Effects Of NME2 Silencing On Hepatoma Carcinoma Cells Proliferation,invasion And Metastasis

Objetive:Protein products of members of the NME gene family are involved in cell growth,differentiation,signal transduction,etc.,and play an important role in the occurrence and development of cancer.Among them,NME1 and NME2 genes have 88%homology.NME1 protein is a metastatic inhibitor,which has been proved to inhibit the metastasis of tumor cells in human tumors such as liver cancer,breast cancer,colorectal cancer and ovarian cancer.Studies have shown that NME1 and NME2 are highly expressed in HCC,and the expression of NME1 mRNA is inversely proportional to the incidence of intrahepatic metastasis and TNM staging.However,the role of NME2 in large expression in HCC remains unclear.Therefore,in this study,the effects of NME2 on the biological functions such as proliferation,apoptosis,invasion and metastasis of hepatocellular carcinoma cells were preliminarily investigated through gene interference technology.Methods:In this study,sk-hep-1 cells showed low expression of NME2 protein by recombinant lentivirus infection.The experiment was divided into sh-NME2 group and sh-NC group,namely plv-hu6-nme2 virus group and negative control virus group.After 72h of cell infection in the two groups,protein western blot and cellular immunofluorescence were used to detect the expression of NME2 protein in the two groups.The sh-NME2 group was compared with the sh-NC group,and the expression of NME2 protein decreased significantly,indicating successful transfection.Successful transfection cells of two groups,with CCK-8 test cell activity,clone formation and soft agar cloning experiments with the tablet to detect cell proliferation and colony formation ability,flow cytometry to detect cell cycle and apoptosis,scratch test cell migration ability,Transwell Chambers method to detect cell invasion ability,in the end,western blot was used to detect the changes of relevant oncogene proteins.Results:1.Western blot results showed that the expression of NME2 protein in sh-NME2 group was significantly decreased compared with that in sh-NC group.Through immunofluorescence,it can also be seen that NME2 protein is expressed in the cytoplasm and nucleus,and the NME2 fluorescence of the sh-NME2 group is significantly weaker than that of the sh-NC group,indicating that the expression of NME2 protein in the sh-NME2 group is significantly lower than that in the NC group,which is consistent with the western blot results of the protein,thus indicating the success of sk-hep-1 cell line construction with low expression of NME2.2.The results of CCK-8 method showed that the growth rate and proliferation capacity of cells in sh-NME2 group decreased compared with sh-NC group.3.The results of plate cloning and soft agar cloning experiments showed that the colony forming ability and cell proliferation ability of sh-NME2 group were significantly reduced,compared with sh-NC group.4.Flow cytometry results showed that the s-phase cells in the sh-NME2 group increased significantly compared with the sh-NC group,and the proportion in the G0/G1 phase decreased significantly,indicating that the silencing of NME2 gene caused sk-hep-1 cells to block in the s-phase,5.Flow cytometry results showed that the apoptosi’s rate of cells in the sh-NME2 group was higher than that in the sh-NC group,indicating that NME2 gene silencing promoted the apoptosis of sk-hep-1 cells.6.The results of the scratch experiment and the Transwell chamber experiment showed that the migration ability of cells in the sh-NME2 group was weaker than that in the sh-NC group,and the cell invasion ability was also weaker than that in the sh-NC group,indicating that the NME2 gene silencing reduced the migration and invasion ability of sk-hep-1 cells.7.Western blot results showed that in the sh-NME2 group,c-myc,NFκB and Cyclin-D1 decreased significantly compared with the sh-NC group,indicating a positive correlation between NME2 and c-myc,NF-κB and Cyclin-D1.Conclusion:1.The proliferation of sk-hep-1 cells was decreased by NME2 gene silencing.2.The silencing of NME2 gene blocks sk-hep-1 cells in the S phase.3.NME2 gene silencing reduced the invasion and metastasis ability of sk-hep-1 cells.4.NME2 may promote cancer by regulating the expression of c-myc,NFκB and Cyclin-D1.