Effect Of RNA Interference Of SNCG Expression On Radiosensitivity Of Breast Cancer MCF-7Cells

BackgroundBreast cancer is one of the most common malignant tumor of women, and themost common and the second-leading cause of cancer death in American women. InChina, the incidence and mortality of breast cancer shows a clear upward trend,which has a serious impact on women’s physical and mental health and even threatentheir lives. The traditional treatment of breast cancer includes surgery, chemotherapyand radiotherapy. By the research,40%to60%of breast cancer patients need toaccept radiotherapy. Breast cancer cells have moderate sensitivity to radiationtherapy, however, the survival rate has not been improved significantly. There will bethe possibility of recurrence or metastasis. In recent years, a new idea aboutgene-radiotherapy of tumor is put forward for the treatment of tumors. This studywill be an organic combination of gene therapy and radiotherapy, aiming to researchthe sensitization effect of gene therapy on breast cancer radiotherapy and to exploreits mechanism.SNCG (gamma-synuclein) was originally found in human breast cancer genecDNA databases[1]. Due to the specific expression of SNCG in advanced breastcancer, it is called breast cancer-specific gene1(BCSG1). SNCG gene is localized onchromosome10q23and encodes a smaller123amino acid protein[2]. Studies showthat over-expression of wild-type SNCG protein in cancer might be associated withmalignancy[3]. Thus, SNCG expression leads to over-riding the mitotic arrestallowing cells to continue progression through the cell cycle resulting in aneuploidy.The study of SNCG gene sequences shows that SNCG expression in tumor cell linesis mainly because of activated transcription, not gene amplification or mutations[4].Recent studies have found that SNCG shows high expression in breast cancer[5], uterine cancer[6], prostate cancer[7], pancreatic cancer[8], colorectal cancer[9]andmany other malignancies, and its abnormal expression is closely related to theclassification and malignant progress of tumor. SNCG over-expression can stimulatebreast cancer cell proliferation and induce metastasis[10], and can be used as anindependent adverse prognostic factor for breast cancer[11], thus it is expected tobecome a target for molecular therapy of breast cancer.In the experiment, shRNA was transfected into breast cancer MCF-7cells vialipofectamine to cut SNCG gene expression, and to establish SNCG low expressionof MCF-7cell lines by stable transfection. Cells were irradiated with different dosesof X-ray, in order to observe the influence of down-regulated SNCG gene in breastcancer on cell apoptosis and radiation sensitivity.ObjectiveTo study the effect of RNA interference on SNCG mRNA and protein levels, aswell as SNCG-shRNA combined with different doses of X-ray irradiation on breastcancer cell apoptosis and radiosensitivity, and to explore possible mechanisms of theeffect of down-regulated SNCG gene on radiotherapy sensitization of breast cancercells.Materials and Methods1According to the known SNCG gene cDNA sequence, four specificinterference sequences (SNCG-shRNA-1, SNCG-shRNA-2, SNCG-shRNA-3,SNCG-shRNA-4) and a non-specific sequence (SNCG-shNC) were designed andsynthesized.2Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)and Western blotting technique was used to detect the changes in the expression ofmRNA and protein levels of SNCG gene interfered after shRNA was transfected intoMCF-7cells of breast cancer via liposome-mediated transfection techniques.3Clone formation assay was used to observe the influence of SNCG-shRNA onradiosensitivity of breast cancer cell.4Flow cytometry was used to detect the influence of SNCG-shRNA combinedwith X-ray irradiation on cell apoptosis.5Statistical Methods: SPSS17.0software was used to analyze the collected data. All data were expressed as mean±standard deviation（x s）,and the quantitativedata were analyzed by one-way ANOVA, P<0.05showed statistically significantdifference.Results1ShRNA was successfully transfected into breast cancer MCF-7cells vialiposome-mediated transfection techniques.2RT-PCR and Western blot showed that mRNA and protein levels of SNCGgene were significantly reduced after SNCG-shRNA was transfected into breastcancer MCF-7cells (P<0.05).3Clone formation assay showed that surviving fraction of positive transfectiongroup was significantly lower than that of the negative transfection group and theblank control group (P<0.05), and surviving fraction of each group showed adeclining trend with increasing radiation dose.4Flow cytometry showed that apoptosis rate of positive transfection groupcombined with irradiation was significantly higher than that of irradiation group andthe negative transfection group (P<0.05), while the difference between the apoptosisrate of negative transfection group combined with irradiation and that of irradiationgroup was not statistically significant (P>0.05).Conclusion1The mRNA and protein expression of SNCG gene was significantlydown-regulated after SNCG-shRNA was successfully transfected ino breast cancerMCF-7cells.2SNCG-shRNA combined with X-ray irradiation could increase the apoptosisof MCF-7cells, reduce the survival fraction of cells, and ultimately enhanceradiosensitivity of breast cancer MCF-7cells.