The Effection Of RNA Intefrerence HER2Gene On Sensitivity Of Ionizingradiation, Autophagy And Apoptosis In Breast Cancer

Breast cancer is a serious affect women’s physical and mental healthand even life-threatening one of the most common malignant tumor, withthe change in environment, diet and living habits, presents an increasingtrend in the incidence of breast cancer in China.HER-2gene amplificationand protein over expression and HER-2overexpression with poorprognosis of breast cancer found in the25%-30%of breast independentrisk factor. With monoclonal antibodies into clinical revolutionaryachievements for HER-2positive breast cancer. HER-2/neu onlyabnormal expression in tumor tissue, which have become a hot researchtargeted cancer therapy, and the rise of RNAi technology-specificinterference HER-2gene provides a very important way in the study ofbreast cancersingle method often can not achieve very satisfactory results,RNAi combined radiotherapy and chemotherapy for HER-2positivebreast cancer is more promising treatment.Objective:In this study, full use of the means of molecular biology and cellbiology, select breast cancer development, transfer and discharge mostclosely related to the chemotherapy resistance Her2gene study.Application of RNAi principle, construct siRNA lent viral expressionvector to overcome the shortcomings of conventional breast cancertreatment. Taking into account the importance of radiation therapy incancer therapy, optimization of radiotherapy program, will provideimportant clues and new ideas for the treatment of breast cancer. Research content:1.Build Her2proto-oncogene siRNA lentiviral expression vector,transfection of human SKBR3breast cancer cell lines, detection ofHER-2protein and mRNA silencing effect.2. Successfully constructed cell lines with different doses of ionizingradiation, the observed cell sensitivity to ionizing radiation, autophagyand apoptosis changes.Research methods:1.Her2target molecule of the siRNA design, build, siRNA lentiviralexpression vector. For the Her2design siRNA, build siRNA lentiviralexpression vector, lentiviral into breast cancer cells, the establishment ofa negative control (negative interference fragment virus). Of Her2proteinexpression (western blot) and the level of transcription (RT-PCR)2.By colony formation assay radiosensitivity3.MDC assay the autophagy and DAPI detection of apoptosis andthe relationship of the HER2gene.Results1.SiRNA-1siRNA-2and NC SKBR3cells were infected, theapplication westernblotting assay after treatment of SKBR3cells in theamount of expression of the HER2protein, the results show the HER2protein in the cells compared with the CON group and NC group cells,siRNA groupthe expression levels were significantly lower (P <0.05), cellprotein expression levels of the CON group and NC group showed nosignificant difference (P>0.05).2. siRNA-1siRNA-2and NC SKBR3cells were infected with theextraction of total cellular RNA by RT-PCR assay. Compared with theCON group and NC group, siRNA-1cells HER2mRNA expression levelswere significantly lower (P <0.05). Compared to the CON group, NC group cell mRNA expression levels slightly lower, both compared to thenon-statistically significant (P>0.05).3. Three groups of cells after irradiation, the number of cell clonesare reduced interference group compared to the normal group, andsignificantly reduced the number of cell clones8Gy when there is astatistically significant (p <0.05).4.After after4Gy irradiation, the percentage of autophagic vesicles ofthe three cell lines significantly increase was statistically significant (p<0.05) showed that ionizing radiation promote autophagy interferencegroup was significantly lower than the normal group autophagy vesiclesPercentagewas statistically significant (p <0.05).5. Three groups of cells after after4Gy irradiation, the number ofapoptotic cells increased interference group compared to the normalgroup, the number of apoptotic cells increase was statistically significant(p <0.05)Conclusion1.SKBR3cells lentivirus-mediated RNAi can effectively silence theexpression of HER2。2. Silence the expression of the HER2gene, the cells increasedsensitivity to ionizing radiation。3. SKBR3cells after ionizing radiation, can upregulate autophagyactivities, protection of the killing effects of ionizing radiation on cells4. Silence HER2gene expression after ionizing radiation to promoteapoptosis.