| Part I The correlation research between the HMGB1 expression and radio-sensitivity in human breast cancer cellsObjective To investigate the relationship between the HMGB1 expression and radio-sensitivity in human breast cancer cells.Methods To construct the human breast cancer cell models MCF-7-sh-HMGB1 and MCF-7-NC. Synthesized shRNA were cloned into pGPU6/GFP/Neo carrier, the carrier were named after the synthesis as pGPU6/GFP/Neo-HMGB1 and pGPU6/GFP/Neo-shNC. Low HMGB1 expression and the negative control stable cell lines were cultivated with 600ug/ml G418 for 5 weeks, selected the clones and then amplificated, the stable transfection cells were named as MCF-7-shHMGB1 and MCF-7-NC. We used RT-PCR and WB to test the interference efficiency of HMGB1 in stable transfection cell lines, clone formation to test radiation sensitivity in each group.Results The fluorescence purity of the stable transfection cell lines were 99%, the mRNA level of HMGB1 in MCF-7-sh-HMGB1, MCF-7-NC and MCF-7 were (0.12975±0.0314) VS (0.9625±0.0476) VS (1). The mRNA level of HMGB1 was interferenced as 12.5% of the parental cell line, and no difference between the MCF-7 and MCF-7-NC cell lines. The protein expression of HMGB1 in MCF-7-shHMGB1 was significantly lower than in the MCF-7-NC. Radiation sensitivity was significantly increased when HMGB1 was down regulated. The SF2 value of the MCF-7-NC and MCF-7-shHMGB1 were 0.7756±0.0016,0.5732±0.0031 (p<0.01); DO value were (2.7555±0.0810) vs. (2.4807±0.0331*) (p<0.05); Dq value (3.1689±0.1431) vs. (0.7225±0.0210*)(p<0.05).Conclusion We constructed human breast cancer cell lines named as MCF-7-sh-HMGB1 and MCF-7-NC Successfully, the HMGB1 expression was stably knockdown in MCF-7-shHMGB1, no significantly changes were found in the MCF-7-NC compared with the MCF-7 cells, HMGB1 downregulation resulted in increased radio-sensitivity in human breast cancer cells.Part II Mechanism research about HMGB1 downregulation increased the radio-sensitivity of human breast cancer cellsObjective To explore the mechanism about how HMGB1 affect the radiosensitivity in human breast cancer cells, and to provide a new effective target for breast cancer radiotherapy, at the end, to give out a theory basis for clinical treatment.Methods In this study, we established stably transfected control (MCF-7-NC) and HMGB1 knockdown (MCF-7-shHMGB1) cell lines. The expression of HMGB1 mRNA and the relative telomere length were examined by real-time PCR. Radiosensitivity was tested by clone formation assay. The protein expressions were detected by western blot. The telomerase activity was detected by PCR-ELISA. Proliferation ability was determined by CCK-8 assay. The level of cell cycle and apoptosis rate were tested by flow cytometry. DNA damage foci in each group were detected by immunofluorescence confocal.The level of reactive oxygen species were detected by using ROS detection kit.Results Down-regulation of HMGB1 increased the radiosensitivity of MCF-7 cells, and inhibited the accumulation of hTERT and cyclinDl. Telomere length in MCF-7, MCF-7-shHMGB1 and MCF-7-NC cell lines were 0.8574±0.0812,0.6763±0.0610 and 1, there were significant differences between MCF-7-shHMGB1 and MCF-7-NC(***p<0.001), also differences between MCF-7-shHMGBl and MCF-7. The telomerase activity in MCF-7-NC and MCF-7-shHMGB1 were (1.6155+0.1512) and (1.169±0.0924), knockdown of HMGB1 in MCF-7 cells inhibited telomerase activity and cell proliferation. Under no irradiation, the percentage of S cycle phase in MCF-7-NC and MCF-7-shHMGB1 were (44.090±5.78)% and(29.080±4.234)%; while suffered to 6Gy irradiation, the percentage of G2/M cycle phase in MCF-7-NC and MCF-7-shHMGB1 were (29.790±3.291)%, (23.324±4.580)% and (28.592±2.673)% vs (34.293±4.231)%, (31.959±3.265)% and (35.653±5.297)% at the 6h,12h,24h from the irradiation time point. The apoptosis level of MCF-7-NC and MCF-7-shHMGB1 were (5.00±0.848)% and (25.500±1.272)%, down-regulation of HMGB1 increased the extent of apoptosis(**p<0.01). Down-regulation of HMGB1 modulated telomere homeostasis by telomere-binding proteins, such as TPP1 (PTOP), TRF1 and TRF2. HMGB1 knockdown also inhibited the ATM and ATR signaling pathways; and enhanced the level of ROS.Conclusion HMGB1 downregulation breaks telomere homeostasis, promotes radio-sensitivity, and inhibits the repair of DNA damage in breast cancer cells. These results suggested that HMGB1 might be a potential radiotherapy target in human breast cancer.Part ? Relationship research between HMGB1 expression and the character of clinical pathologyObjective To explore the differences of HMGB1 expression between the breast invasive ductal carcinoma and adjacent tissues, and to analyze the correlation between the HMGB1 protein expression and pathologic stage, lymph node stage, overall prognosis and other clinical pathological features, to explore new targets for the clinical treatment in breast cancer.Methods To collect the breast cancer tissues from oncology department, zhongnan hospital affiliated by wuhan university. The standards to set were as follows:July, 2001-August,2011; no preoperative neoadjuvant radiochemotherapy; all patients were pathologically diagnosed as infiltrating ductal carcinoma; no primary tumors in other organs; deadline of follow-up time is Jan,2013. HMGB1 protein in tumor tissues and tissue adjacent to carcinoma were detected by immunohistochemical method, and analyze the relationship among the HMGB1 protein expression and age, menopausal status, tumor size, pathologic stage, hormone receptor level, lymph node metastasis status, metastasis status and overall survival rate in patients. To analyze the correlation of protein expression of HMGB 1 with cumulative overall survival rate by using Kaplan-Meier methods.Results There were no significant correlation among the expression of HMGB 1 and age, menopausal status, pathologic stage, estrogen receptor status, HER-2 level, lymph node metastasis and metastasis stage, there was a significant correlation with tumor growth status and progesterone receptor status and cumulative overall survival status(p< 0.05).Conclusion HMGB1 may promote breast cancer proliferation, and likely has a negative correlation with cumulative overall survival. |
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