Experimental Research Of MiR-433 Targets Promoting Apoptosis And Inhibiting The Proliferation And Migration In Gastric Cancer Cells Targeting JNK1

Objective: The purpose of this study is to explore the relationship between miR-433 and gastric cancer,and to elucidate the role of miR-433 in the development of gastric cancer and its possible intrinsic mechanism of action.The further objective of this study is to provide further ideas for the etiology and molecular pathology of tumors,in the hope of finding new tumor therapeutic targets,and providing new ideas and strategies for clinical treatment of gastric cancer.Methods: Firstly,human normal gastric mucosal epithelial cells(GES-1)and human BGC-823,MNK-28 and MNK-45 gastric cancer cell lines were cultured.Total RNA was extracted by TRIzol kit and qRT-PCR was performed by TaqMan microRNA assay to detect the level of microRNA-433 in gastric cancer cell lines.Bioinformatics revealed that JNK1 mRNA contained potential binding sites of miR-433.The 3’UTR end of human JNK1 gene was inserted downstream of the luciferase gene.The activity of luciferase and renin luciferase was detected by double luciferase reporting system.The expression of JNK1 protein was detected by miR-433 transfection,co-transfection of miR-433 and JNK1 to MNK-45,respectively.Secondly,after miR-433 transfection,miR-433 and JNK1 co-transfection to MNK-45,CCK-8 method was used to detect cell proliferation,PI staining was used to detect cell cycle,Annexin V-FITC/PI staining was used to detect cell apoptosis,and Western blotting method was used to detect the expression of apoptosis-related factors Bcl-2,Bax,PCNA and Caspase-3,and to observe the effects of miR-433 on proliferation,cell cycle and apoptosis of MNK-45 cells through JNK1.Subsequently,cell migration and invasion tests were carried out by conventional methods.The expression of MMP-2 and MMP-8 was detected by Western blotting method,and the migration and invasion of MNK-45 cells by Mi-433 through JNK1 were observed.Results: The results showed that the expression levels of miR-433 in BGC-823,MNK-28 and MNK-45 gastric cancer cells were all significantly lower than those in normal GES-1 cells;miR-433 significantly inhibited luciferase activity downstream of JNK1 3’UTR,suggesting that JNK1 may be a target gene of miR-433 in gastric cancer cells;The expression of JNK1 protein in MNK-45 cells was significantlyinhibited by the co-transfection of miR-433 and JNK1,which was abolished by the co-transfection of miR-433 and JNK1.Subsequently,the results showed that miR-433 gene transfection could significantly inhibit the proliferation of MNK-45 cells,induce cell cycle arrest,promote cell apoptosis,inhibit the expression of PCNA protein,up-regulate Caspase-3 protein expression and increase the Bax/Bcl-2 ratio;co-transfection of miR-433 and JNK1 significantly inhibited the beneficial effects of miR-433 gene transfection on the proliferation,cell cycle and cell apoptosis of MNK-45 cells.Finally,miR-433 gene transfection significantly inhibited the migration and invasion capability of MNK-45 cells,inhibited the expression of MMP-2 and MMP-9 proteins.Similarly,these effects were reversed by JNK1 transfection.Conclusions: The down-regulation of the expression of miR-433 in gastric cancer cell line is negatively correlated with the expression of JNK1.Increasing the expression of miR-433 can inhibit the proliferation and induce apoptosis of gastric cancer MNK-45 cells,thereby reducing the invasion and migration ability.In conclusion,miR-433 participates in the occurrence and progression of gastric cancer.Increasing the expression of miR-433 plays a biological role in promoting apoptosis and inhibiting the migration and invasion of gastric cancer cells by targeting JNK1.MiR-433 has become a new potential target for the diagnosis and treatment of gastric cancer.