNPs and miRNAs involved in the development of NSOCs.Results:13,830 SNPs in the miRNA promoter elements and 75,375 SNPs in the miRNA enhancer elements were respectively identified and their associations with risk of NSCL/P were evaluated in stageⅠ.Among them,two most-promising SNPs in the stageⅠ（P<1.0E-04:rs3830766 in the enhancer elements and rs11564745 in both promoter and enhancer elements）were further investigated in stageⅡ.We found that rs3830766（enhancer of hsa-miR-4260）was significantly associated with cleft lip only（NSCLO）under recessive model（P=0.007;OR=0.66,95%CI=0.49-0.89）.The luciferase reporter assay showed that deletion allele had a lower enhancer activity in HEPM（Human embryo palatal mesenchyme）cells and HOEC（Human oral epithelial cell）compared with G allele(P_HEPM=0.003,P_HOEC=6.45E-05).In addition,significantly decreased expression of hsa-miR-4260 in the serum of NSCLO cases compared to the controls was detected（P=0.026）.Over-expression of miR-4260mimics inhibited the proliferation and increased the apoptosis of HOEC and HEPM cells.UBB was identified as target gene of hsa-miR-4260 in HOEC and HEPM cells.Conclusion:Our study identified a miRNA regulatory SNP hsa-miR-4260/rs3830766that was probably associated with susceptibility of NSCLO by interacting with UBB.