The Functions And Mechanisms Of Agmatinase In Colorectal Carcinoma

Objective:Colorectal carcinoma(CRC)is one of the most common gastrointestinal malignant tumors in humans.Agmatinase(AGMAT)served as a key enzyme in the alternative pathway of polyamine metabolism,mainly located in mitochondria.As a oncogene,the abnormal expression of AGMAT in various tumor tissues influenced the role of Agmatine in polyamine biosynthesis by regulating the substrate of Agmatine,which promoted the occurance and development of tumors.AGMAT is a target gene of miR-151a-5p through the database analysis,however,the potential biofunction and molecular mechanism of miR-151a-5p and AGMAT are needed to be uncovered,and further studied in CRC.Methods:1、The relative expression levels of AGMAT in various tumor tissues and adjacent normal tissues were analyzed using the Timer database.The expression levels of AGMAT in CRC via the GEPIA database and HPA.2、The correlativity between AGMAT and miR-151a-5p was analyzed by the starbase database and miRmap database.3、The relative mRNA and protein expression levels of AGMAT in CRC cell lines were detected by RT-qPCR and Western blot.4、The overexpression/knockdown plasmids of AGMAT was designed to establish the AGMAT stable expression CRC cells.miR-151a-5p si RNA were designed to transfect into the AGMAT stable expressing CRC cell lines.si NC or Empty plasmid served as a negative control.5、To investigate the biofunctions of AGMAT in CRC,the proliferation,migration,and invasion ability of CRC cells were detected in CCK-8 and Transwell assay.6、To investigate the interactive relationships of miR-151a-5p and AGMAT in CRC,The dual-luciferase reporter analysis assay was used.7、To investigate the molecular mechanisms of AGMAT in CRC,the effect of AGMAT on EMT-related proteins and the pivotal signaling molecules in the TGF-β/Smad pathway was analyzed by the Western blot.8 、 The rescue assay of miR-151a-5p proved that miR-151a-5p regulates the progression of CRC cells via targeting AGMAT.Results:1、The expression of AGMAT was abnormal in various tumor tissues from the Timer database.Compared with adjacent normal tissues,the expression levels of AGMAT in human CRC tissues were significant up-regulated from the analysis of GEPIA database and Timer database.2、The potential binding sites of miR-151a-5p and AGMAT were provided by the Starbase database and miRmap database.The positive correlativity between miR-151a-5p and AGMAT was revealed by the starbase database。3、Among the 7 human CRC cell lines,the m RNA and protein expression level of AGMAT was the highest in SW480 cells and the lowest in HCT116 cells.Thus,SW480 cells were selected to determine the effects on CRC cells after AGMAT knockdown,whereas HCT116 cells were selected to explore the effects of the overexpression of AGMAT.4、The results of the Dual-luciferase gene reporter assay showed that miR-151a-5p has a targeting binding relationship with the 3’-UTR terminal of AGMAT,and there was a positive correlation between miR-151a-5p and AGMAT.5、the over-expression of AGMAT promoted the proliferation,migration,and invasion ability of CRC cells,while the opposite trend was demonstrated in the knockdown of AGMAT.6、Following the over-expression of AGMAT,the protein levels of ZO-1 and Ecadherin were decreased by Western blot,additionally,the protein expression of Ncadherin,Vimentin,and β-Catenin,MMP2,and MMP9 was increased.On the contrary,the knockdown of AGMAT demonstrated the opposite trend.7、Western blot showed that the up-regulation of AGMAT enhanced the protein levels of Smad7,Smad4,p-Smad2/3 and TGF-β1,while the down-regulation of AGMAT significantly reduced the protein levels of Smad7,Smad4,p-Smad2/3 and TGF-β1.8、The rescue assay of miR-151a-5p revealed that miR-151a-5p promotes the proliferation,migration,and invasion ability of CRC cells via targeting AGMAT.Conclusion:In conclusion,as a directly target gene of miR-151a-5p,the expression of AGMAT was positively associated with miR-151a-5p,and dsyregulated in various tumors,included CRC.Functationally,miR-151a-5p promoted the proliferation,migration,and invasion ability of CRC cells via targeting AGMAT.Additionally,AGMAT enhanced EMT in CRC cells via activating the TGF-β/Smad pathway.These results suggested that miR-151a-5p/AGMAT axis may serve as a potential biomarker for the early diagnosis or a new therapeutic target for CRC patients.Figure [15] Table [27] Reference [87]...