The Mechanism Of Antitumor Of MiR-199a-3p In Male Germ Cell Tumors

Testicular germ cell tumor(TGCT) is a kind of tumor, which originated form testis or near the testis and attracted much attention becuase of the increased incidence in recent years. The formation of TGCT was connected with the congenital dysplasia of testis, genetic factors, some gene mutations and signaling pathway abnormality. The current understanding of its pathogenesis is still very limited. microRNA-199a-3p(miR-199a-3p), which was regulated by TGF ?1 and participated in the male reproductive development, was screened in GC- 1 spg cells in our previous work. Current research showed that the mi R-199a-3p played an important role in the process of cancer development.In order to explore the function and regulatory mechanism of miR-199a-3p in TGCT, this study carried out the following several works:1. Discussed the biological effects of miR-199a-3p in testicular germ tumor cellsHuman malignant testis tumor cells Ntera-2 and mouse spermatogonial cells GC-1 spg were choosed as study materials in this study. Through MTT, wound healing and flow cytometry experiment, cell proliferation, migration and the distribution of cell cycle and apoptosis caused by mi R-199a-3p were detected. The results showed that miR-199a-3p can inhibit the proliferation, migration of both Ntera-2 and GC-1 spg cells, but had no effectt on cell apoptosis. In addition, the phenomenon of cell cycle arrest caused by miR-199a-3p was observed in Ntera-2 cells. The above results suggestted that miR-199a-3p palyed tumor suppress role in testicular germ cell tumor.2. Found that the miR-199a-3p target regulating the gene expression of Nme2Nme2 gene is a tumor metastasis suppressor,which was closely related with tumor invasion and metastasis. By bioinformatics analysis, dual luciferase reporter gene detection and quantitative PCR experiments, we concluded that miR-199a-3p target to 3 'UTR sequence of Nme2 gene. And the inhibition of mi R-199a-3p could obviously downregulate expression of Nme2 gene.3. Preliminary confirmed that miR-199a-3p and Nme2 had functional correlation in TGF-?1 pathway.After treated GC-1 spg cell by TGF-?1, the results showed that the expression of mRNA level of Nme2 and mi R-199a-3p were upregulated with the concentration increase of TGF-?1. We also investigated that the expression of Nme2 increased significantly after GC-1 spg treated by both miR-199a-3p and TGF-?1. Current research has shown that Nme2 play a negative feedback regulating role in TGF-?pathway. We hypothesized that some functions of the miR-199a-3p may be compensated by Nme2 in TGF-?1 pathway.4. Discussed the effect of mi R-199a-3p on cell metabolism in Ntera-2Biochemical analysis results showed that inhibition of miR-199a-3p significantly increased the content of lactate in Ntera-2 cells, suggesting that miR-199a-3p can influence the metabolism process of Ntera-2 cells. Then 12 metabolism genes regulated by miR-199a-3p were selected by high through-put qPCR array technology. Of these,2 genes were upregulated,10 were downregulate.5. Verified the relationship of miR-199a-3p and selected metabolism genes in clinical specimens of testicular tumors.Using GEO database analysis, the results of miR-199a-3p expression abundance in different normal tissues and its corresponding tumor tissue showed that miR-199a-3p expression was tissue-specific and it played a role of either tumor suppressor or tumor promoter in different tissues, respectively. In human TGCTs tissue, it acts as tumor suppressor. Quantitative PCR analysis of the clinical specimens showed that miR-199a-3p downregulated in testicular cancers, and suggestted that it indeed plays a role of tumor suppressor. Further analysis found that expression of 4 metabolism genes(LDHA, MCT1, PGK1 and TIGAR) were inverse with that of miR-199a-3p in testicular tumors. This result was consistent with the result in Ntera-2 cells and showed that LDHA, MCT1, PGK1 and TIGAR might be target genes of miR-199a-3p in metabolic pathway.In conclusion, we verified that miR-199a-3p acts as tumor suppress in testicular tumors, and Nme2 is its direct target gene. The mi R-199a-3p and Nme2 had some functional correlation in TGF-? pathway. We also found that miR-199a-3p was related to glycometabolism process in testicular tumor, and 12 differentially expressed metabolism genes regulated by miR-199a-3p were screened. Of them, LDHA, MCT1, PGK1 and TIGAR might be its potential target genes. As a result, we conclded that miR-199a-3p palyed tumor suppressor function in the male germ cell tumor through different way,it may be a potential biomarker for prognosis and treatment of testicular neoplasm.