The Biological Activity Effects Of RNA Interference For ADAMs Gene Family Silencing On Tumor Cells

BACKGROUNDThe tumor cell microenvironment is important in the proliferation and invasion of the tumor, ADAMs(a disintegrin and metalloprotease), a family of zinc-dependent proteolytic enzymes, play an important role in the modulation of the tumor cell microenvironment. They are multifunctional proteins that are involved in the proteolytic processing of other transmembrane proteins, cell adhesion and cell signaling events. They are called ADAMs for structure of metalloproteinase domain and disintegrin domain. We have been taking up with the subject of inflammation and AD mediated by APP pathway, which is characterized by ADAM17 and ADAM10. The proprotein convertases (PCs) are implicated in the activation of various precursor proteins that play an important role in tumor cells, which is characterized by PC7 predominantly. We found that the relationship between tumor cells and ADAMs is very close excitely, then. It is reported that ADAM17 and ADAM10 are highly expressed in tumors and is predominantly localized on the surfaces of most tumor cells including cancers of breast, ovary, kidney, colon and prostate. Also ADAMs has been reported to play an important role in tumor formation, invasion and metastasis in both animal models and cancer patients. We hypothesized that knockdown of ADAMs inhibit tumor information and invasion, and that ADAMs would be a good molecular target for tumor therapy.RNAi is a sequence-specific post-transcriptioned gene silencing mechanism, inducing by double-stranded RNA (dsRNA) and caused degradation of mRNA homology in sequence to the dsRNA. RNAi has distinct advantages over other methods that inhibit gene expression, such as its high specifity, high efficacy, high stabilization and hereditablity, and is gradually becoming a substitute anti-sense RNA a new research point. Originally, chemically presynthesised siRNAs were used for silencing, whereas more recently, to overcome the transient nature of siRNA, DNA vector-based strategies have been developed to delivery short hairpin RNAs(shRNA) that are processed intracellularly, giving rise to siRNAs. Vector-based RNAi has become a popular approach for analyzing gene function in mammalian cells. Recent studies have reported that effective knockdown of gene can be achieved by short hairpin RNAs(shRNA) in a single vector. In this study, we target ADAM17,ADAM10 and PC7 for the treatment of tumor cells with shRNA, because these tumor cells were found to express high levels of these genes and to assess the possibility whether ADAMs would be a molecular target for cancer gene therapy.Part I Effects of ADAM17 silence by RNA interference on the proliferation and apoptosis of many kinds of tumor cellsObjective: To investigate whether short hairpin RNA expression vector targeting ADAM17 gene can have influence on proliferation and apoptosis of tumor cells, as is a potential research target, which provide a kind of method to resist human tumor cells.Methods: The constructed ADAM17 gene interfering eukaryotic expression plasmid were transfected into tumor cells ( Hela,HepG2,SGC-7901,MCF-7 ) via lipofectamine mediation.Green fluorescent protein expression and transfection efficiency were observed under fluorescent microscopy. The growth of tumor cells was detected in a week. MTT assay was performed to detect the state of tumor cells proliferation. Flow cytometry was performed to detcect the state of tumor cells apoptosis.Results: The interfering eukaryotic expression plasmid of ADAM17 gene was transfected into the tumor cells successfully. Fluorescence microscopy showed that the transfection efficiency was about 80%. MTT detection showed that cell proliferation in RNAi group was significantly lower than in both the controls (P < 0. 01 or 0.05). The apoptosis percentage in RNAi group (43.68±5.62%%) was significantly higher than that in the two negative controls (21.96±0.76 and 20.66±0.70%) after 48 hours (each P<0.01).Conclusion: The silencing of ADAM17 by means of eukaryotic expression plasmid can promote the apoptisis of tumor cells, suppress their proliferation and slow their growth significantly, which will provide a novel therapeutic target for the resistance by RNAi in tumor cells. Part II Small interfering RNA inhibits expression of ADAM10 and PC7 gene in N2a cellsObjective: To investigate whether short hairpin RNA(shRNA)of targeting ADAM10 and PC7 gene can inhibit the expression of target gene in mammalian cells,which may provide a useful profile for futher investigation and a promising gene therapy approach for tumor treatment by RNA interference.Methods: The target gene were designed, synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase to construct eukaryotic expression plasmid of small hairpin RNA( shRNA) targeting ADAM10 and PC7 gene.They were proved by the methods of enzyme digestion and sequence analysis. The transfection efficiency was determined by using fluorescence microscopy.The recombinant plasmids were transfected into N2a cells via lipofectamine mediation.The mRNA silence of target gene was detected by semi-quantitative RT-PCR and the expression level of protein was determined by Western Blot and immunofluorescence staining, respectively. The recombinant plasmid (pGensil-1-PC7) was injected into the hippocampus of mouse. The results were compared between the transfected and the normal control cells.Results: The interfering eukaryotic expression plasmids of ADAM10 and PC7 were constructed successfully. Fluorescence microscopy showed that the transfection efficiency was about 90%. After transfection, target gene expression in N2a cells was 1.35±0.09 for control group, 0.35±0. 03 for recombinant plasmid transfection group, respectively. The ulteration status of target gene protein levels were similar to that of target gene mRNA levels. The recombinant plasmid (pGensil-1-PC7) was transfected into mouse successfully, which was observed by using immunofluorescence. effects on RNA transcription and protein expression so that suppress the growth of tumor cells and induce apoptosis of the cells possibly, which offer a reliable base for the further in vivo animal experiment. It will be a rational approach in gene therapy for tumor.In brief, shRNA's inhibiting role is successful. It proves that we use siRNA to interfere the expression of target gene in tumor cells effectively so that provide the base that we can cure tumor in the future. In virtue of dramatic inhibitory effect of ADAM family gene (ADAM17,ADAM10,PC7) on tumor cells, it might be a potential target of tumor treatment, which can lay the foundation for further research on gene therapy in vivo experiment.