MiR-199a-3p Inhibits Metabolism Of Testicular Tumor Cell By Targeting Sp1

Aerobic glycolysis is one of the characteristics of tumor metabolism.More and more experiments have shown that the enhancement of aerobic glycolysis contributes to the development of the tumor,so the inhibition of aerobic glycolysis can suppress the tumor development.MicroRNA(miRNA)is an endogenous non-coding RNA in multicellular organism with length about 21 to 25 nucleotides,and it is widely distributed and highly conserved in species,and involved in many important biological processes.In recent years,the study have found that miRNA plays an important role in glucose metabolism of tumors,through directly or indirectly regulating glucose metabolism related enzymes,oncogenes or tumor suppressor genes.As important regulators of tumor metabolism,microRNAs are expected to become the breakthrough of the disease treatment.The previous work found that miR-199a-3p is regulated by TGF-β1 pathway in spermatogonia and participates in the male reproductive development.In recent years,some researches have shown that the expression of miR-199a-3p is low and might be an inhibitor in tumors.In Ntera-2 cells,human testicular teratoma cell line,the study showed that overexpression of miR-199a-3p inhibits cell proliferation,migration and invasion.Because of its complexity of regulation function,the role of miR-199a-3p in testicular tumor remains to explore.Before,our laboratory proved that miR-199a-3p regulates the lactic acid production and intake of glucose in Ntera-2 cells,and four significant genes(LDHA,TIGAR,PGK1,MCT1)regulated by miR-199a-3p through QPCR array technology were selected.Through bioinformatics prediction,we found that there is no binding site of miR-199a-3p in the 3’UTR of four genes,suggesting that it is an indirect regulation.Subsequently,all target genes of miR-199a-3p were predicted by Targetscan databases and named as A data set;Then transcription factor binding sites of four genes were predicted using software Genomatix transcription factor analysis and named as B data set.Finally integrating data sets A and B,the transcription factor Spl was selected to a common molecule in both data sets.Thus we speculated that Spl might be linker between miR-199a-3p and metabolic genes.In order to verify the above hypothesis,in this article,we used Ntera-2 cells as experiment material,and discussed the possibility of miR-199a-3p/Spl signal pathway and identified the downstream factors of this pathway.Our works include the following several aspects:1.Analysis the effects of miR-199a-3p on metabolism of Ntera-2 cellsThrough transfection of miR-199a-3p mimics,we detected the production of lactic acid,glucose intake,ATP level and the content of ROS in Ntera-2 cells.The results showed that in the Ntera-2 cells,miR-199a-3p has inhibitory effect on lactic acid production,glucose intake and ATP levels,and had no obvious effect on ROS.2.Definite transcription factor Sp1 as the direct target of miR-199a-3pFirstly,we successfully constructed double luciferase report gene vectors with Sp13’UTR of wild type or mutant type,respectively;Then the vectors were respectively transfected into Ntera-2 cells.After 48 h,by double luciferase report gene experiment,we found that:in the Sp1 3 ’UTRwt group,compared with Negative Control(NC),the fluorescence signal of 199a-3p mimics is significantly lower;while in the Sp1 3’UTRmut group,there was no significant change about fluorescence signal between 199a-3p mimics and NC.It confirmed that the miR-199a-3p directly targeted Sp1.3.miR-199a-3p regulates the cell metabolism by inhibiting the Sp1Because the high expression of Sp1 in Ntera-2 cells,we transfected Sp1 siRNA to reduce the level of Sp1.Subsequently,we tested the production of lactic acid,glucose intake,ATP level and the content of ROS,and found that the down-regulation of Sp1 can reduce lactic acid production,glucose intake and ATP level and the content of ROS in Ntera-2 cells.The results showed that in the process of Ntera-2 metabolism,in exception of the content of ROS,the function of Sp1 and the function of miR-199a-3p have a reciprocal relationship.Through the functional rescue experiment,we found that the over-expression of Sp1 can abate the inhibition of miR-199a-3p on glucose metabolism,and can not ease the inhibition of miR-199a-3p on ATP levels.4.Identify the down stream metabolism gene of miR-199a-3p/Sp1 signaling pathwayTransfecting Sp1 SiRNA into Ntera-2 cells to reduce the expression of Sp1,then we detected the mRNA expression of glucose metabolism related genes LDHA,MCT1,PGK1,TIGAR by fluorescence quantitative PCR technique.The results showed that when the Sp1 was knocked down,the expression of LDHA and MCT1 is extremely significantly decreased,expression of PGK1 is significant decline,but there was no significant difference in the expression of TIGAR.Combined with our previous work,we speculated LDHA for miR-199a-3p/Sp1 downstream target.Finally by Western Blotting,the results confirmed that both over-expression of miR-199a-3p and the knock-down of Spl could decrease LDHA protein levels.Thus,LDHA was confirmed to be a downstream target of miR-199a-3p/Sp1.In conclusion,this thesis found and verified that the miR-199a-3p inhibits aerobic glycolysis process in Ntera-2 cell;the Spl is the functional target gene of miR-199a-3p in Ntera-2 cells.And LDHA was confirmed to be a downstream target of miR-199a-3p/Sp1.Thus,we confirmed the existence of miR-199a-3p/Sp1/LDHA signal pathway in Ntera-2 cells,and this pathway inhibits the process of aerobic glycolysis in Ntera-2 cells.