| Part I The relevance of the expression of STUB1 and hTERT cytoplasmic protein to the prognostic in human cervical cancerObjective To investigate the effect of the expression of STUB1 and hTERT cytoplasmic protein on the significance of clinical in cervical cancer.Methods The expressions of STUB1 and hTERT were detected by using SP immunohistochemical staining assay. Data between two groups were analyzed by chi-square test. Kaplan-Meier analysis was used to detect the influence of STUB1 and hTERT protein level on the overall survival (OS). The independent factor of the STUB1 and hTERT protein level on the OS was analyzed by COX regression model.Results Expression of STUB1 protein was significantly downregulated in cervical cancer tissues compared to the chronic cervicitis and CIN tissues (P<0.05), while expression of hTERT protein was significantly upregulated (P<0.05). The highly expression of STUB1 protein showed no significant statistical differences in defferent ages, abortion status, menopause status, tumor sizes, histological types, FIGO stage, pelvic lymph node metastasis, vaginal invasion, parametrial infiltration, adjuvant radiotherapy and recurrence status in cervical cancer patients. The highly expression of STUB 1 expression with high differentiation grade and good survival status were significantly higher than that of patients with low differentiation grade and bad survival status (P<0.05). The positive expression of total hTERT protein showed no significant statistical differences in defferent ages, abortion status, menopause status, tumor sizes, histological types, pelvic lymph node metastasis, vaginal invasion, parametrial infiltration, adjuvant radiotherapy and OS in cervical cancer patients. The positive expression of total hTERT expression with high International Federation of Gynecology and Obstetrics (FIGO) stage, low differentiation grade and recurrence were significantly higher than that of patients with low International Federation of Gynecology and Obstetrics (FIGO) stage, high differentiation grade and no recurrence (P<0.05). The 5-year survival rate of total hTERT high expression group was lower than that of the total hTERT low expression, but the difference was not significant (P=0.128). The 5-year survival rate of STUB1 high expression group was significantly higher than that of the STUB1 low expression (P=0.016). The 5-year survival of the patients with high expression of hTERT in the nucleus not in the cytoplasmic are shorter than that of the low expression of hTERT in the nucleus (cytoplasmic 68.6% vs 71.7%, P=0.070; nucleus 61.8% vs 67.2%, P=0.016). The multivariate analysis showed that the STUB1 expression and the expression of hTERT in the cyplasmic were independent prognostic factors, not the total hTERT expression or in the nucleus.Conclusion The expression of STUB1 and hTERT in the cytoplasmic might be a predictor of poor prognosis and expected to become a new therapeutic target in cervical cancer.Part ? The relevance of the expression of STUB1 and hTERT cytoplasmic protein to radiosensitivity in human cervical cancer cell linesObjective To investigate the relationship between STUB1 protein level and the expression level of hTERT in the cytoplasmic and radiosensitivity of cervical cancer cell lines.Methods C33A and Hela cell lines were repeatedly irradiated by 6MV X-ray,600cGy/d,10 and 12ds, respectively. Clonogenic survival assay were used to detect the radiosensitivity. The expression level of STUB1 and hTERT protein were measured by Western blotting. Real-time PCR and TeloTAGGG telomerase PCR ELISA Kit (TRAP) were performed to examine telomere length and telomerase activity. The cell cycle distribution and cell apoptosis were examined by flow cytometry. The cell invasion ability was performed by transwell. The immune fluorescence detection was used to analyze the capacity of DNA damage repair. The ubiquitination of hTERT was measured by Co-Immunoprecipitation (Co-IP).Results The results of clonogenic survival assay showed that C33AR and HelaR cells were significantly more radioresistant than their parental cells. The western blotting showed that the total hTERT protein was highly expressed in radioresistant cell lines, while STUB1 protein was lower expressed. Moreover, telomerase activity, telomere length, G2/M phase arrest and invasion ability were increased, whereas the spontaneous and radiate induce apoptosis, and DNA damage foci were decreased in radioresistant cell lines. Furthermore, compared to C33A and Hela cells, the expression of hTERT in the cytoplasmic was much higher in C33AR and HelaR cells, but in the nucleus, the expression was much lower. More notably, the ubiquitination of hTERT protein in C33AR cells were much lower than that of C33A cells.Conclusion The radioresistant cell lines C33AR and HelaR were established successful. The intrinsic radiosensitivity between cervical cancer cells might be result from the increased of telomerase acitivity, telomere length and G2/M phase arrest, and the decresed of the apoptosis and DNA damage foci, which might be mediated by the decreased of the ubiquitination of hTERT expression in the cytoplasmic. STUB1 might participate in these process. Further study will be focused on the mechanisms of STUB 1 regulate radiosensitivity, and the effect of the increased the hTERT ubiquitination in the cytoplasmic on the radiosensitivity.Part III The effects of STUB 1 overexpression on the radiosensitivity of human cervical cancer cells to X-rays and its mechnismsObjective To explore the potential mechanisms of the STUB1 affect on the radiosensitivity in cervical cancer cells.Methods C33AR and HelaR cells stably overexpressing STUB1 were established by puromycin. Clonogenic survival assay were used to detect the radiosensitivity. The protein expression levels were measured by Western blotting. Real-time PCR and TeloTAGGG telomerase PCR ELISA Kit (TRAP) were performed to examine telomere length and telomerase activity. The cell cycle distribution and cell apoptosis were examined by flow cytometry. The immune fluorescence detection was used to analyze the capacity of DNA damage repair. The ubiquitination of hTERT was measured by Co-Immunoprecipitation (Co-IP).Results C33AR and HelaR cells stably overexpressing STUB1 were established successful. The results of clonogenic survival assay showed that STUB1 overexpression were significantly increasd the radiosnsiivity in both C33AR and HelaR cells. The results of TRAP and real-time PCR showed that telomerase activity, telomere length is decreased after STUB1 overexpressing in C33AR cells. The western blotting showed that the total hTERT protein, hTERT expression in the cytoplasmic, the expression of PI3K, AKT, p-AKT, TRF2, PTOP1, POT1 and TIN2 are decreased; while the expression of hTERT expression in the nucleus, TRF1 and RAP1 are inceased after STUB1 overexpression. The Co-IP showed that the ubiquitination of hTERT protein in the cytoplasmic of C33AR-STUB1 cells were much higher than that of C33AR-NC cells. The cell cycle assay showed that the G2/M phase distribution is increased after STUB1 overexpression, while the G2/M phase arrest is decreased after STUB1 overexpression. The spontaneous and radiate induce apoptosis, and DNA damage foci are enhanced in C33AR-STUB1, respectively.The western blotting showed that the expression of ATM, ATR, Chk1, Chk2, p-Chk1,p-Chk2 and Bcl-2 are decreased after STUB1 overexpression, but p-ATM, p-ATR, CDC25C, Bax and ? H2AX are increased.Conclusions After STUB1 overexpression in C33AR cells, first, the expression of hTERT in the cytoplasmic is decreased via increasing the ubquitilytion of the hTERT protein in the cytoplasmic, the hTERT protein activation by phosphorylation is decreased through downregulating the expression of PI3K/AKT. Secondly, the activation of hTERT in the nucleus is decreased, which lead to an decreased of telomere activity, telomere length and downregulation most telomere binding proteins. Thirdly, shorten the G2/M phase arrest mediated by ATM/ATR-Chkl/Chk2-CDC25C pathway, but enhanced cell apoptosis via increasing the ratio of Bax/Bcl-2. Finaly, enhanced DNA damage foci and at the end promote the radiosensitivity. In conclusion, STUB1 represents a potential target to promote radiotherapy effects in human cervical cancer. |
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