Differential Expression of Noncoding RNAs Revealed Enhancer RNA AC016735.2 as a Potential Pathogenic Marker of Congenital Microtia Patients

Abstract

Purpose: Congenital microtia is a complex maxillofacial malformation with various risk factors. This study aimed to find potential pathogenic noncoding RNAs for congenital microtia patients. Methods: We collected 3 pairs of residual ear cartilage samples and corresponding normal ear cartilage samples from nonsyndromic congenital microtia patients for microarray experiments. The differentially expressed RNAs were screened, and enrichment analysis and correlation expression analysis were performed to elucidate the function of the differentially expressed genes (DEGs). We further investigated the most significantly differentially expressed long noncoding RNA (lncRNA), AC016735.2, through follow-up analyses including RT-qPCR and Western blotting, to validate its differential expression in residual ear cartilage compared with normal ear cartilage. SiRNA was designed to study the regulatory role of AC016735.2, and cell proliferation experiments were conducted to explore its impact on residual ear chondrocytes. Results: Analysis of the microarray data revealed a total of 1079 differentially expressed RNAs, including 305 mRNAs and x lncRNAs, using a threshold of FC>1.5 and P<0.05 for mRNA, and FC>1.0 and P<0.05 for lncRNA. Enrichment analysis indicated that these genes are mainly involved in extracellular matrix regulation and embryonic development. AC016735.2 showed the highest differential expression among the eRNAs, being upregulated in residual ear cartilage. It acts in cis to regulate the nearby coding gene ZFP36L2, indirectly affecting downstream genes such as BMP4, TWSG1, COL2A1, and COL9A2. Conclusion: Significant differences were observed in the expression of lncRNAs and mRNAs between residual ear cartilage and normal auricular cartilage tissues in the same genetic background of congenital microtia. These differentially expressed lncRNAs and mRNAs may play crucial roles in the occurrence and development of microtia through pathways associated with extracellular matrix regulation and gastrulation. Particularly, AC016735.2, an eRNA acting in cis, could serve as a potential pathogenic noncoding gene.