The Research Of Screening And Verification On The Differentially Expressed Genes Related To Congenital Microtia

Objectives:In this study, the purposes are to screen the differentially expressed genes in the residual ear cartilage tissue and the normal ear cartilage tissue of patients with congenital microtia by gene chips, and to choice some genes closely related to congenital microtia by bioinformatics analysis, and to further verify their expressions in the residual ear cartilage tissue and the normal ear cartilage tissue by qRT-PCR and Western blotting assay, and provide a scientific basis for the study of molecular mechanism of congenital microtia deeply and its control and prevention.Methods:The specimens were collected from the unilateral congenital microtia patients admitted to Plastic Surgery HosptiaL, Chinese Academy of Medical Sciences. There were no infection and ear chondritis. The residual ear cartilage tissue (removed part in operation, about 100mg) and the normal ear (OTO cranial angle larger side) cartilage tissue (discarded part in operation, about 100mg) were obtained in the process of operation. The residual ear cartilage tissue was used as experimental group, the normal ear cartilage tissues was selected as control group. After drawing material, the cartilage tissues were stored in liquid nitrogen immediately or total protein and total RNA were extracted directly.The differentially expressed genes in the residual ear cartilage tissue and the normal ear cartilage tissue of patients with congenital microtia were screened by the United States Agilent company Arraystar Human LncRNA Microarray V3.0. ZNF44, Wdr82 and FGFR2 genes closely related to congenital microtia were chosen by bioinformatics analysis. The expressions of the three genes were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting assay.Results:1. The results from RNA quantification and quanlity assurance showed that, ① OD260/280 values of 6 samples were 1.95,1.86,1.85,1.87,1.88 and 1.88, respectively. ② 28S and 18S bands were clear. ③ All samples had nogenomic DNA contamination. The extracted RNA had good integrity and purity.2. The results from fluorescence images of microarray hybridization indicated that, ① The Cy3 green fluorescence was round, no overlap, ① The image background was low, the noise free signal.3. Microarray data analysis results suggested that, ① 553 differentially expressed genes were found, containing 433 down-regulated genes (For example, zinc finger protein 44 gene and WD40 repeat protein 82 gene were down-regulated 2.00 and 3.65 times, respectively) and 430 up-reuglated genes (For example, fibro-blast growth factor 2 gene was up-reuglated 2.64 times). ② The differentially expressed genes involved in the functions of embryogenesis, limb formation, skeleton development and signal transduction.4. The results from qRTPCR indicated that, compared with control group, ZNF44 (t=5.398, P<0.01) and Wdr82 (t=5.519, P<0.01) expressions were significantly reduced, while FGFR2 expression was significantly increased (t=4.588, P<0.01) in the cartilage tissue of experimental group. The results were the same with that in gene chip.5. The results from Western blotting demonstrated that, compared with control group, ZNF44 (t=4.396, P<0.05) and Wdr82 (t=6.910, P<0.01) expressions were significantly reduced, while FGFR2 expression was significantly increased (t=13.187, P<0.01) in the cartilage tissue of experimental group. The results were the same with that in gene chip and qRTPCR.Conclusions:1. The differentially expressed genes related to congenital microtia were screened and verified.2. The changes of FGFR2, ZNF44 and Wdr82 expressions might be related to development of congenital microtia.