Identification of key genes in cleft lip with or without cleft palate regulated by miR-199a-5p

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Abstract

Background

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects, which etiology involves both genetic and environmental factors. Previous studies have shown that miR-199a-5p may mediate the occurrence of CL/P. However, the key target genes regulated by miR-199a-5p are not clear. In this study, we employed a systematic bioinformatics analysis of target genes regulated by miR-199a-5p which may be involved in CL/P.

Methods

The miRBase, Human miRNA tissue atlas, miRecords, miRpathDB, miRWalk, miRTarBase, DIANA-TarBase (v7.0), Literature search, DAVID software, Cytoscape plugin ClueGO + Cluepedia app, MalaCards, TargetScanhuman7.1, Venny 2.1, STRING and GEO databases were comprehensive employed to identify the key genes regulated by miR-199a-5p associated with CL/P.

Results

Total 429 experimentally validated target genes were obtained from five miRNAs related databases. Expressions of miR-199a-5p and its experimentally validated target genes were elevated in bone, brain and skin. KEGG pathway analysis revealed that the target genes were enriched in focal adhesion, microRNAs in cancer and hippo signaling pathway. Biological process categorization revealed that significant portions of the target genes were grouped as transcription, DNA-templated. Total eight intersection genes were identified by using MalaCards and TargetScanhuman7.1. The target gene transforming growth factor alpha (TGFA) of miR-199a-5p involved in CL/P is screened and verified.

Conclusion

MiR-199a-5p may mediate CL/P by regulating key target gene TGFA. The study may contribute to a better understanding of the etiology of CL/P.

Introduction

Cleft lip with or without cleft palate (CL/P) is the most common form of birth defect in humans, occurring frequently in Amerindians and Asians [1]. Approximately 70% of oral clefts exist as a non-syndromic form, and the remaining 30% are related to Mendelian disorders or teratogenic, chromosomal and sporadic conditions. The incidence is about 1.82 per 1000 live births in China, while the global incidence of CL/P is estimated to be 1.43 per 1,000, with a wide variability among ethnic groups and regions [2]. Clinically, CL/P causes facial deformity in children. The anomaly influences sucking, swallowing and speaking. Furthermore, individuals with CL/P have higher morbidity and mortality throughout life than do unaffected children [3]. Therefore, CL/P is a major public health problem having direct impacts on the health of children and the quality of life.

The etiology of CL/P involves complicated heredity and environmental factors, such as geographic origin, genetics and socioeconomic status. Gene-environment and/or gene-gene interactions may contribute to CL/P. Some gene positions in different chromosomal regions and genes related to CL/P have been reported from association and linkage analysis [4].

MicroRNAs (miRNAs) are kinds of small non-coding RNAs that regulate embryonic development by binding to mRNAs to control their post-transcriptional expression and inhibit their translation, leading to the destabilization of their target mRNAs [5]. The roles of miRNAs in cell differentiation and embryonic development were investigated in the past few years [6]. Increasing evidences suggest that miRNAs are functionally important for the regulation of CL/P [7,8]. The genetic variants of miRNA processing genes such as DROSHA may increase the risk of CL/P [9]. MiRNA-mRNA interactions play a crucial role in orofacial development. However, these interactions may be influenced by genetic variations in the 3′-UTR of human genes that can alter the thermodynamic interplay between them. For instance, L. Ma et al. found that miRNA-binding single nucleotide polymorphism (SNPs) of MSX1 is associated with the increased risk of CLP in Chinese Han population [5].

The miRNA-199a hairpin precursor gene is located on human chromosome19, and its orthologous gene resides on mouse chromosome 9 [10]. Previous studies have revealed that miR-199a-5p play an important role in various biological processes including development, growth, the response to hypoxia and anti-cancer effect [11,12]. Evidence has been found that miR-199a-5p may be associated with CL/P [13]. The expression of miR-199a-5p has been found to increase with the development of the upper lip of mice [14]. Hai-Lei Ding et al. carried out miRNA sequencing on miRNAs extracted from mouse embryonic. The result demonstrated that miR-199a-5p was differentially expressed between palatal shelves, frontonasal prominence and maxillary prominence [15]. These studies indicate that miR-199a-5p may be associated with the formation of CL/P.

Whether the key genes regulated by miR-199a-5p are involved in CL/P has not yet been explored. This article primarily used bioinformatics to find the main genes of miR-199a-5p, and clarify the possible pathogenesis of the CL/P induced by miR-199a-5p.

Section snippets

The mature sequence of miR-199a-5p

MiR-199a-5p is from pre-miR-199a-1 and pre-miR-199a-2, which are transcribed from DNA sequences on different chromosomes. MiR-199a-5p is the highly homologous miRNA of miR-199 b-5p. In order to understand the mature sequences of miR-199a-5p and miR-199 b-5p, the miRBase (http://www.mirbase.org/) [16] and TargetScanhuman7.1 (http://www.targetscan.org/vert_71/) [17] were used. MiRNA research revealed different sets of miRNAs expressed in diverse cell types and tissues. Thus, the Human miRNA

Mature sequences of miR-199a-5p and the expression in tissues

The sequence and structure of miRNA can affect its function. MiR-199a-5p comes from two different pre-miR-199a-1 and pre-miR-199a-2, which are transcribed from DNA sequences on different chromosomes. Fig. 2A shows the mature sequences of miR-199a-5p and miR-199 b-5p, which are highly homologous. The picture showed that miR-199a-5p and miR-199 b-5p share common target gene and similar mature sequences. Therefore, it can be speculated that miR-199a-5p and miR-199 b-5p may have similar functions.

Discussion

Although a lot of studies focus on the etiology of CL/P, there are little research on the relationship between miRNAs and CL/P. Disorder of miRNAs conduces to pathogenesis of all kinds of human diseases [29]. Several studies have revealed that miR-199a-5p may increase the risk of CL/P. However, the genes regulated by miR-199a-5p which may mediate the occurrence of CL/P are still not clear. Therefore, we conducted this study by using bioinformatics.

Primary sequence determinants as well as

Conflicts of interest

The authors deny any conflicts of interest related to this study.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No.81273103), the Education Department of Jiangsu Province (No. 16KJB330010), the China Postdoctoral Science Foundation funded project (No. 2016M601892) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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