The Role Of Oncogene RMP Expression In Epithelial-Mesenchymal Transitions In Hepatocellular Carcinoma And It’s Relationship With Tumor Invasion And Metastasis

Objective：In this study, we investigate that overexpression and depletion of RMPcould affect cellular morphology, cell migration and invasion, and eventually induceEMT in hepatocellular carcinoma cell.Methods：Vectors of pCDNA3.1-RMP and pGPU6-RMPi were identified bydouble restriction enzyme digestion followed by transfection. Identified the suitableG418concentration in the selection of stable transfection HepG2cell. Obtained theRMP overexpression and depletion in HepG2cell lines by G418selection method.Transfected HepG2cell was divided into different groups: RMP overexpression groupand RMP depletion group. After different treatment, we verified the integrated plasmidin genomic DNA by RT-PCR. RMP expression level in each group was detected byqRT-PCR. Stable transfected HepG2cell proliferation viability was measured by CCK-8assay. Cell adherence assay was used to detect the adhering capacity of each cells. Cellability to migrate was measured by wound healing assay. Invasion and chemotaxis ofeach cell was detected by transwell assay followed by transfection with different dose ofpCDNA3.1-RMP plasmid. MMPs activities were measured by RT-PCR and zymograph.E-cadherin, N-cadherin, vimentin protein expression were assessed by Western blot.Results:1. We successfully detected1619bp band in pCDNA3.1-RMP plasmid and53bp hairpin in pGPU6-RMPi plasmid after double restriction enzyme digestion2. HepG2was cultured in DMEM which contained different concentrations ofG418for14days. After obeservation we confirmed that the600ng/μl is the bestconcentration of G418for this experiment.3. HepG2was cultivated in DMEM medium which contained G418aftertransfection, We obtained the stable transfection cell line after single clone selection andidentification.4. The cellular morphology of RMP stable expressed HepG2cell changed tospindle shape, and lost the cell to cell adhesion.5. RMP overexpressed cell line shows higher anchor dependent growth ability insoft agar clone formation assay, but it was decreased followed by RMP depletion.Through futher analysis, we found that the morphology of RMP overexpressed cellclusters were loose and lost the cell-cell adhesion, in contrast to RMP depleted cellclusters which formed tight and well junction just like the normal HepG2cell.6. RMP overexpression reduced cell adhesion ability but RMP depletion didn’t.7. RMP overexpression promoted cell migration and invasion.8. RMP expression in HepG2cell increased the MMP-2and MMP-9expression, incontrast to RMP depletion,which downregulated the expression of MMP-2and MMP-9.9. RMP is essential for the activity of secreted MMP-2in zymograph.10. Western blot analysis demonstrated that the overexpression of RMPsignificantly reduced the E-cadherin expression which is epithelial phenotype guard andalso promoted the expression of mesenchymal marker N-cadherin and Vimentin.Interestingly, depletion of RMP expression will affect none of them.Conclusion：1. In vitro, RMP can promote metastasis of hepatocellular carcinoma, and increasecell invasion through upregulated and activated MMPs. However, RMP depletion canrestrain such phenomenon.2. RMP can promote EMT in hepatocellular carcinoma cells by downregulating E-cadherin and upregulating N-cadherin and Vimentin. Also RMP can affect cell andcell cluster morphology in three dimensional culture.