The Relationship Between BZAP45 And Mucoepidermoid Carcinoma

Mucoepidermoid carcinoma (MEC) is the most common malignacy in salivary glands and accounts for about 30% of malignant tumors in salivary glands in Chinese population. Although it sometimes shows a slow growth resembling a benign lesion, this neoplasm can be highly aggressive with a dismal prognosis. A 5-year disease-free survival rate between 0 and 43% has been demonstrated in the patients with high-grade tumors.There is little reports about BZAP45, up to now, we only know that it serves as cell cycle regulator. Whether this gene has other funtions or its relationship with tumors has no reports.RNA interference (RNAi) is a cellular mechanism in which double-stranded RNA triggers the sequence-specific"silencing"or"knockdown"of gene expression. In mammalian cells this double-stranded RNA may be either a small hairpin RNA (shRNA) or a simple RNA duplex with two unpaired nucleotides on the 3'-ends (siRNA). The transfection of synthetic siRNAs causes only transient suppression of target genes, which is often limited to the cell lines that are easy to be transfected. However, compared with siRNA, shRNA is more efficient to achieve stable long-term RNAi effects.There are many vectors used in RNAi, but it is a better way to study gene funtion by construction of lentivirus-RNAi vector.Based on our previous work, in this study, we constructed lentivirus-RNAi vector to silence BZAP45 gene in Mc3 cells. The biofeatures changes of Mc3 cells in vivo and in vitro and the possible mechanisms were studied, including four parts as follows:1. To confirm the expression level of BZAP45 in MEC tissue and cells was higher than that in normal tissue and cellsWe designed specific primers of BZAP45 to verify the results of cDNA microarray by real time PCR. Rabbit antibody specific for BZAP45 was generated by immunizing rabbits with recombinant human BZAP45 protein and purified by using standard protocols. The expression level of BZAP45 in MEC and normal salivary gland tissue was detected by immunohistochemistry and real time PCR. The expression level of BZAP45 MEC cells was detected by real time PCR. The results showed that the expression level of BZAP45 in MEC tissue, Mc3 cells were much higher than that in normal fibroblast cells and normal salivary gland tissue2. To construct BZAP45 siRNA expression lentivirus and to infect cellsTo generate lentivirus expressing RNAi specific for the BZAP45 gene, the four siRNAs were designed based on two conservative cDNA fragments within the coding region of human BZAP45 gene. The most effective one was chosed for later study. After Mc3 cells were infected, limiting dilution assay was used to pick positive clone, and real time PCR was used to identify the infected cells. The cells expressed BZAP45 siRNA were got and were used for later study.3. To study the changes of biofeatures in Mc3 cells after BZAP45 scilenced in vitro and in vivoMTT assay, cell counting assay, colony formation assay, monolayer wound healing assay, transwell invasion assay, HE staining and transmission electron microscopy were used to detect the changes in Mc3 cells in vitro after BZAP45 scilenced, Tumorigenesis assay was used to to detect the changes in Mc3 cells in vivo after BZAP45 scilenced. The results showed that the proliferation, the ability of clone formation and migration were decreased notably. Cells doubling time of Mc3 and Mc3-RNAi was 23h and 48h, the clone forming efficiency of Mc3 and Mc3-RNAi was 78% and 20%. Following incubation of physically wounded cells for 48 h, the Mc3 and Mc3-NC migrated insignificantly at 65.833±4.940μm or 64.733±2.684μm, respectively. In contrast, the Mc3-RNAi only migrated about 45.667±3.066μm and the mobile distance of Mc3-RNAi was significantly shorter than that of controls. In transwell invasion assay, average 85±6.1 Mc3 and 82.0±7.8 Mc3-NC cells per high power field had migrated onto the filter surface while only 24.0±3.7 Mc3-RNAi cells reached on the filter. The development of Mc3-RNAi cell-related solid tumors grew slowly and the mean volume of solid MEC tumors in Mc3-RNAi group decreased by about 60%, as compared with that in control groups . As a result, the mean weight of tumors in Mc3-RNAi group was significantly lighter than that in control groups The ultrastructure of Mc3 cells also changed after BZAP45 silenced.4. The possible mechanisms of BZAP45 effects the biofeatures of Mc3 cellsThe cell cycle distribution was detected by flow cytometry. Also some factors related to cell cycle, apoptosis and cell proliferation were detected by immunofluorescence. The expression levels of P53, c-myc, P21 seem no change after BZAP45 silenced, but the expression level of caspase3 was higher in Mc3-RNAi cells than in the other two groups, while the expression levels of cyclin-D1 and PCNA seem lower in Mc3-RNAi cells than that in the other two groups.Conclusions:1. The expression level of BZAP45 in MEC tissues and cells was higher than that in normal salivary gland tissues and cells2. BZAP45-RNAi-lentivirus was constructed and Mc3 cells were infected successfully.3. The biofeatures in Mc3 cells after BZAP45 scilenced were changed in vitro and in vivo notably, the malignancy of Mc3 cells was decreased after BZAP45 gene scilenced.4. BZAP45 gene may be related to the genesis and/or development of MEC...