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.

FIGURE 1

(A) PCA plots of the samples for the microarray experiment. The yellow triangle represents the normal group, which corresponds to the normal ear cartilage, whereas the blue dot represents the abnormal group, corresponding to the residual ear cartilage. The larger triangle and dot indicate the weight of the 2 groups in PC1 and PC2 dimensions. (B) Volcano plots illustrate the differentially expressed mRNAs and lncRNAs. RNAs with a fold change (FC) >1.5 and P value of <0.05 are labeled with colors, while RNAs with FC>2.5 are annotated with names. (C) Heatmaps display the differentially expressed mRNAs and lncRNAs in the 2 groups. (D) Bar plot showing the proportion of upregulated and downregulated mRNAs and lncRNAs in the residual ear cartilage compared with the normal ear cartilage.

FIGURE 2

GO enrichment results of all the differentially expressed mRNAs (A), upregulated mRNAs (B), and downregulated mRNAs (C) separately.

FIGURE 3

(A) Volcano plots depicting the differentially expressed eRNAs, eRNAs with FC >1 and P<0.05 are labeled with colors, and RNAs with FC >1.5 are presented with names. (B) Heatmaps represent the differential expression of eRNAs in the 2 groups. (C) GO enrichment results of highly correlated DEGs with AC016735.2. (D) GSEA enrichment results of highly correlated DEGs with AC016735.2. (E) Correlation analysis of the expression levels of AC016735.2 and its nearby coding gene ZFP36L2.

FIGURE 4

(A) AC016735.2 and ZFP36L2 mRNA expression quantification by qPCR in microtia residual cartilage samples (n=12) and normal ear cartilage (n=12). (B) Western blotting results for BRF2 protein in microtia residual cartilage samples (n=3) and normal ear cartilage (n=3). And ACTIN was tested for quality control. Band densities of BRF2 were normalized to ACTIN. (C) Efficiency measurement of siRNAs by qPCR in residual ear chondrocytes. siRNA1 was selected for downstream experiments. (D) qPCR measurement of ZFP36L2 expression in residual ear chondrocytes after knocking down AC016735.2. Measurements were carried out in triplicate, and experiments were repeated 3 times.*P<0.05, **P<0.01, and ***P<0.001.

FIGURE 5

(A) qPCR measurements of genes related to extracellular matrix components and metabolism in residual ear chondrocyte after knocking down of AC016735.2. (B) qPCR measurements of genes related to TF binding in residual ear chondrocyte after knocking down of AC016735.2. (C) qPCR measurements of genes related to BMP pathway and Wnt Pathway in residual ear chondrocyte after knocking down of AC016735.2. (D) Proliferation measurement of normal ear chondrocytes (normal chondrocytes group), residual ear chondrocytes (microtia chondrocytes), AC016735.2-knocking down residual ear chondrocytes (eRNA-siRNA group) and siRNA normal control residual ear chondrocytes (siRNA-NC group) by CCK-8 assay. The OD value was measured at 5 different time points labeled on the x-axis. Measurements were carried out in triplicate, and experiments were repeated 3 times.*P<0.05, **P<0.01, and ***P<0.001.