Expression Profiling Of Long Non-coding RNA And Message RNA In Congenital Microtia

Objectives:In our study, the purposes are to analyze LncRNAs and mRNAs expression profiles in microtia by gene chips. According to bioinformatics analysis, some functional LncRNAs and mRNAs would be found. Furthermore, we choice some genes closely related to congenital microtia, and verify their expressions in the residual ear cartilage tissue and the normal ear cartilage tissue by qRT-PCR and Western blotting assay.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 tissue were stored in liquid nitrogen immediately or total protein and total RNA were extracted directly.The differentially expressed LncRNAs and mRNAs 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. Funtional annotations and prediction of the differential expression of LncRNAs and mRNAs were obtained by KEGG biological pathway analysis and GO analysis. Construction of the coding-non-coding gene co-expression network is also one of the channel in the bioinformatics analysis. LncRNA-NR₀28308, LncRNA-uc003jsd.1, LPL, TNFRSF11B, LncRNA-ENST00000449766, ZFP36L2 genes closely related to congenital microtia were chosen by bioinformatics analysis. The expressions of the six genes were further verified by quantitative real-time polymerase chain reaction （qRT-PCR） and Western blotting assay.Results:Gene chips used in this study included 30586 IncRNAs and 26109 mRNAs probe; Compared with normal cartilage,180 IncRNAs differential expression were detected in the residual ear cartilage 74 up-regulated expression IncRNAs and 106 down-regulated expression. Signal pathway analysis revealed 5 up-regulation pathways and 27 down-regulation pathways, which are involved glyceride metabolism, osteoclast differentiation, tumor growth, etc. In the GO analysis,321 up-regulation genes and 553 down-regulation genes in the biological process. At the same time, the coding-non-coding gene co-expression network was contructed by 15 LncRNAs and 120 mRNAs.The results from qRT-PCR indicated that, compared with control group, LncRNA-NR₀28308,LncRNA-uc003jsd.,LP,TNFRSF11B,LncRNA-ENST00000449 766 and ZFP36L2 expressions were significantly increased in the cartilage tissue of experimental group. The results were the same with that in gene chip.Conclusion:Microtia have obvious IncRNAs differential expression, these IncRNAs and certain signaling pathways may play an important role in the occurrence and development of microtia.The changes of LncRNA-NR₀28308, LncRNA-uc003jsd.1, LPL, TNFRSF11B, LncRNA-ENST00000449766 and ZFP36L2 expressions might be related to development of microtia.