The Study On Changes Of MHCC97-H Cells Biological Behaviors And KAI1 Gene Expression By Small RNA Interference Of Ubiquitin Ligase (E3) Gp78

Background and ObjectiveHepatocellular carcinoma (HCC) is the second world wide malignant disease that causes human death,especially its metastasis out of the liver is difficult for the clinical therapy. Hepatocellular carcinoma usually metastasis to neighboring or distant tissues and organs early through the blood, so the liver cancer cells have strong invasive ability. The mechanism of invasion is very complicated. More and more studies suggest that the migration and invasion capabilities of tumor cell, is one of the important mechanism of tumor metastasis. Previous studies in animal cell lines have shown that gp78, as an ubiquitin ligase E3 of endoplasmic reticulum (ER) associated degradation, may participate in the mechanism of metastasis. Its overexpression can induce the change of the phenotype of tumor cells, and this change may enhance the ability of cell survival and proliferation, potency of cell invasion. Recent studies have reported that gp78 mRNA expression in tumor tissues was significantly increased when compared with adjacent normal tissue. Meanwhile, some other studies have demonstrated that the metastasis suppressor factor KAI1 is the specific substrate of gp78. The E3 activity of gp78 can promote the metastasis of sarcoma through facilitating the degradation of KAI1. However, the role of gp78 in the process of HCC cell invasion and metastasiscurrently is poorly understood. Especially, the effects of gp78 on the biological characteristics of HCC cells in vitro remains unclear.In this study, we assume that downregulation of gp78 expression may lead to increase KAI1 gene expression in HCC cells. Further more, it will decrease the ability of tumor cell proliferation, colony formation, migration and invasion. And then we may infer that the metastatic ability of HCC cells will be reduced. In order to test this hypothesis, the expression of gp78 in hepatoma cells MHCC97-H was inhibited by RNAi. The changes of biological behaviors, including: cell proliferation, migration and invasion, and also the expression of KAI1 were observed.Methods1. Three siRNAs targeted gp78 were designed by a program that is available online (www.genscript.com). siRNA-expressing plasmids were constructed by cloning the siRNA fragment into pRNAT-U6.1/Neo after BamHI and HindIII enzyme digestion. The plasmids were extracted following the manufacturer's instruction and then sequenced to confirm the correct insertion; the new plasmids were named pRNAi-1, pRNAi-2 and pRNAi-3. MHCC97-H cells were seeded on 6-well culture plates and grown to 80% ~ 90% confluences before the transfection. The recombinant pRNAi-1, pRNAi-2 and pRNAi-3 were used for the transfection in the corresponding experimental groups. LipofectamineTM 2000 was treated in the blank control group, and the empty plasmid pRNAT-U6.1/Neo was transfected into the negative control group. The transfected cells were selected by G418. Until positive clones were discovered after several days (7-10days),the cells were cultured and finally selected by G418 for another 10 days. Single clones were selected to build a stable transfected cell line.2. Changes of biological behaviour of MHCC97-H,including cell proliferation, clone formation, the ability of migration and invasion after gp78 RNA interference were investigated.3. Detection of expression of gp78 and KAI1 in MHCC97-H cells before and after gp78 silenced by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot analysis.Results1. The recombinant plasmids were identified to have correct sequences by DNA sequencing analysis, and the result confirmed that the DNA chains had ligated to the vectors. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. Green fluorescene of the stable transfected MHCC97-H cell lines could be observed under laser scanning confocal microscope.2. Gp78 expression were down-regulated significantly in MHCC97-H cells after transfection with pRNAi-1, pRNAi-2 (all P<0.05). Since the pRNAi-2 was the most effective vector, it was selected for further experiment on the effect of RNAi on the expression of KAI1. 3. MTT result showed that cell proliferation ability of pRNAi-2 cells was significantly lower than that of empty plasmid group and untreated controls. Plate clone formation experiment demonstrated that single cell clone formation capacity of pRNAi-2 cells was obviously lower than that of empty plasmid group and untreated controls. The experiment of cell scratch Healing revealed that the migration capacity of pRNAi-2 was obviously lower than that of both controls.The experiment of Transwell insert invasion indicated that the invasion capacity of pRNAi-2 was obviously lower than that of both controls.4. The KAI1 expression in cells transfected with pRNAi-2 was significantly increased compared with that in MHCC87-H cells transfected with pRNAT and untransfected MHCC97-H cells by RT-PCR and Western blot analysis.ConclusionsIn vitro, gp78-specific RNA interference can signifcantly inhibit the expression of gp78 in MHCC97-H cells, and increased the expression of KAI1 gene, which may reduce the proliferation, colony formation, migration and invasion capacity of MHCC97-H cells, and thus may weaken the hepatoma cells metastasis, and revers its malignant phenotype.