Targeting Breast Cancer Stem-like Cells With Human Telomerase Reverse Transcriptase Promoter-driven Oncolytic Adenovirus RCA-TERT-Ad35

Object:To Study of RCA-TERT-Ad35 target killing cancer cells and provide a theoretical basis for the thearaputic of breast cancer.Methods:1.Breast cancer cell line MCF-7 was subcultured by suspension culture to form MCF-7 microspheres.The expression of hTERT mRNA in MCF-7 cells and MCF-7 microspheres was detected by RT-PCR.2.MTT assay was used to detect the survival rate of MCF-7 cells treated with ’different concentrations of paclitaxel in MCF-7 microspheres and control group.3.RIA-LACZ,RIA-LACZ-RGD and RIA-LACZ-Ad35were used to amplify MCF-7 cells and MCF-7 microspheres(MOIs 0,0.2,1,5,20,100)Cell lines were in vitro transfection experiments.After 48 hours,P-galactosidase staining was performed to assess the transfection efficiency of the three viruses at different cells and at different concentrations.4.RCA-TERT-Ad35 observation group and RCA-Ad35 control group were infected with MCF-7 cells,after 7 days hatching MTT solution treat MCF-7 cell line.Detection of the killing effect.5.In vitro oncolysis experiments:Three cell lines(MCF-7,MCF-7 microspheres and paclitaxel-resistant HG cells),each cell was equally divided into four.All three viruses were treated with RCA-TERT-Ad35,RCA-Ad35,WT-Ad5(wild type)and blank control group.After 4 days,the ability of each group of virus to kill in different cell lines was evaluated with crystalline violet reagent.6.In the mouse model of transplanted tumor,4 weeks of mice,the tumor volume in 100 mm3,were randomly divided into three groups,each group of six mice.RCA-Ad35 group and RCA-TERT-Ad35 group,PBS group was injected into the tumor every 2 days.Results:1.The expression of hTERT mRNA in MCF-7 microspheres was significantly higher than that in MCF-7 cell lines.The expression of hTERT mRNA was also highly expressed in MCF-7 microspheres.2.MCF-7 microsphere cell lines are more resistant to paclitaxel chemotherapy than MCF-7 cell lines.In vitro transduction of MCF-7 cell line and MCF-7 microsphere cell line The RIA-LACZ-Ad35 group had stronger transduction rate than RIA-LACZ-RGD group-7 and RIA-LACZ group Such as MCF-7 microspheres.The RCA-TERT-Ad35 group showed stronger lethality of MCF-7 cell line than RCA-Ad35 group.Compared with WT-Ad5 group and RCA-Ad35 group,the RCA-TERT-Ad35 group was used in a variety of cell lines(MCF-7 cell line,MCF-7 microsphere cell line,HG-paclitaxel resistance group)Drug cell lines)showed the strongest oncolytic effect.In RCA-TERT-Ad35 group,RCA-Ad35 group has stronger inhibitory effect on tumor than RCA-Ad35 group.Conclusion:1.The oncolytic adenovirus RCA-TERT-Ad35 is effective in killing breast cancer carcinogens in vitro and in vivo.2.It provides theoretical basis for the therapeutic of breast cancer.