Study Of The Relationship Between The Expression Of A Telomere-binding Protein CTC1and Radiosensitivity Of Human Breast Cancer

Part I Study on the relationship between the expression of telomere binding protein CTCl and the radiosensitivity of human breast cancer cellsObjective To construct a human radiosensitive/radioresistant breast cancer cell model and to study the correlation between the expression of telomere binding protein CTCland the radiosensitivity of human breast cancer cells.Methods1.We use the human breast cancer cell line MDA-MB-435as the research object. Cells were irradiated by X ray in fractionated external beam radiation pattern, with600cGy dose a week and12times in total, then cultured for more than30generations until the cell character is stable. In this way we acquire the progeny cell named MDA-MB-435R with radiation induced resistance traits. Clonogenic survival assay were used to detect the cell survival fraction of these two cell lines treated with different doses of X rays and to acchieve the cell survival curves. We identified the radiosensitivity of these two cell lines by analyzing the radiobiology parameters of them.2. We use CTCl siRNA to interfer the above cell model for48h and then identify the effect of interference in order to determine the best interference sequence by realtime PCR. Then we construct the shCTCl plasmid according to the best interference sequence and screen out shCTCl stable transfected human breast cancer cell lines. Realtime PCR and Western blot were used to detect the changes of mRNA and protein level of CTCl in these two cells before and after interference by the optimum interfering sequence.3. Clnogenic survival assay were used to detect the differences of radiobiological parameters in these two cells before and after interference by the optimum interfering sequence.Results1. The progeny cell named MDA-MB-435R induced by fractionated irradiation of X ray in MDA-MB-435cell line were in good condition, and obtained a significant and stable radioresistance. The results of cloning formation assay showed that MDA-MB-435R cells were significantly more radioresistant than MDA-MB-435cells. The DO values were (3.266±0.072) vs.(2.093±0.131)(P<0.01); Dq values were (2.379±0.108) vs.(2.088±0.046)(P>0.05); SF2values were (0.701±0.016) vs.(0.574±0.018)(P<0.01). The construction of the radioresistant cell of human breast cancer was successful.2. Among the three siRNA against CTCl, siCTCl#3is the best interference sequence, which can significantly decrease the CTCl mRNA level. The positive clones of shNC and shCTCl were observed by the day of16th and19th after transfection. However, the cells of shCTCl group died31-33days post-trasfection, while the cells of shNC group survived. The mRNA and protein level of CTCl in these two cells treated with siCTCl#3was significantly lower than the control groups (P<0.05). In the MDA-MB-435cell lines,(23.42±8.05)%and (46.29±9.56)%than that of the Mock group. In MDA-MB-435R cell line, the CTCl mRNA and protein level of siCTCl#3group were respectively (18.25±6.74)%and (72.04±4.26)%than that of the Mock group. CTCl mRNA level and protein level in MDA-MB-435R cell line were1.6±0.15and1.36±0.18times than that of the MDA-MB-435cells (P<0.05).3.The SF2values of siCTCl#3groups in these two cell lines dramatically decreased than siNC and Mock groups respective ly(P<0.05), while siNC group and Mock group of these two cells showed no significant differences (P>0.05).Conclusion We successfully constructed a human radiosensitive/radioresistant breast cancer cell model-MDA-MB-435/MDA-MB-435R cells. MDA-MB-435R cells are more radioresistant than the MDA-MB-435cells. Telmere-binding protein CTCl was negatively correlated to the radtosensitivity of human breast cancer cells. We did not construct the shCTCl stable transfected human breast cancer cell lines successfully, while the control group of shNC stable transfected human breast cancer cell lines were constructed and survived. Part II Preliminary study of the mechanism of the radioresistance imparted by telomere-binding protein CTC1in human breast cancerObjective To investigate the potential mechanisms of radioresistance imparted by telomere binding protein CTC1in human breast cancer cells in order to provide a new target for breast cancer prevention and therapy, and to provide an important theoretical basis for more effective individualized treatment plan of clinical patients.Methods siRNA (siCTCl#3and siNC) was transfected into MDA-MB-435/MDA-MB-435R cells by liposome for48h:1. CCK8kit were used to detect the cell proliferation and cytotoxicity;2. Trypan blue staining was used to determine cell proliferation kinetics;3. Realtime PCR detection were used to analyze the cell relative telomere length;4. Immune fluorescence detection was used to analyze the capacity of DNA damage repair and co localization of CTC1and yH2AX;5. Detection of cell cycle and apoptosis were conducted by flow cytometry;6. Caspase-3activity was detected to verify the trigger actiocn of the early apoptotic events.Results1. The450nm absorbance values of siCTCl#3group were significantly lower than the two control groups (siNC and Mock group), which suggested that the proliferation was markedly reduced (P<0.05).2. The cell population doubling time of MDA-MB-435cell and MDA-MB-435R were30h and44.5h, which was prolonged for14.5h in radio resistant cells than the parental cells. The logarithmic proliferation rate of MDA-MB-435R was significantly reduced (P<0.01).3. The relative telomere length of MDA-MB-435R cells is2.06±0.23times of MDA-MB-435cells (P<0.01). The relative telomere length in Mock group and siNC group of two kinds of cells showed no significant difference (p>0.05), while the relative telomere length in siCTCl#3group was significantly shorter than the two control groups (P<0.05).4. The number of γH2AX foci in the radioresistant MDA-MB-435R cells was significantly fewer than the radiosensitive MDA-MB-435cell line (P<0.05). In addition, in MDA-MB-435cells, the percentage of γH2AX foci positive cells in irradiation group and irradiation combined with interference group were (73.14±4.74)%and (92.35±3.22)%(P<0.05); the numbers of γH2AX foci positive per cell were (2.7±0.5) and (5.8±0.3)(P<0.01). In MDA-MB-435R cells, the percentage of γH2AX foci positive cells in irradiation group and irradiation combined with interference group were (25.36±3.92)%and (56.72±5.34)%(P<0.01); the numbers of γH2AX foci positive per cell were (1.3±0.2) and (2.8±0.3) a (P<0.01). The γH2AX foci showed no apparent colocalization phenomenon with CTC1 protein.5. In contrast to the two control groups (Mock group and siNC group), siCTCl#3group did not dramatically induced cell cycle G2/M arrest. In MDA-MB-435cells, the apoptotic rate of irradiation group and irradiation combined with interference group was (26.84±3.91)%and (49.32±5.67)%(P<0.01), and the necrosis rate of the two groups was (9.05±2.32)%and (19.15±4.26)%(P<0.01); in MDA-MB-435R cells, the apoptotic rate ofirradiation group and irradiation combined with interference group were (21.26±6.75)%and (46.21±4.24)%(P<0.01), while the necrosis rate of the two groups was was (4.68±2.05)%and (5.14±2.17)%(P>0.05).6. Caspase-3activity of these two cells greatly increased12h after transfection, reached the peak at24h post-transfection, and then decreased gradually48h after transfection, and dropped to very low levels96h after transfection. At24h post-transfection, the Caspase-3activity of siCTCl#3group in MDA-MB-435cells and MDA-MB-435R cells were4.36±0.53and3.21±0.24times than that of the Mock groups respectively (p<0.01).Conclusion The potential mechanisms of radioresistance imparted by telomere binding protein CTC1in human breast cancer cells are related to enhancing tumor cell proliferation, promoting DNA damage repair, inhibition of telomere shortening and apoptosis, but may not be accociated with the regulation of cell cycle progression. Part III Analysis of the expression of telomere binding protein CTCl in human breast cancer tissues and the clinical pathological characteristicsObjective To compare the expressions of a telomere-binding protein named CTCl in human infiltrating ductal carcinoma tissues and the adjacent non-neoplastic tissues. To analyze the relationship between the expression of CTCl and the clinical and pathological features, such as pathological grade and lymphatic metastasis status in order to search for new therapeutic targets for the treatment of breast cancer.Methods We collected57cases of human breast cancer tissue of the Pathology Department in Wuhan University Zhongnan Hospital. Inclusion criteria:patients first diagnosed as infiltrating ductal carcinoma in our hospital between2007.6~2008.12; without neoadjuvant chemotherapy and any other primary tumor. The follow-up deadline is Dec.31,2013. We compare the expressions of a telomere-bind ing protein named CTCl in human infiltrating ductal carcinoma tissues and the adjacent non-neoplastic tissues by immunohistochemical assay and analyze the expression of CTCl in patients with different ages, menopause status, tumor sizes, pathological grades, hormone receptor status, lymphatic metastasis.5-year regional recurrence status and5-year survival status. Kaplan-Meier analysis was used to detect the influence of CTCl protein level on the5-year regional cumulative recurrences and the5-year survival rates of breast cancer patients.Results In the tumor tissues of the57human breast cancer cases, the expression of CTCl protein was positive in38cases. The positive expression rate was66.67%. Among the22cases of the adjacent non-neoplastic tissues, the expression of CTCl protein was positive in3cases, the positive rate was13.64%. There was significant difference between them (P<0.01). The positive expressions of CTCl protein showed no significant statistical differences in different ages, menopause status, tumor sizes, hormone receptor status,5-year regional recurrence status, and5-year survival status in breast cancer patients. The positive expression of CTCl protein with high pathological grade of breast cancer patients was significantly higher than that of the patients with low pathological grade (P=0.023). The positive expression of CTCl protein of breast cancer patients with various lymphatic metastasis status showed significant differences (P=0.003). Kaplan-Meier analysis showed that the5-year regional cumulative recurrence of CTCl high expression group and low expression group was6.46%and0%respectively(P=0.279,95%CI=0.116~3.621). The5-year survival rate of CTCl high expession group was lower than that of the CTCl low expession group, but the difference was not significant (P=0.194,95%CI=0.236-1.340). The mean median survival rates of CTC1high expession group and low expession group were50and58months respectively.Conclusion Expression of telomere binding protein CTCl of human breast cancer tissues was significantly higher than that of the adjacent non-neoplastic tissue, and it was accociated with pathological grade and lymphatic metastasis status, while it was not related to the5-year regional cumulative recurrence and the5-year survival rate.