The Mechanism Of MiRNA-92a' Regulating The Proliferation Of Prostate Cancer Through EBAG9 Pathway

Objective:Ous aim is to investigate the influence and the mechanism of microRNA-92 a on the expression of EBAG9 in prostate cancer cells.Methods:Predict the target gene of microRNA-92 a by the database miRanda?TargetScan?PicTar. The 210 genes of intersection was selected by software miRwalk,from which10 target genes related to cancer was screened.The mi RNA-92 a mimic?miRNA-92 a mimic NC?miRNA-92 a inhibitor ?miRNA-92 a inhibitor NC was transfected into the PC3 cells,from which the total RNA was respectively extracted after 24 h. The expression of mRNA above the screening target gene were detected by RT-PCR method,and the results were statistically test.According to the statistical results,the target genes EBAG9 was selected.Then the miRNA-92 a mimic?miRNA-92 a mimic NC?miRNA-92 a inhibitor?miRNA-92 a inhibitor NC was transfected into the PC3 cells,from which the total protein of EBAG9 was extracted after 48 h by Western blot.40 cases of prostate cancer and benign prostate hyperplasia was collected by Tianjin Institute of Urology,from which the total RNA was extracted and the RT-qPCR was carried out.The distribution of EBAG9 in tumor tissue and normal tissue was detected combined with Oncomine database.The potential binding sites of EBAG9 with mi RNA-92 a was predicted by microRNA database software.Primers of EBAG9 was designed,the 3'UTR fragments of wild-type and mutant-type were amplified by PCR.And the agarose gel electrophoresis was used to identify each other respectively.And the target gene and plasmid with fluorescent was digested by restriction endonuclease.finally, the plasmid with the target gene were connected and the 3'UTR luciferase reporter plasmids containing the EBAG9 was constructed.Agarose gel electrophoresis was used to verify after the digestion of restriction enzyme.Then the gene sequence was used to verify its correctness.The recombinant plasmid containing the fragment of EBAG9 and miRNA-192 a were transfected into 293 T cells respectively.Luciferase activity was detected after transfection by fluorescence report detection system.Results:1.10 tumor-associated target gene was successfully screened out by miRNA software with database miRanda, TargetScan, PicTar, miRwalk. 2.After the transfection of mi RNA-92 a mimic?miRNA-92 a mimic NC?mi RNA-92 a inhibitor?miRNA-92 a inhibitor NC 24 h,the EBAG9 gene has significant statistical significance on mRNA levels by the validation of RT-PCR in each experimental group. 3.After the transfection of mi RNA-92 a mimic?miRNA-92 a mimic NC?mi RNA-92 a inhibitor?miRNA-92 a inhibitor NC 48 h,the EBAG9 gene has significant statistical significance on protein levels by Western Blot in each experimental group.And the EBAG9 gene change of mRNA levels and protein levels are consistent. 4.The expression of EBAG9 gene was significantly higher in prostate cancer cells and tissues compared with normal prostate hyperplasia tissue and cell. 5.The fragment containing EBAG93'UTR was successfully constructed and its correctness was verified by agarose gel electrophoresis. 6.The recombinant plasmid containing the wild-type 3'UTR region of EBAG9 was successfully constructed and its correctness was verified by agarose gel electrophoresis.The vector containing dual luciferase reporter gene was linked successfully,the plasmid was amplifed to obtain high concentration.The digested fragment was verified by properly agarose gel electrophoresis. 7.the plasmid containing the wild-type 3'UTR region of EBAG9 was amplifed to obtain high concentration.and the result were sequenced and verified successfully. 8.The recombinant plasmids containing the wild-type 3'UTR region of EBAG9 and miR-92 a fragment were successfully transfected into 293 T cells using Roche.Dual Luciferase activity was determined using a microplate reader.The results show miR-92 a can significantly reduce the luciferase activity of the recombinant plasmid containing the 3'UTR of EBAG9 and it has significant statistical significance related to the control group.Conclusions:1. The expression of EBAG9 gene was significantly higher in prostate cancer cells and tissues compared with normal prostate hyperplasia tissue and cell.It is positively related to tumor grade and stage significantly by analyzing the clinical andpathological data.2. The mi RNA-92 a regulate proliferation of prostate cancer cells in the 3'UTR of EBAG9. 3. It is very important to seek further major gene transcription regulator of EBAG9 with further research, and then discuss the complex regulation mechanism.