LncMEG3,miR-9-5p And MiR-96-5p Cooperate In Modification Of NDRG1 And Induce The Progression Of Prostate Cancer

ObjectiveN-myc downstream regulatory gene 1(NDRG1)is a significant regulatory gene in cancer progression,while the molecular mechanisms may be varied in different cancers.We have demonstrated that non-coding RNA affected the expression of NDRG1 in prostate cancer(PCa).However,the function and mechanism of non-coding RNA regulating NDRG1 on the progression of PCa remain largely unclear.MethodOncomine,TCGA,GEO,and other databases were used to compare the expression of NDRG1,miR-96-5p,and lncMEG3 in PCa tissues and adjacent non-tumor tissues.Quantitative real-time PCR was used to determine the expression level of target genes.Western-blot and immunohistochemistry were used to detect protein level changed.Immunofluorescence(IF)and fluorescence in situ hybridization(FISH)were conducted to identify NDRG1 and lncMEG3 locations,respectively.Lentivirus and siRNA were utilized to construct overexpressed or knocked down cell lines.The changes in cell proliferation,cell cycle,cell apoptosis,cell migration,and invasion were observed by colony-forming assay,CCK-8 assay,Ed U assay,flow cytometry analysis,wound-heal assay,and transwell assay.Target Scan,Starbase,miRcode,and other databases were applied to found the micro RNA that may interact with lncMEG3 and NDRG1.The full-length of lncMEG3 and NDRG1 3’-UTR plasmids were constructed.The Luciferase reporter gene experiment was performed to confirm interaction.At the same time,the interaction between lncMEG3 and miR-9-5p was also confirmed by RNA Immunoprecipitation Assay(RIP).Overexpressed NDRG1 or lncMEG3 cells that contained luciferase genes were inoculated subcutaneously in nude mice.The ability of proliferation,migration,and invasion was observed by tumor formation through the animal imaging system.The tumor in situ and the metastatic site were observed by HE staining.The changes of related proteins were verified by immunohistochemistry and western-blot.Spearman correlation analysis was used to calculate the relationship between target genes.The Kaplan-Meier method was utilized for patients survival analysis.ResultOur studies have illustrated that the expression of NDRG1 in PCa was decreased,and it was closely related to the Gleason score and prognosis.NDRG1 could inhibit the epithelial-mesenchymal transformation(EMT)of tumor cells in vitro and in vivo.The expression of miR-96-5p was increased in PCa,which promoted the migration and invasion of cells.MiR-96-5p could target the 3’-UTR region of NDRG1 to regulate the expression and affect the EMT process of cells.Down-regulation of NDRG1 would activates the NF-k B pathway and cause changes in related genes.In addition,we also found that the expression of lncMEG3 in PCa was decreased.Our study showed that lncMEG3 could inhibit the proliferation of tumor cells and accelerate apoptosis.The expression of miR-9-5p was increased in PCa,which promoted the proliferation and reduce the apoptosis of tumor cells.Lnc MEG3 acted as a “ce RNA” to relieve the repressive effect of miR-9-5p on its target NDRG1,then suppressed the proliferation of tumor cells.ConclusionNDRG1 functions as a tumor suppressor.MiR-96-5p can modulate the activity of the NF-k B pathway by regulating the expression of NDRG1,which also promotes the process of cell migration,invasion,and EMT;Lnc MEG3 also functions as a tumor suppressor in affecting the proliferation and apoptosis of tumor cells through competitively binding miR-9-5p with NDRG1.