高迁移族蛋白HMGB1对端粒稳态的调控及与放射敏感性的相关性研究

发布时间：2018-05-20 13:32

本文选题：乳腺癌 + HMGB1　；参考：《武汉大学》2015年博士论文

【摘要】：第一部分高迁移族蛋白HMGB1的表达与人乳腺癌细胞放射敏感性的关系研究目的：研究HMGB1的表达对人乳腺癌细胞放射敏感性的影响。方法：构建人乳腺癌MCF-7-sh-HMGB1及阴性对照模型MCF-7-NC稳转细胞模型。将shRNA进行合成克隆入pGPU6/GFP/Neo shRNA载体中,将合成之后的载体命名为pGPU6/GFP/Neo-HMGB 1和pGPU6/GFP/Neo-shNC。HMGB1低表达和阴性对照稳定转染细胞系采用600ug/ml G418筛选5周,挑选克隆扩增获得,获得的稳定转染细胞命名为MCF-7-shHMGB1和MCF-7-NC,采用RT-PCR和WB检测其HMGB1的干扰效率,克隆形成实验检测MCF-7-shHMGB1和MCF-7-NC细胞系的放射敏感性。结果：稳定转染细胞系的荧光纯度为99%,MCF-7-sh-HMGB1与阴性对照组MCF-7-NC和空白亲本组相比,mRNA相对表达量为(0.12975+0.0314)vS.(0.9625±0.0476) vs.(1)。.HMGB干扰后其mRNA表达为亲本的12.5%,亲本细胞与MCF-7-NC间无明显差异；蛋白表达方面HMGB1显著低于MCF-7-NC组,克隆形成显示HMGB1干扰组的放射敏感性显著提高,MCF-7-NC和MCF-7-shHMGB1的F2为(0.7756±0.0016) vs. (0.5732±0.0031) (p0.01); DO 为(2.7555±0.0810) vs. (2.4807±0.0331*) (p0.05); Dq为(3.1689±0.1431) vs. (0.7225±0.0210*) (p0.05)。结论：成功构建了人乳腺癌MCF-7-sh-HMGB1及MCF-7-NC稳转细胞模型,MCF-7-shHMGB1稳定下调HMGB1的表达,MCF-7-NC无明显下调；干扰HMGB1的表达导致MCF-7细胞的放射敏感性显著增强。第二部分下调IMGB1的表达增强了人乳腺癌细胞放射敏感性的机制研究目的：探讨HMGB1调控人乳腺癌细胞放射敏感性的分子机制,为乳腺癌的防治提供新的有效靶点,从而为临床治疗提供理论依据。方法：采用稳定干扰细胞模型MCF-7-sh-HMGB1及阴性对照模型MCF-7-NC进行机制研究,相对端粒长度采用realtime-PCR,目的蛋白表达采用Western blot,端粒酶活性采用PCR-ELISA。细胞增殖采用CCK-8检测。细胞周期与凋亡采用流式细胞术检测,DNA损伤灶点的表达采用免疫荧光-共聚焦检测。活性氧ROS采用活性氧检测试剂盒(荧光法)检测。结果：下调HMGB1的表达增加了MCF-7细胞的放射敏感性,减少了hTERT和cyclinD1的集聚。MCF-7、MCF-7-shHMGB1和MCF-7-NC的相对端粒长度分别为0.8574±0.0812、0.6763±0.0610和1,MCF-7-shHMGB1组与MCF-7-NC相比,相对端粒长度明显缩短(***p0.001)；与MCF-7组相比结果也类似(*p0.05)。MCF-7-NC与MCF-7-shHMGB1二者端粒酶活性分别为1.6155±0.1512和1.169±0.0924,干扰HMGB1后导致MCF-7端粒酶活性明显下降(*p0.05),而且显著抑制了细胞增殖。在未照射时,MCF-7-NC和MCF-7-shHMGB1细胞的S期的比例分别为(44.090±5.78)%和(29.080±4.234)%；在6Gy照射后6h,12h,24h进行检测,MCF-7-NC和MCF-7-shHMGB1细胞的G2/M分别为(29.790±3.291)%、(23.324±4.580)%、(28.592±2.673)%VS(34.293±4.231)%、(31.959±3.265)%、(35.653±5.297)%,两者差异具有显著性意义,而其余细胞周期间无明显差异。MCF-7-NC和MCF-7-shHMGB1的凋亡比例分别为(5.00±0.848)%和(25.500±1.272)%,下调HMGB1增加了凋亡水平(**p0.01)。在早期阶段,HMGB1也参与了活性氧的调控。HMGB1通过改变端粒结合蛋白的水平来调控端粒稳态,如TPP1、TRF1、TRF2。HMGB1的下调同时抑制了ATM和ATR信号通路。结论：以上结果显示下调HMGB1的表达破坏了人乳腺癌的端粒稳态,增强了放射敏感性,抑制了DNA损伤修复,抑制了细胞生长、促进其凋亡,HMGB1可能是人乳腺癌放射治疗潜在的新靶点。第三部分人乳腺癌组织中HMGB 1的表达与临床病理特征相关性分析目的：比较HMGB1在乳腺浸润性导管癌患者的肿瘤组织与癌旁组织中的表达差异,并分析HMGB1蛋白表达水平与病理分级、淋巴结转移、总体预后等相关临床病理特征的关系,为乳腺癌的治疗寻找新的靶标。方法：收集武汉大学中南医院病理科存档的72例乳腺癌患者的组织标本切片,入组标准：我院肿瘤科2001-7至2011-8收治的存有完整蜡块组织的首诊乳腺癌患者；未行术前新辅助化疗,所有患者均行病理诊断确诊为浸润性导管癌；接受标准术后辅助治疗；无其他部位原发肿瘤；随访时间截止为2013-1。利用免疫组化法检测患者癌组织与癌旁组织中HMGB1的表达差异,并分析HMGB1蛋白的表达与乳腺癌患者的年龄、绝经状态、肿瘤大小、病理分级、激素受体水平、淋巴结转移、M分期、总体生存状态的相关性。Kaplan-Meier方法分析HMGB1蛋白的表达对累积生存率的影响。结果：HMGB1的阳性表达与年龄、绝经状态、病理分级、雌激素受体状态、HER-2受体状态、淋巴结转移分期及M分期无明显相关性,与肿瘤的增殖能力、孕激素受体状态正相关,与累积生存率负相关,具有统计学意义(P0.05)。结论：HMGB1的表达与乳腺癌的增殖正相关,其高表达促进肿瘤生长,且与预后负相关,但需要进一步扩大样本量及延长部分样本的随访时间。
[Abstract]:Part 1 the relationship between the expression of high mobility group protein HMGB1 and the radiosensitivity of human breast cancer cells: Objective: To study the effect of HMGB1 expression on the radiosensitivity of human breast cancer cells. Methods: to construct human breast cancer MCF-7-sh-HMGB1 and negative control model MCF-7-NC stable cell model. ShRNA was synthesized and cloned into pGPU6/GFP/N In the EO shRNA carrier, the synthesized carrier was named pGPU6/GFP/Neo-HMGB 1 and pGPU6/GFP/Neo-shNC.HMGB1 in the low expression and negative control stable transfection cell line for 5 weeks by 600ug/ml G418, and the cloned amplification was selected. The stable transfected cells were named MCF-7-shHMGB1 and MCF-7-NC, and RT-PCR and WB were used to detect the interference of HMGB1. Results: the radiosensitivity of MCF-7-shHMGB1 and MCF-7-NC cell lines was detected by cloning. Results: the fluorescence purity of the stable transfected cell line was 99%. Compared with the negative control group MCF-7-NC and the blank parent group, the relative expression of mRNA was (0.12975+0.0314) vS. (0.9625 + 0.0476) vs. (1)..HMGB interference, and the mRNA was expressed as a parent. In 12.5%, there was no significant difference between the parent cells and MCF-7-NC; the expression of protein HMGB1 was significantly lower than that of the MCF-7-NC group, and the clonal formation showed that the radiosensitivity of the HMGB1 interference group was significantly increased, the F2 of MCF-7-NC and MCF-7-shHMGB1 was (0.7756 + 0.0016) vs. (0.5732 + 0.0031) (P0.01), DO was (2.7555 + 0.0810) vs. (2.4807 + 0.0331*). Dq was (3.1689 + 0.1431) vs. (0.7225 + 0.0210*) (P0.05). Conclusion: successfully constructed the MCF-7-sh-HMGB1 and MCF-7-NC stable cell model of human breast cancer, MCF-7-shHMGB1 stably downregulated the expression of HMGB1, and there was no obvious downregulation of MCF-7-NC. The expression of interference HMGB1 resulted in a significant increase in the radiosensitivity of MCF-7 cells. Second down regulated the expression of IMGB1. The mechanism of enhancing the radiosensitivity of human breast cancer cells is to explore the molecular mechanism of HMGB1 regulating the radiosensitivity of human breast cancer cells, providing new effective targets for the prevention and control of breast cancer, and providing a theoretical basis for clinical treatment. Methods: a stable interfering cell model MCF-7-sh-HMGB1 and a negative control model MCF-7-N are used. The mechanism of C was studied. The relative telomere length was realtime-PCR, the expression of the target protein was Western blot, the activity of telomerase was detected by CCK-8 in PCR-ELISA. cell proliferation. The cell cycle and apoptosis were detected by flow cytometry. The expression of DNA damage focal point was detected by immunofluorescence confocal detection. Reactive oxygen ROS was tested by active oxygen test. Results: the expression of HMGB1 decreased the radiosensitivity of MCF-7 cells and reduced the concentration of hTERT and cyclinD1, and the relative telomere length of MCF-7-shHMGB1 and MCF-7-NC was 0.8574 + 0.0812,0.6763 + 0.0610 and 1 respectively. Compared with MCF-7-NC, the relative telomere length of MCF-7-shHMGB1 group was significantly shorter (***p0.001). Compared with the MCF-7 group, the telomerase activity of *p0.05.MCF-7-NC and MCF-7-shHMGB1 two were 1.6155 + 0.1512 and 1.169 + 0.0924 respectively. After interfering HMGB1, the telomerase activity of MCF-7 decreased significantly (*p0.05), and the cell proliferation was significantly inhibited. The S phase ratio of MCF-7-NC and MCF-7-shHMGB1 cells was 44.090, respectively (44.090). (+ 5.78)% and (29.080 + 4.234)%, 6h, 12h, 24h were detected after 6Gy irradiation. The G2/M of MCF-7-NC and MCF-7-shHMGB1 cells was (29.790 + 3.291)%, (23.324 + 4.580)%, (28.592 + 2.673)%VS (34.293 +%)%, (31.959 + 3.265)%, (31.959 + 3.265)%, and the difference was significant, but there was no significant difference between the remaining cell cycles. The apoptosis ratio of MCF-7-shHMGB1 was (5 + 0.848)% and (25.500 + 1.272)% respectively. Down regulation of HMGB1 increased the level of apoptosis (**p0.01). At the early stage, HMGB1 also participated in the regulation of reactive oxygen species (.HMGB1) by changing the level of telomere binding protein to regulate telomere homeostasis, such as TPP1, TRF1, TRF2.HMGB1 and inhibition of ATM and ATR signaling. Road. Conclusion: the above results show that the expression of HMGB1 down-regulation destroys the telomere homeostasis of human breast cancer, enhances radiosensitivity, inhibits the repair of DNA damage, inhibits cell growth, and promotes its apoptosis. HMGB1 may be a potential new target for radiation therapy in human breast cancer. The expression of HMGB 1 in third human breast cancer tissues and clinical pathology Objective: To compare the difference in the expression of HMGB1 in the tumor tissues of the patients with invasive ductal carcinoma of the breast, and to analyze the relationship between the expression of HMGB1 protein and pathological classification, lymph node metastasis, the overall prognosis and other related clinicopathological features, to find a new target for the treatment of breast cancer. Methods: collect the Wuhan big 72 cases of breast cancer in the pathology department of the central and southern hospital were examined for tissue specimens. The standard of the group: 2001-7 to 2011-8 of the Department of oncology in our hospital were treated with complete paraffin tissue for the first diagnosis of breast cancer patients; no preoperative neoadjuvant chemotherapy was performed. All patients were diagnosed with a dipped ductal carcinoma by pathological diagnosis; the standard postoperative adjuvant treatment was accepted. There was no primary tumor in other parts; the time of follow-up was 2013-1. using immunohistochemical method to detect the difference in the expression of HMGB1 in the cancer tissues and para cancerous tissues, and to analyze the expression of HMGB1 protein and the age, menopause, tumor size, pathological grade, hormone receptor level, lymph node metastasis, M staging, overall survival state of the patients with breast cancer. The correlation.Kaplan-Meier method was used to analyze the effect of HMGB1 protein expression on the cumulative survival rate. Results: the positive expression of HMGB1 was not associated with age, menopause, pathological grade, estrogen receptor status, HER-2 receptor status, lymph node metastasis staging and M staging, and was positively related to the proliferation of tumor and progesterone receptor status. The negative correlation of cumulative survival rate was statistically significant (P0.05). Conclusion: the expression of HMGB1 is positively related to the proliferation of breast cancer. Its high expression promotes tumor growth and has a negative correlation with the prognosis, but it is necessary to further expand the sample size and prolong the follow-up time of some samples.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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