Experimental Study Of ¹⁸F-FDG Micro PET/CT Imaging To Evaluate BRCA1 Gene Silencing On MDA-MB-231 Breast Cancer Cell Radiosensitivity

ObjectiveBRCA1 gene expression in human breast cancer MDA-MB-231 cell line was silenced by short hairpin RNA（sh RNA）mediated by lentivirus as an interference vector.To investigate the effect of BRCA1 gene silencing on the radiosensitivity of breast cancer MDA-MB-231 cells from in vivo and vitro experiments,respectively.And fluorodeoxyglucose Positron emission tomography/Computed tomography(¹⁸F-FDG PET/CT)imaging was used to evaluate the efficacy of early radiosensitization in the MDA-MB-231 breast cancer model in nude mice.Methods1.In vitro experiments:Human breast cancer MDA-MB-231 cells were cultured in vitro,and three lentivirus-mediated sh RNAs were designed and used to construct BRCA1 gene silencing cell lines of MDA-MB-231 cells,sh BRCA1#1,sh BRCA1#2 and sh BRCA1#3,and sh NC cells for negative control.Western blot assay was used to detect the BRCA1protein expression in each group of MDA-MB-231 cells after different lentiviral transfection,and the cell lines with the lowest BRCA1 protein expression were screened by quantitative analysis and used for subsequent experimental studies.MDA-MB-231cells were grouped into a negative control group（sh NC）and a silencing group（sh BRCA1）,and the absorbance values of cells in the sh NC and sh BRCA1 groups were measured by CCK-8 assay at 24h,48h and 72h after vertical irradiation with 0 or 4Gy doses of X-rays,and cell viability was calculated.In addition,the cells of both groups were irradiated with 0,2,4,6 and 8Gy X-rays,and the clonogenic number of cells in sh NC and sh BRCA1 groups was measured by clone formation assay,and the survival curves were fitted to calculate the Sensitization enhancement ratio（SER）.2.In vivo experiments:The MDA-MB-231 breast cancer animal model was constructed in the right upper extremity axilla of nude mice,and according to the random number table method,all nude mice were divided into negative control group（sh NC group）,negative control combined with radiotherapy group（sh NC+RT group）,silent group（sh BRCA1 group）and silent combined with radiotherapy group（sh BRCA1+RT group）,a total of 24 animals,and each group had 6 animals.¹⁸F-FDG Micro PET/CT imaging was performed on each group of nude mice 24 hours before radiotherapy and 24 hours after the end of the four radiotherapy sessions,respectively.After analyzing the micro PET/CT images of each nude mouse,the highest ¹⁸F-FDG uptake level was selected and the area of interest was outlined to obtain the maximum standardized uptake value of tumor（SUVmax-tumor）,The maximum standardized uptake value of the contralateral muscle（SUVmax-muscle）,which was obtained by simultaneously sketching the normal muscle tissue next to the contralateral spine at the same level as the area of interest,and thus the tumor muscle ratio（TMR）was calculated,which was used as an index to evaluate the efficacy of radiotherapy.When the tumor could be observed with the naked eye,the length and diameter of the tumor were measured at 2 days intervals to calculate the tumor volume.After the imaging of all nude mice were completed,the tumor tissues were weighed,and then HE staining for tumor tissues,the expression levels of Hypoxia inducible factor-1α（HIF-1α）,Glucose transporter-1（Glut-1）and the cell proliferation nuclear antigen Ki-67 were measured by immunohistochemical staining.3.Statistical analysis:The paired t-test was used to compare the pre-and post-radiotherapy of nude mice in the same group,and the independent samples t-test was used to compare the differences between two groups;the one-way ANOVA was used to compare the differences between multiple groups,and the least significant difference t-test（LSD-t）was used to compare the data between two groups.The correlation between the TMR values of tumor tissues and the expression of each immunohistochemical index was compared by Pearson correlation analysis.Results1.In vitro experiments:BRCA1 silencing breast cancer cells were successfully constructed.The results of Western blot showed that the BRCA1 protein expression in all three silenced groups was lower than that in the sh NC group,in which the sh BRCA1#1sequence had the most obvious silencing effect and was used for subsequent experiments.The cell proliferation level of sh BRCA1+RT group was significantly lower than that of sh NC+RT group in a time-dependent manner,and the cell viability of sh BRCA1+RT group was significantly lower than that of sh NC+RT group,and the difference was statistically significant（P<0.001）.The cell clone formation assay suggested that silencing BRCA1 gene expression combined with radiotherapy could significantly inhibit the clone formation and proliferation ability of cells,and after receiving graded doses of X-ray irradiation,the cell survival fraction in the sh BRCA1 group was significantly lower than that in the sh NC group（P<0.001）in a dose-dependent manner,and the SER in the sh BRCA1 group was 1.41.2.In vivo experiments:¹⁸F-FDG Micro PET/CT imaging results showed that the TMR values in the sh NC,sh NC+RT,sh BRCA1 and sh BRCA1+RT groups before radiotherapy were 2.256±0.116,2.269±0.145,2.033±0.364 and 2.079±0.304,respectively,with no statistically significant differences（F=1.354,P>0.05）.After radiotherapy,TMR values were 3.063±0.297,1.696±0.270,2.933±0.242 and 1.177±0.098 in each group,respectively,with statistically significant differences（F=89.982,P<0.001）,and TMR values increased in both the sh NC and sh BRCA1 groups compared with those before radiotherapy（t=-7.464,P=0.001;t=-4.254,P=0.008）;sh NC+RT TMR values in both sh NC+RT and sh BRCA1+RT groups were lower than before radiotherapy（t=4.228,P=0.008;t=8.740,P<0.001）,and TMR values in sh BRCA1+RT group were significantly lower than those in sh NC+RT group after radiotherapy（t=4.395,P=0.001）.The tumor growth curves and tumor weight showed that the sh BRCA1+RT group had a significantly slower growth rate in tumor volume compared to the other 3 groups.Pathological results showed that the tumor cells in sh NC and sh BRCA1 groups had large deep-stained nuclei and obvious heterogeneity.The number of tumor cells in both the sh NC+RT and sh BRCA1+RT groups was significantly reduced with the formation of lamellar necrotic areas.sh BRCA1+RT group had significantly lower expression of HIF-1α,Glut-1 and Ki-67 than the other 3 groups（F=104.466,33.711,23.283,all P<0.001）,in addition,TMR values of tumor tissues were significantly and positively correlated with the expression of HIF-1α,Glut-1 and Ki-67（r=0.804,r=0.774,r=0.687,all P<0.01）.ConclusionBRCA1 gene silencing enhanced the radiosensitivity of MDA-MB-231 breast cancer cells and downregulated the expression levels of HIF-1α,Glut-1 and Ki-67.¹⁸F-FDG Micro PET/CT imaging can be used to evaluate the efficiency of early radiosensitization in a nude mouse model of MDA-MB-231 breast cancer by effectively monitoring tumor glucose metabolism.