The Anti-tumor Effect And Mechanism Of Chi-29b Chimera In Ovarian Cancer

Objective:To establish targeting therapy for inhibiting the growth of ovarian cancer through tumor tissue-specific delivery of miRNA-29b into the tumor cells aimed to effect the re-expression of PTEN tumor-suppressor gene.Methods:The chimera of MUC1aptamer-miR-29b (Chi-29b) was synthesized while the MUC1aptamer could specifically bind to the surface of OVCAR-3cells. Chi-29b was digested by Dicer enzyme to examine whether the Chi-29b could release miR-29b in vitro. The cell internalization efficacy of Chi-29b was assayed using fluorescence-activated cell scanning (FACS). In OVCAR-3cells, western-blot was performed to detect the expression levels of DNMT1, DNMT3A, DNMT3B and PTEN protein, with methylation technology applied to detect the methylation ratio of the PTEN promoter, RT-PCR used to assay the expression level of PTEN mRNA, and cell apoptosis observed by Hoechst33342staining.Results:Dicer digested the Chi-29b chimera and released miR-29b efficiently. The chimera of Chi-29b was delivered into the OVCAR-3cells in a concentration-dependent manner and a peak inflection of about81%internalization_efficacy could be established at a concentration of250nM with the higher doses dependence plateaued when the Chi-29b concentration reached500nM and1000nM. In OVCAR-3cells, when compared to controls, Chi-29b treatment significantly reduced protein levels of Dnmtl, Dnmt3A, and Dnmt3B expression (P<0.001), decreased the methylation ratio of PTEN promoter (P<0.01), induced the up-regulation of PTEN mRNA and protein (P<0.001,P<0.01,respectively) and cell apoptosis (P<0.001)Conclusion:The Chi-29b chimera can be specifically delivered into OVCAR-3cells in a concentration-dependent manner through the targeting effect of MUC1protein. In OVCAR-3cells, Chi-29b chimera can effectively induce cell apoptosis and up-regulate the expression of PTEN gene and protein through methylation mechanism.Part2The anti-chemoresistant effect and mechanism of Chi-29b chimera in ovarian cancerObjective:To determine the anti-tumor effect of MUC1aptamer-miR-29b chimera (Chi-29b) in xenografted models and chemo-resistance tumor models of ovarian cancer and to further explore the associated mechanism.Methods:The OVCAR-3-taxol cell line was established and the IC50value was examined by MTT method. The cell proliferation was analyzed by MTT assay when OVCAR-3-Taxol cells were incubated respectively with1mg/mL of paclitaxel,500nM of Chi-29b, or both the same dose of paclitaxel and Chi-29b. The tumor models of xenografts with OVCAR-3, OVCA420and OVCAR-3-Taxol were established by subcutaneous injection of OVCAR-3, OVCA420and OVCAR-3-Taxol tumor cells into the right hind limbs of nude mice, and they were randomly divided into different groups. In xenografted tumor models,the growth of tumor and its effect on ALDH1-positive cell were evaluated after the nude mice were intraperitoneal injection of Chi-29b, with methylation technology applied to detect the methylation ratio of the PTEN promoter, RT-PCR used to assay the expression levels of PTEN and ALDH1mRNA, western-blot performed to detect the expression levels of PTEN. MAPK4、MAPK10、IGF1、AKT、Bax and angiotomin protein and cell apoptosis observed by Tunnel method.Results:The IC50value of OVCAR-3-taxol cell line was5764±143μg/L and Chi-29b chimera significantly inhibited the cell proliferation of OVCAR-3-taxol cells in vitro (P<0.001). In OVCAR-3-xenografted tumor models, when compared to controls, intraperitoneal injection of Chi-29b significantly inhibited the tumor growth (p<0.001),up-regulated the expression of PTEN mRNA (p<0.001),and decreased the methylation of PTEN and the protein expression profiles of MAPK4and IGF1(p<0.001). In contrast, the Chi-29b cherma inhibited tumor growth through reducing the protein expression profiles of MAPK4. MAPK10and IGF1without any effect on PTEN expression in OVCA420-xenografted tumor models (p<0.01). In OVCAR-3-taxol-xenografted tumor models, when compared to controls, intraperitoneal injection of Chi-29b significantly inhibited the tumor growth and induced cell apoptosis (P<0.001), upregulated the mRNA levels of PTEN and the protein levels of PTEN and Bax (p<0.001) and reduced protein levels of Akt、MAPK4、MAPK10and IGF1(p<0.001). The ALDH1mRNA in OVCAR-3-taxol xenografts was significantly higher than that in OVCAR-3xenografts (p<0.001). Intraperitoneal injection of Chi-29b chimera significantly reduced the expression level of ALDH1mRNA in the OVCAR-3and OVCAR-3-taxol xenografts (p<0.001) and the amount of ALDH1-positive cells in OVCAR-3-taxol tumor xenografts (p＜0.001)Conclusion:The Chi-29b chimera can effectively exert an anti-tumor effect in xenografted and chemoresistant ovarian tumor models. The appearance of chemoresistant cells are releated to the increasing of ALDH1-positive cells in ovarian cancer. The Chi-29b chimera plays an anti-chemoresistant role through inhibiting the activation of cancer stem cell in ovarian cancer. The Chi-29b chimera inhibits the growth of ovarian cancer cells through PTEN-Akt-Bax, MAPK, IGF signal pathways and may be associated with the ALDH1gene.