Molecular Pathology Of MicroRNA-454-3p And Its Target Genes SYT6 In Type B3 Thymoma And Thymic Carcinoma

Thymic epithelial tumors(TET)are a group of rare tumors,occuring in the anterior superior of mediastinal.The biological behavior,response to treatment and the prognosis of tumor are varied in different subtypes.Among the seven subtypes,type B3 thymoma and thymic carcinoma are the most malignant types,which often lead to misdiagnosis of each other as the histomorphology and biological behavier are often similar.MiRNAs are short non-coding RNAs about 22 nt long which have been raised as candidates for tumor suppression and tumor biomarkeds.miRNAs are also known to be involved in reprogramming and so as to be considered to play important roles in a wide range of biological processes.In this study,we attempted to retrospectively analyze type B3 thymoma and thymic carcinoma by a variety of experimental methods,in order to identify the clinicopathological characteristics,differential diagnosis and their differences in miRNAs.Materials and Methods(1)59 cases of thymic epithelial tumors(including 30 type B3 thymoma and 29 thymic squamous cell carcinoma)were collected from Nanjing Jinling Hospital andAnhui Provincial Hospital South district.Carefully observed the clinical and pathological data were collected and analyzed.The expression of MUC1 and GLUT1 in thymic epithelial tumors was examined by immunohistochemical staining and the data were analyzed.(2)Three cases of type B3 thymoma and three cases of thymic carcinoma were selected for further study for gene chips experiment.Paraffin-embedded tissues were collected and the total RNA was extracted.The miRNA expression profiles were analyzed by miRNA microarray and GO analysis and signal pathway analysis were used for predicting miRNA target gene.(3)The total RNA was extracted from paraffin samples of three type B3 thymomas and three thymic carcinoma by using real-time quantitative PCR.(4)The differential expression of microRNA and its target genes between type B3 thymoma and thymic carcinoma was detected by dual luciferase reporter assay.The antibodies related to target genes were detected by immunohistochemistry to further confirm the expression of the target protein.Results.(1)Type B3 thymoma and thymic carcinoma have the similar pathology morphology.Immunohistochemical stain of MUC1 and GLUT1 showed the two proteins were found to be valuable in the differential diagnosis of thymic carcinoma and type B3 thymoma,with a high sensitivity and specificity.(2)Three patients of type B3 thymoma and three cases of thymic squamous cell carcinoma were analyzed by miRNA microarray.Thirty-six up-regulated miRNAs and nineteen down-regulated miRNAs were found in thymic carcinoma compared with type B3 thymoma.(3)MiR-192-5p,miR-454-3p and miR-148a-3p were validated by RT-PCR,and indicated that the result of miR-454-3p and miR-148a-3p were in accordance with that of microarray.(4)Through bioinformatics analysis,GOs analysis showed that one of the target genes of miR-454-3p and miR-148a-3p was predicted to be SYT6 by target gene prediction.The relationship between miR-454-3p and miR-148a-3p and their target gene SYT6 was detected by double luciferase reporter assay.The results showed that miR-454-3p had significant inhibitory effect on SYT6.The immunohistochemical results showed that SYT6 protein was positive in thymic squamous cell carcinoma,the positive region was in the cytoplasm and nucleus,but not in the B3 thymoma,suggesting that SYT6 is up-regulated in thymic carcinoma expression.ConclusionsType B3 thymoma and thymic carcinoma are the two most malignant tumors of thymic epithelial neoplasms,which are similar both in the morphology and biological behavior.When it difficult to differentiate,they can be identified by new immunohistochemical markers MUC1 and GLUT1.The downregulation of miR-454-3p/miR-148a-3p is a common event in thymic carcinoma.MiR-454-3p is a tumor suppressor gene and its target gene SYT6 may be a potential proto-oncogene in thymic squamous cell carcinoma and play an important role in the pathogenesis of thymic carcinoma by promoting proliferation and differentiation,and it can be used as a diagnostic marker and a potential therapeutic target in future.