Inhibition Of Invasiveness Of Ameloblastoma By Suppression Of MMP-2 Gene Expression Using RNA Interference

Human ameloblastoma is the most frequently encounted odontogenic epithelium tumor in oral and maxillofacial region. About 59.3-63.2% of odontogenic epithelium tumors are ameloblastoma. Although it is usually considered as benign tumor, it has the potential of local invasion and bone destruction. The tumor cells of ameloblastoma often invade into bone trabecula. The recurrence rate of ameloblastoma is significantly high after surgical treatment, which might reach to 50%～90%. Repeated surgical operation is often harmful to patient and may cause severe facial deformity. For the sake of these, it is important to study the mechanisms of the invasion of ameloblastomas.The invasion of tumor mainly means the process that tumor cells invade and destroy the normal tissue around tumor cells. Usually, tissue around tumor is connective tissue, such as basement membrane and extracellular matrix. It is an important step of tumor invasion that degradation and destruction of these structure. The main tissue structures of basement membrane and extracellular matrix including collagen, laminin and fibronectin. To decompose and deconstruct these structure need the effect of resolvase. Studies indicated that matrix metalloproteinase (MMP) and its corresponding tissue inhibitors had taken an important role in the process of tumor invasion and metastasis. Among all of the MMPs discovered recently, MMP-2 is one of MMP which has the closest relationship with tumor invasion. Many studies shown that many kinds of malignant tumor has high expression of MMP-2, and the expression level of MMP- 2 has close relationship with the degree of tumor invasion and poor prognosis. The previous study of our group shows that MMP-2 was positive stain in the cytoplasm of peripheral cylindrocellular around tumor. MMP-2 mRNA also expressed in ameloblastoma, and the expression level was significant higher than that in normal dental follicle tissue.Usually, there are three levels of modulation for MMP-2 expression and activation, namely, the level of gene transcription, proenzyme activation and inhibitor. Recently, the highlight studies are mainly in latter two aspects. However, the study of the modulation for MMP-2 on transcription level is very few. In the previous study of our group, MMP-2 activity was significantly inhibited by MMP-2 specific inhibitor Ro31-9790, and so did the growth of xenograft of ameloblastoma in submembrane of kidney. This kind of modulation for MMP-2 was based on MMP-2 inhibitor level.RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that is thought to inhibit the replication and expression of selfish DNA elements and viruses. RNAi was mediated by the generation of 21-23nt dsRNA molecules, termed small interference RNA (siRNA). dsRNA molecules are processed by the RNaseIII-like enzyme Dicer to generate siRNA, and the siRNAs direct the recognition and subsequent degradation of homologous mRNAs by a multiprotein complex, and the expression of mRNA was silenced. Now, RNAi is a useful method for gene study. It has very important effect in the area of study gene function, gene knock out, adjustment of gene expression and gene therapy.The main purpose of this study is to suppress MMP-2 expression using RNA interference, and regulate MMP-2 expression on the gene transcript level. The invasive behavior of ameloblastoma after MMP-2 gene silenced by siRNA was studied. Then we further to study the mechanisims of the invasive growth of ameloblastomas. Therefore, this study including five parts a following: PartⅠPrimary Culture and Growth Characteristic of Ameloblastoma CellsUp to now, the mechanisims of invasive growth behavior into bone of ameloblastoma are not very clear. So, it is an important method to study the molecular mechanisims of invasiveness and biological characteristic of ameloblastoma by culture ameloblastoma cells in vitro. However, due to the short life span and slow growth of ameloblastoma cells and difficulty to purification in routing culture medium such as DMEM, it is not easy to study the biological behavior of ameloblastoma. In order to find a better culture medium for ameloblastoma cells'growth, serum free culture medium was adopted to culture ameloblastoma cells in this experiment. The growth characteristic of ameloblastoma cells was studied to provide a basis for further study of biological behavior of ameloblastoma.Methods: Defined Keratinocyte-serum free medium (DK-SFM) and DMEM medium were used for ameloblastoma cell culture in vitro. Cytokeratin 14, 16, 18 and Vimentin monoclonal antibodies were used for immunohistochemistric analysis to identify the origin of cultured cells. The growth rate in different culture medium was observed. The morphology and passages of ameloblastoma cell in different medium were studied. The SPF value and PI value of cells were detected by flow cytometry. Results: Cytokeratin 14 and 16 was positive stain in cultured cells, however, cytokeratin 18 and Vimentin was negative stain in culture cells. In DK-SFM medium, the morphology of cultured cells was distinct. Only some sporadic fibroblast was seen in it.Ameloblastoma cells were taken through 3 to 6 passages and the average passages were 4.8. The survival time of the cells was maintained 48 days to 97 days, and the average survival time was 73.8 days. The average value of SPF and PI of cells cultured in DK-SFM was 6.8% and 15.9%, respectively. However, in DMEM medium, the morphology of the cells cultured was not clear. On the other hand, a lot of fibroblast was seen in it. The cells were only taken through 2 to 4 passages and the average passages of it were 3.2. The survival time of the cells was maintained 27 days to 61 days, and the average survival time was 46.7 days. The average value of SPF and PI of cells cultured in DMEM are 5.4% and 11.0%, respectively. Conclusion: Ameloblastoma cells have long survival time in DK-SFM medium, which is more suitable for the growth of ameloblastoma cell than that of DMEM medium.PartⅡConstruction and Sequence Optimization of MMP-2 Gene Specific siRNA Expression PlasmidRNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that is thought to inhibit the replication and expression of selfish DNA elements and viruses. RNAi was mediated by the generation of 21-23nt dsRNA molecules, termed small interference RNA (siRNA). dsRNA molecules are processed by the RNaseIII-like enzyme Dicer to generate siRNA, and the siRNAs direct the recognition and subsequent degradation of homologous mRNAs by a multiprotein complex, and finally, the expression of mRNA was silenced. It is belong to post transcription silence. In RNAi experiment, there are two methods of constructing siRNA, namely, construct siRNA in vitro and siRNA expression in vivo. The former is to synthesis siRNA in vitro, and then the siRNA was transfected into cells. There are some problems in this kind of method to construct siRNA, such as short gene silence time and unstable transfection effect. The latter is to construct siRNA expression vector such as plasmid vector or virus vector. After tansfecting this vector into cells, it will produce siRNA automatically. It has long gene silence time and stable transfection effect. So, in present experiment, MMP-2 specific siRNA expression plasmid will be constructed, and the specific sequence of siRNA will be optimized in order to provide basis for next experiment study related to MMP-2 gene.Methods: Two items of siRNA expression plasmids, namely P1 and P2, were constructed by directed cloning and transfected into Hela cell by Lipofectamine 2000. The transfection effect was confirmed by fluorescence microscope. The transfection rate was checked by flow cytometry. Reverse transcript polymerase chain reaction (RT-PCR) was used to detect the expression of MMP-2 mRNA. Results: All of the two items of siRNA expression plasmids were successfully tranfected into Hela cells. The transfection rate at 72h of P1 and P2 was 41.8% and 93.2%, respectively. The transfection rate of P2 was significant higher than that of P1. The inhibition rate of MMP-2 mRNA expression of P1 and P2 was 38.6% and 68.9%, respectively. Conclusion: The MMP-2 specific siRNA expression plasmid constructed in vitro was successfully transfected into Hela cells, but it had sequence difference. The trasfection rate and gene silence rate of P2 is better than that of P1.PartⅢInhibition of Invasiveness of Ameloblastoma by Suppression MMP-2 Gene Expression Using MMP-2 Gene Targeting siRNAUsually, there are three levels of modulation for expression and activity of MMP-2, namely, the level of MMP-2 gene transcription, activation of proenzyme and inhibitor of MMP-2. In our previous study, the activity of MMP-2 was obviously inhibited by using MMP-2 specific inhibitor Ro31-990, and so did the growth of xenograft of ameloblastoma in submembrane of kidney. This adjustment of MMP-2 was based on MMP-2 inhibitor level. In present experiment, MMP-2 targeting siRNA expression plasmid constructed in Part III was transfected into ameloblastoma cells in order to silence MMP-2 gene and to regulate the expression of MMP-2 on the level of MMP-2 gene transcription. The invasive behavior of ameloblastoma after MMP-2 gene silencing by siRNA was studied.Methods: Ameloblastoma cells were cultured in Defined Keratinocyte-SFM medium. MMP-2 specific siRNA expression plasmid constructed in vitro was transfected into ameloblastoma cells. Fluorescence microscope was used to detect the result of transfection. Flow cytometry was used to check the transfection rate, the cell cycle and apoptosis. Gelatinolytic zymography analysis was used to investigate the activity of MMP-2 in supernatant of culture medium. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to check the expression of MMP-2 mRNA. Western-blot analysis was performed to investigate the expression of MMP-2 protein. The invasiveness of ameloblastoma cells was detected by three dimension culture system. Transwell analysis was done to measure the invasion of ameloblastoma cells. Cell adhesion rate on fibronectin was detected by cell adhesive analysis. The analysis of students t test was used to analyze the data. Results: There is no significant difference of apoptosis between experimental study and control study. MMP-2 specific siRNA expression plasmid was successfully transfected into ameloblastoma cells. The expression of proferment MMP-2 and active MMP-2 in supernatant of experimental group were decreased by 39.6% and 56.5%, respectively. The transfection rate at 96h after transfection was 63.6%. At the same time, MMP-2 mRNA expression was reduced to 66.0%. MMP-2 protein expression was reduced to 64.6%. The inhibition rate of the invasiveness of ameloblastoma cells was 61.3%. The inhibition rate of adhesion of ameloblastoma cell was 48.3%. P<0.05. Conclusion: MMP-2 gene was successfully silenced by MMP-2 targeting siRNA. MMP-2 has close relation with the invasiveness of ameloblastoma.PartⅣInhibition Against Xenografts of Ameloblastoma in Nude Mice Using MMP-2 Targeting siRNAIn previous experiment, it has been demonstrated that MMP-2 gene could be silenced by MMP-2 targeting siRNA in vitro, and the expression of MMP-2 was inhibited, and then, the invasive behavior of ameloblastoma cells had been inhibited. In order to study the effects of RNAi in vivo, in present experiment, subcutaneous xenograft of ameloblastoma in nude mice was established and MMP-2 targeting siRNA expression plasmid was injected around the xenograft. Methods: Fresh ameloblastoma tissue was minced into 1-2 mm3 size and subcutaneous transplanted into the back of the two anterior extremities root and the posterior back of nude mice. Self control was designed. Namely, left anterior limb side was taken as blank group and 250μl Opti-MEMR I culture medium was injected around the xenograft, posterior back side was taken as liposome group and compounds of liposome was injected around the xenograft, right anterior limb side was taken as experiment side and MMP-2 specific siRNA expression plasmid was injected around the xenografts. Two weeks after transplantation of the ameloblastoma tissue lump, the experimental factor was exerted on control side and experimental side according to the group design. The volume of the xenografts and the weight of xenografts in the termination time of the experiment were recorded. The students t test of SPSS11.0 software was taken as statistic analysis. If the P value is less than 0.05, the result has significance. The pathological analysis was performed to study the xenografts. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to check the expression of MMP-2 mRNA in xenografts. Western-blot analysis was performed to investigate MMP-2 protein in xenografts. Results: All of the xenografts were survival. The volume of xenografts in control side was significant larger than that in experimental side. So did the weight of xenografts. P<0.05. There was some follicular structure of ameloblastoma in the connective tissue around the xenografts of all of the control side. The follicular structure had the typical V-G standard. There was no follicular structure in connective tissue of the experimental side, and only some residual follicular structure scattered in connective tissue. The expression of MMP-2 mRNA and MMP-2 protein in control group was significant higher than that in experimental group, respectively. Conclusion: Nude mice model of xenograft of ameloblastoma was successfully established. The invasion and growth of xenograft of ameloblastoma was suppressed might due to the reason of genesilence effect of MMP-2 targeting siRNA.