The Preliminary Research On The Relationship Between Wnt/PCP Signal Pathway And The Epigenetic Etiology Of Congenital Microtia

ObjectivesWith an epigenetic comparison with the methylation status of total genome of congenital microtia and non ear deformity patients and a deep analysis on the DEP and ROI methylation changes on the MeDIP chip, the signal pathways most likely related to the growth and development were screened. To investigate the probable mechanism of the influence of the signal pathway and epigenetic abnormality on the congenital microtia, we verified the related wnt1 and wnt11 gene expression level and evaluated the prolifertation and the apoptosis of the ear cartilage cells which were blocked the Wnt/ PCP signal pathway.Methods1. The total genome methylation status of congenital microtia and non ear deformity patients was achieved by the MeDIP chip technique. Deep analysis with GO and Pathway Analysis was done to screen the signal pathways and genes that probably related to the congenital microtia.2. The wnt1 and wnt11 gene expression level was evaluated by Real-time PCR in the cartilage tissues from the congenital microtia and non ear deformity patients, as well as the malformated and normal cartilage tissues from the same congenital microtia patients, which was to verify the consistent gene expression with the richment of Wnt signal pathway in the results of methylation chips.3. CCK-8 method was used to test the cellular proliferation of normal and deformity chondrocytes which came from the same congenital microtia patient’s ears, and Annexin Ⅴ method to test the cellular apoptosis of those chondrocytes.4. The JNK inhibitor Ⅱ and the JNK inhibitor Ⅱ with 5-Azacytidine were applied to normal and deformity chondrocytes which came from the same congenital microtia patient’s ears. The Real-time PCR was used to test the variation of wnt1 and wnt11 gene expression in the different groups, and Western blot was used to test the variation of Wnt1 and Wnt11 protein expression in the different groups. Then CCK-8 method was used to test the cellular proliferation variation of normal and deformity chondrocytes and Annexin Ⅴ method to test the cellular apoptosis of those chondrocytes in different groups after the Wnt/PCP signal pathway was blocked.Results1.With the deep GO and Pathway Analysis of the DNA methylation chips of congenital microtia and normal ear cartilage tissues, Wnt signal pathway was screened to have the most rich scores and most likely related to the congenital microtia. The wnt1 and wnt11 genes were with higher methylation in the promoter region and CpG islands in normal group than microtia group. We made the analog Network of Wnt signal pathway according to the results of GO and Pathway Analysis, which was abnormal in DNA methylation, and fixed our research on the wnt1 and wnt11 genes and their specific signal pathways, which were also abnormal in DNA methylation.2. There was no difference in wnt1 and wnt11 gene expression in microtia and non ear deformity patients. But the higher wnt11 gene expression was detected in the deformity cartilage tissues than normal which came from the same congenital microtia patients. 3.It showed no difference in the biological character between difference otic chondrocyte generations, and no difference in cellular proliferation and apoptosis between chondrocytes from the deformity and normal cartilage of the same congenital microtia patients.4. After the JNK inhibitor Ⅱ and the JNK inhibitor Ⅱ with 5-Azacytidine were applied to normal and deformity chondrocytes which came from the same congenital microtia patients, no difference in the biological character was showed in both normal and deformity chondrocytes compared to each control group. The lower wnt11 gene expression was detected in the JNK inhibitor Ⅱ group in both normal and deformity chondrocytes by Real-time PCR. And the lower wnt11 protein expression was also detected in the JNK inhibitor Ⅱ group in both normal and deformity chondrocytes by Western blot. With an effective block of Wnt/PCP signal pathway, the cellular proliferation of both normal and deformity chondrocytes increased, which was consistent with the growth curve fitted. In the cellular apoptosis tests, the higher percentage of live cells and lower percentage of total apoptosis cells in the JNK inhibitor Ⅱ group than control was detected. There was no difference in cellular proliferation and apoptosis in both normal and deformity chondrocytes which were applied with JNK inhibitor Ⅱ with 5-Azacytidine. ConclusionsThe abnormal DNA methylation of Wnt signal pathway was detected in the total genome DNA methylation chips of the congenital microtia patients. To verify the wnt1 and wnt11 genes expression which was with abnormal methylation in Wnt signal pathway, a higher wnt11 gene expression was detected in deformity cartilage than the normal cartilage which came from the same patient. It is consistent with the DNA methylation status in the promoter region and CpG islands of congenital microtia patients. The deformity chondrocyte has the same proliferating ability with the normal chondrocyte from the congenital microtia patient, which could be a choice of seed cells for auricular tissue engineering. With applying of JNK inhibitor Ⅱ to both normal and deformity otic chondrocytes, the Wnt/PCP signal pathway is blocked effectively and wnt11 gene expression decreases, which promotes the cellular proliferation and reduce the cellular apoptosis of otic chondrocytes. It could be inferred that the Wnt/PCP signal pathway plays a important role on cellular proliferation and apoptosis of otic chondrocytes, and the over expression of wntll in embryo might relate to the etiology of congenital microtia. The mechanism of how the abnormal methylation of wnt11 plays on the etiology of congenital microtia needs further research in the future.