Expression Of HepaCAM And Its Effect On Invasion Of Tumor Cells In Renal Cell Carcinoma

PART ONEObjective: To investigate the relationship between downexpression of hepaCAM (hepatocyte cell adhesion molecule) and renal cell carcinoma (RCC).Methods: The gene expression of hepaCAM was examined by semi-quantitative RT-PCR in 6 normal renal tissues and 30 paired RCC renal tissues specimens,and its relationship with clinicopathological characteristics of tumor was analyzed.Results: hepaCAM mRNA expression was very strong in 6 normal renal tissues, while in 30paired RCC renal tissues,the expression of hepaCAM was significantly lower in RCC tissues than that in their adjacent normal renal tissues (P<0.01). But no correlation with different grades and different stages of RCC was found(P>0.05).Conclusion: the expression of hepaCAM was high in normal renal tissues and low in RCC tissues, but different grades and stages couldn't be evaluated by hepaCAM.PART TWOObjective: The identification of eukaryotic expression plasmid of hepaCAM. To observe the localization of hepaCAM in 786-0cells and establish a stable and efficient transfection method of renal cancer cell line786-0.Methods: The plasmids were digested by BamHI/HindIII and sequenced to confirm the inserted sequence. After the constructed plasmids had been transfected into renal cancer cell line 786-0,transfection efficiency were calculated by fluorenscence microscope at different time and different plasmid-liposome proportion. Then positive cell clones were selected with G418 and amplified. The localization of hepaCAM in 786-0 cells was observed with fluorenscence microscope and RT-PCR analysis was taken to know about increase of hepaCAM expression in 786-0cells.Results: It was confirmed by endonuclease digesting and DNA sequencing that the recombinant plasmids had been constructed successfully. And the stable and efficient transfection method was established,the best transfection time was 48h and the most suitable plasmid-liposome proportion was 1:2. hepaCAM protein was localized on the perinuclear membrane,cytoplasm and at the tip of cell surface protrusions.The result of RT-PCR showed that pEGFPN2/hepaCAM plasmids had obviously increased hepaCAM expression of 786-0 cells,suggesting that stable selection was successful.Conclusion: pEGFPN2/hepaCAM was stably transfected into 786-0cells which lay the foundation for further studies of hepaCAM in RCC.PART THREEObjective: This study was to evaluate the effect of hepaCAM on invasion in human renal cancer cell line 786-0.Methods: MTT and colony formation were performed to examine cell growth activity.The expression of hepaCAM and MMP2,9 mRNA before and after stable transfection into 786-0cells was detected by RT-PCR.The activities of MMP2,9 were analyzed by gelatin zymography. The invasiveness of 786-0 cells in vitro was determined by Transwell chambers methods.Results: When hepaCAM was transfected into 786-0 cells, MTT showed the growth inhibition rates on the fourth,fifth,sixth day of culturing were 26.5%,38.1%,35.7%respectively,and the number of colony formation was largely reduced to 5 folds according to colony formation assay. The mRNA expression and activities of MMP2,9 in 786-0cells were inhibited by hepaCAM (P <0.05).The number of cells migrating through the Transwell chamber membrane was significantly reduced after stable transfection of hepaCAM(P <0.01).Conclusion: hepaCAM exhibits antiproliferative effect on 786-0 cells and may play a crucial role in the invasion of renal cell carcinoma through downregulating expression of MMP2,9 and inhibiting their activities, so increasing hepaCAM may be a potential strategy in preventing the invasion of renal cell carcinoma.