Protein profile of ear auricle cartilage and the important role of ITGB1/PTK2 in microtia.

Background: Microtia is a congenital malformation of the external ear that involves anything from a small reduction in size to a complete absence. The external ear is composed of elastic cartilage which is also the important skeleton of the outer ear. However no previous study explored the difference between abnormal elastic cartilage and normal cartilage in the molecular level.

Methods: Microtia cartilage and normal cartilage tissue samples from patients subjected to autologous costal cartilage reconstruction were obtained in surgery. Total proteins were extracted and purified, and then proteomic analyzed via LC-MS/MS using DDA/DIA data collection methods. Proteins were also isolated with lysis beads and then analyzed via antibody chip. Differentially expressed proteins were identified in both experiments and further analyzed with functional enrichment analysis and KEGG pathway analysis. Valuable regulatory gene expression level was verified by RT-PCR.

Results: A total of 4178 protein types were identified in the DDA experiment. A total of 2154 proteins were quantified, 172 of which were significantly upregulated and 82 downregulated in the microtia group (P < 0.05). Antibody chip detection allowed identification of 584 protein phosphorylation sites with 102 upregulation sites and 9 downregulation sites (P < 0.05). Differentially altered proteins were annotated to 143 KEGG pathways, while differentiated phosphate site-associated genes were annotated into 21 KEGG pathways. Two intersecting pathways, the PI3K/AKT/mTOR pathway and the focal adhesion pathway, may paly important role on ear auricle cartilage development. One item is significant in both differential protein expression and phosphorylation. Integrin beta-1, that is downregulated in protein quantification of the microtia group. The mean ITGB1 mRNA level of the microtia patient group was significantly lower than in the healthy control group (P = 0.0007 < 0.05). And the gene expression of downstream gene PTK2 was also decreased. (P = 0.0288 < 0.05).

Conclusion: The research locates the key protein Integrin Beta-1, and verified it at the mRNA level. The increasing level of ITGB1 and decreasing of PTK2 may play an important role in congenital ear deformity. This research will inspire more otolaryngologists and orthopedics doctors to pay attention to the etiology and mechanism of microtia.