| Part Ⅰ The effect of down regulated of HK2 gene on the radiotherapy efficacy for triple negative breast cancer in vitroObjective:As the first rate limiting enzyme in the glycolytic pathway,hexokinase2（HK2）controls the energy metabolism of tumor cells.HK2 is expressed at high levels in a variety of cancer cells and cell lines and is involved in cancer proliferation,invasion,metastasis,and chemoradiotherapy sensitivity through several pathways.Targeting HK2 to improve tumor therapeutic effects has shown great promise for clinical use,suggesting that HK2 may be a potential therapeutic target to improve tumor therapeutic sensitivity.While triple negative breast cancer（TNBC）as a kind of highly malignant tumor,whether HK2 can serve as a potential target to improve the radiotherapy sensitivity of TNBC needs further study.This part of the experiment was designed to investigate in vitro the effects on the sensitivity of TNBC cells to radiotherapy through lentivirus mediated downregulation of HK2.Method:To study the relationship between silencing HK2 gene and radiotherapy sensitivity of MDA-MB231 breast cancer cells were detected in vitro by constructing of HK2 shRNA lentiviral vector,CCK-8 assay,flow cytometry assay and cell cloning assay.Construction of slow virus mediated short hairpin RNA（shRNA）:The HK2 shRNA gene sequences（HK2 shRNA）and the negative control sequence（Negative shRNA）were designed by Shanghai han heng company.The HK2 shRNA and the Negative control sequences was connected with a plasmid respectively,and transfected to 293T cells,the target genes were implanted into slow virus vector,then transfected to breast cancer cell line MDA-MB231.Breast cancer cell lines MDA-MB231 with low expression of HK2 were screened by the antibiotic purinomycin RT-PCR and Western blotting were used to detect the silencing effect of transfection from mRNA and protein levels.CCK-8 experiment:MDA-MB231 cells were divided into three groups:blank group（Control）,negative Control group（shNC）and silent group（shHK2）.The cells in the three groups were radiated by X-ray with dose gradient（0,2,4,6,8Gy）,the absorbance of three groups were detected by enzyme plate apparatus,and the survival rate of each group at different doses was calculated.Flow cytometry and cell clone formation experiments:The cells of the three groups were cultured and irradiated under the dose of 4Gy X-ray.The apoptosis of three groups were detected by Annexin V-FITC/PI double staining by flow cytometry,and the proliferation of cells were monitored by cell clone experiment.Results:A sequence plasmid pHBLV-U6-scramble-ZSgreen-Purowas was constructed and transfected into breast cancer MDA-MB231 cells.The cell lines with the highest transfection efficiency were screened by adding purinomycin with green fluorescence（GFP）under the microscope.The transfection efficiency was further verified by RT-PCR and Western blotting.About 90%of the cells were successfully infected with lentiviruses when viewed under a 400-power microscope.The results of RT-PCR and Western blotting showed that the expression of HK2 mRNA and protein was significantly decreased（P<0.01）,and the difference was statistically significant.CCK-8 experiments showed that the cell viability of shHK2 group decreased in a time and dose-dependent manner.After radiation of 0,2,4,6 and 8Gy,respectively,the survival rate of cells in the three groups in the same dose of shHK2 group was significantly lower than that in the control group and the shNC group（P<0.05）.The results of cell cloning-formation experiment showed that silting HK2 expression at 0G and 4Gy could inhibit the cloning-formation ability of MDA-MB231 cells in triple negative breast cancer,and the difference between shHK2 group and control group and shNC group was statistically significant（P<0.01）.Flow cytometry showed that the apoptosis rate of shHK2 group was significantly higher than that of control group and shNC group,and the difference was statistically significant（P<0.01）.Conclusion:In vitro,lentivirus mediated RNA interference was used to silence HK2 gene.CCK-8,cell cloning experiment and flow cytometry proved that down-regulation of HK2 enhanced the sensitivity of TNBC to radiotherapy.Part Ⅱ PET/CT evaluation of down-regulated HK2 gene on radiotherapy efficacy of triple negative breast cancer in vivoObjective:To monitor the radiotherapy effect of down-regulated HK2 on TNBC by 18F-FDG and 18F-FLT PET/CT.To explore the application value of 18F-FDG and 18F-FLT in early radiotherapy of TNBC.To detect the histological changes of TNBC after radiotherapy by pathology.Method:1.To investigate the effects of silencing the HK2 gene on radiosensitivity in a nude MDA-MB231 model,the tumor inhibition rate and the survival of nude mice were used as the efficacy evaluation criteria.To generate the nude mice axillary MDA-MB231 tumor model,the mice were randomly divided into negative control（shNC group）,radiotherapy（shNC+IR group）,silencing（shHK2 group）and silencing combined with radiotherapy（shHK2+IR group）according to the random number method.Six nude mice in each group were exposed to X-ray irradiation at a dose of 2 Gy for 5 times with a cumulative dose of 10 Gy,until the tumor volume reached 6-7 mm,The size of the tumor was measured weekly,and the volume of tumor growth was calculated,and the time of death of the nude mice was recorded for survival analysis.2.18F-FDG and 18F-FLT PET/CT imagingWhen the diameter of tumor bearing mice reached 6-7mm,radiotherapy was carried out.The mice were irradiated with 2Gy X-ray for 5 times with a cumulative dose of 10Gy.18F-FDG and 18F-FLT micro PET/CT imaging were performed before irradiation,1 day and 7 days after irradiation,respectively.Visual analysis method combined with semi-quantitative analysis method was used for micro PET/CT image analysis.The maximal standard-uptake value of tumor（SUVmax-T）was obtained by selecting the level with the strongest radioactive uptake of tumor and delineating the region of interest.At the same time,select the same level of the contralateral normal muslce tissue of the calculate the SUVmax-M of muscle tissue.The uptake ratio of tissue was obtained,and the TMR value was obtained as the curative effect evaluation index.3.Pathological examinationAfter the imaging was completed,the tissue of the nude mice was taken for pathological detection,including observation of the gross tissue of the tumor,HE staining,and immunohistochemical staining,among which the immunohistochemical staining indexes included:Glut-1、Ki67、HIF-1α、P53、caspase-3。4.Statistical analysisSPSS 22.0 software was used to analyz the experiment results.Normal distribution of measurement data was represented as x ± s.Paired t test was used for comparison of each group before and after radiotherapy.One-way ANOVA and least significant difference t test were used for comparison of multiple groups.P<0.05 was considered statistically significant.Results:1.Inhibition rate and survival timeThe nude mice were observed for 28 days after radiotherapy and the growth curve of transplanted tumor in each group was plotted.The results showed that the growth rate of tumor volume in the shHK2 group was significantly slower than that in the shNC group（*P<0.05）,and the growth rate of tumor volume in the shHK2+IR group was significantly slower than that in the shNC+IR group（P=0.0016<0.01）after radiotherapy,the difference was statistically significant.Before treatment,24 hours after treatment and 7 days after treatment,the volume of tumor-bearing mice in shNC group were as follows:（67.45±2.36,69.49±0.97,109.51±5.85）mm3,shNC+IR group:（65.13±4.58,65.00±4.90,84.03±6.14）mm3,shHK2 group:（64.23±5.38,64.78±5.97,86.83±4.44）mm3,shHK2+IR group（63.27±6.22,63.79±6.26,75.26±6.89）mm3.After 24 hours of treatment,there was no significant difference of tumor volume in each group,and there was no significant difference with that before treatment.Seven days after treatment,the tumor volume of shNC+IR group,shHK2 group and shHK2+IR group was lower than that of shNC group,and the tumor inhibition effect was obvious,especially compared with other groups,the tumor inhibition rate of shHK2+IR group was significantly higher,and the difference was statistically significant（P<0.05）.The survival time of nude mice in shNC group was 27.67±2.7（21～35 days）,that in shNC+IR group was 38.03±3.4（28～48 days）,that in shHK2 group was 39.7±3.2（30～50 days）and that in shHK2+IR group was 51.9±4.2（38～64 days）.Kaplan-Meier survival analysis showed statistically significant difference in survival time of tumor-bearing mice in each group（χ2=9.64,P<0.05）.2.18F-FDG PET/CT imagingThere was no significant difference in TMR values between the four groups before treatment.There was no significant difference in TMR values between negative control group（shNC）,radiotherapy group（shNC+IR）and silencing group（shHK2）24h after treatment and before treatment,P>0.05.In the shNC+IR group,TMR value decreased at 7 days after treatment compared with 24 hours after treatment（t=3.75,P=0.03）,and TMR reduction rate was 44.07%,the difference was statistically significant.In shHK2 group,TMR value decreased at 7 days after treatment compared with 24 hours after treatment（t=3.75,P=0.03）,and TMR reduction rate was 28.14%,the difference was statistically significant.In shHK2+IR group,TMR values were significantly decreased 24h and 7d after treatment compared with before treatment（P<0.01）,and TMR reduction rates were 32.25%and 64.51%,respectively,with statistically significant differences.After 7 days,TMR value decreased more significantly in the radiotherapy group than in the silencing group.The TMR value of the radiotherapy group was slightly higher after 24h than before treatment.After treatment,ΔTMR value of shHK2+IR group was the largest,indicating that combined radiotherapy group had better effect.3.18F-FLT PET/CT imagingThere was no significant difference in TMR values between the four groups before treatment.In the shNC group,there was no significant change 24h after treatment compared with before treatment,and TMR value increased significantly 7 days after treatment,the difference was statistically significant（P=0.023）.The TMR values of shNC+IR group were decreased at 24h after treatment compared with before treatment,and at 7 days compared with 24h（P=0.019,P=0.022）.In shHK2 group,TMR values were decreased 24h after treatment compared with before treatment,7 days after treatment compared with 24h（P=0.049,P=0.003）;The TMR values of shHK2+IR group were lower than those of shHK2+IR group 24h after treatment and 24h after treatment（P=0.005,P<0.001）.24h and 7 days after treatment,there were differences among the four groups,and the differences were statistically significant.4.Changes of tumor volumeThere was no significant difference in tumor volume between the first four groups（F=0.18,P=0.92）.24h after radiotherapy,there was no significant difference in tumor volume between the four groups（F=0.26,P=0.86）.On the 7th day after radiotherapy,the tumor volume of the shNC group increased significantly,while that of the shNC+IR group and THE shHK2 group increased slowly and slightly,while that of the shHK2+IR group decreased.5.Pathological resultsThe percentage of Glut-1 positive cells in shNC group,shNC+IR group,shHK2 group and shHK2+IR group was（62.88±6.21,51.56±7.52,49.76±8.89,22.48±6.47）%,respectively.The percentage of Ki67 positive cells were（76.35±3.23,58.49±4.32,57.83±2.89,27.65±3.67）%,respectively.The percentage of HIF-1α positive cells was（76.47±4.21,60.67±6.52,61.56±5.89,26.65±2.47）%,respectively.The percentage of P53 positive cells were（53.48±7.35,40.55±6.24,42.78±7.09,20.17±8.47）%,respectively.The percentage of caspase-3 positive cells were（24.49±4.35,38.80±5.36,40.78±6.29,68.77±5.40）%,respectively.The results of one-way ANOVA showed statistically significant differences among all biological indexes in the shNC+IR group and shHK2 group.The results of comparison between the two groups showed that there was no difference in all indexes in the shNC+IR group and shHK2 group.The positive cell rates of GLUT-1,Ki67,HIF-1α and P53 in the shHK2+IR group were lower than those in the radiotherapy group.The percentage of caspase-3 positive cells was higher than that of radiotherapy group.Conclusion:HK2 silencing combined with radiotherapy can inhibit the growth of tumor volume and prolong the survival time of tumor bearing mice.18F-FDG and 18F-FLT PET/CT used tumor/muscle ratio（TMR value）as semi-quantitative index to monitor the therapeutic effect of silent HK2 on tumor bearing mice for 24h and 7 days.The results showed that:both 18F-FLT and 18F-FDG PET/CT can monitor the therapeutic effect at an earlier stage,while 18F-FLT is not affected by inflammatory cells and has better timeliness than 18F-FDG.The expressions of Glut-1,Ki67,HIF-1α,P53 and Caspase-3 proteins in the tumor biological indicators were all different in the four groups of mice,and these biological indicators pointed to a better effect in the silencing combined with radiotherapy group,on the other hand,suggesting that the silencing of HK2 can increase the sensitivity of radiotherapy. |
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