端粒保护蛋白hPOT1对宫颈癌细胞放射敏感性的影响及其机制研究

发布时间：2018-07-03 21:50

  本文选题：宫颈癌 + 端粒保护蛋白　； 参考：《武汉大学》2014年博士论文

【摘要】：第一部分hPOT1在宫颈癌及癌旁组织中的表达及其临床意义 目的：研究端粒保护蛋白hPOT1在宫颈癌和癌旁正常宫颈组织中的表达差异,以及hPOT1表达与宫颈癌临床病理特征之间的关系,探讨hPOT1在宫颈癌发生、发展中的作用。 方法：应用免疫组织化学方法检测94例宫颈癌组织和12例癌旁正常宫颈组织中hPOT1蛋白的表达情况。参照文献方法,采用hPOT1阳性指数(Positive Index, PI)来进行结果判定。比较hPOT1阳性指数在宫颈癌组织和宫旁正常组织中的差异。并根据hPOT1阳性指数将94例宫颈癌组织分为低表达组(≤30%)和高表达组(30%),分析两组在年龄分布、病理类型、病理分级、T分期以及有无淋巴结转移方面的统计学差异。 结果：免疫组化检测结果显示,hPOT1蛋白主要表达于宫颈癌和正常宫颈组织的细胞核内,染色呈棕黄色颗粒。hPOT1蛋白在宫颈癌组织中的阳性指数为31.52±8.95%,明显高于其在癌旁组织中的阳性指数(15.50±4.26%,P0.001)。hPOT1蛋白低表达组和高表达组的阳性指数分别为23.04±6.36%和40.76±6.49%(P0.001)。两组的年龄分布、病理类型(宫颈鳞癌与非鳞癌)、病理分级(G1-2/G3)、淋巴结转移与否(N0/N+)均无显著性差异(P0.05)。但是,在hPOT1高表达组中T2-3期比例(29/45)明显高于T1期比例(16/45),在hPOT1低表达组中T1期比例(35/49)显著高于T2-3期比例(14/49),两组差异有统计学意义(P0.001),表明T分期越晚,hPOT1表达水平越高。 结论：端粒保护蛋白hPOT1表达升高与宫颈癌的发生、发展过程密切相关,hPOT1可能成为抗肿瘤治疗的潜在靶点。 第二部分抑制hPOT1表达对宫颈癌细胞放射敏感性的影响 目的：建立稳定转染慢病毒载体重组hPOT1-shRNA质粒的宫颈癌C33A细胞模型,以此来研究抑制hPOT1表达对C33A细胞放射敏感性的影响,探讨hPOT1成为宫颈癌放射增敏靶点的可能性。 方法：构建靶向抑制hPOT1的重组慢病毒载体pGMLV-SC5-hPOT1-shRN A及相应的阴性对照空质粒载体pGMLV-SC5-Neg-shRNA,稳定转染C33A细胞,然后分别通过qRT-PCR法和Western blot方法检测hPOT1mRNA含量和蛋白表达水平的变化；通过克隆形成实验,运用GraphPad Prism5.0软件结合单击多靶模型公式拟合细胞存活曲线,得到SF2、Do、Dq、N值,计算放射增敏比SERSF2和SERDq。 结果：本实验成功构建了靶向抑制hPOT1的重组慢病毒载体,并成功转染C33A细胞,建立了稳定转染的实验组细胞模型C33A/hPOT1-shRNA和相应的阴性对照组细胞模型C33A/Neg-shRNA。经qRT-PCR和Western blot方法验证,C33A/hPOT1-shRNA细胞的hPOT1mRNA相对含量和蛋白表达水平显著降低(p0.001)。克隆形成实验结果显示,C33A/hPOT1-shRNA和C33A/Neg-shRNA细胞的SF2分别为0.380±0.059和0.563±0.072(P=0.0003),Do分别为1.428和1.658,Dq分别为0.983和2.393。抑制hPOT1后得到SERSF2和SERDq值分别为1.482、2.432。 结论：靶向性抑制hPOT1表达能够增加宫颈癌C33A细胞对放射线的敏感性,hPOT1有可能成为宫颈癌放射增敏的靶点。 第三部分抑制hPOT1表达对宫颈癌细胞放射增敏作用机制的研究 目的：进一步探讨抑制hPOT1表达提高C33A细胞放射敏感性的机制。 方法：靶向抑制C33A细胞hPOT1表达后,通过Southern Blot方法检测稳定转染细胞传代至8-10代时的平均端粒长度；TRAP-PCR-ELISA方法检测端粒酶活性；流式细胞术检测细胞经过X线照射后不同时间点的细胞凋亡水平和细胞周期分布的变化；细胞免疫荧光法结合激光共聚焦显微镜检测辐射后细胞核内γ-H2AX灶点变化(反映DNA损伤及修复)。 结果：靶向抑制C33A细胞的hPOT1蛋白表达,对端粒酶活性无明显影响；但导致其子代细胞的平均端粒延长(8.78±0.726kb比7.20±0.551kb,p=0.0055)：并增加了C33A细胞的自发性凋亡率(4.07±0.52%比5.16±0.67%,t=2.57,p=0.042)和辐射诱导的凋亡率(43.16+3.11%比34.95%+4.56,t=2.97,p=0.025)；降低了辐射后细胞周期G2/M期阻滞的比例(12h点t=5.995,p0.001；24h点t=9.527,p0.001；48h点t=12.81,p0.001),缩短了G2/M期阻滞的持续时间。通过激光共聚焦显微镜观察辐射后C33A细胞核内的γ-H2AX灶点,我们发现抑制hPOT1蛋白表达导致5Gy X线照射后0.5h γ-H2AX灶点明显增多(P0.001),到照射后12h,C33A/hPOT1-shRNA细胞核中残留的γ-H2AX灶点仍明显多于阴性对照组细胞C33A/Neg-shRNA,表明靶向抑制hPOT1导致辐射诱导的DSB损伤增多且DSB修复能力下降或者延迟。 结论：靶向抑制hPOT1表达增加C33A细胞放射敏感性的机制与端粒酶活性变化无关,而可能与端粒延长但端粒不稳定性增加有关。抑制hPOT1表达减弱和缩短了G2/M期阻滞,可能导致未经修复或修复不完全的细胞进入有丝分裂期,进一步增加端粒的不稳定性,增加细胞凋亡,从而导致放射增敏。另外,辐射后DSB损伤增多和DSB修复能力下降或修复延迟也可能与hPOT1的放射增敏作用有关。总之,抑制hPOT1表达导致C33A细胞放射敏感性增加,hPOT1有可能成为宫颈癌放射增敏的重要靶标之一,本研究为寻找放疗增敏的新靶标提供了有利的理论依据。
[Abstract]:Expression and clinical significance of hPOT1 in cervical cancer and paracancerous tissues
Objective: To investigate the difference in the expression of telomere protective protein hPOT1 in cervical cancer and normal cervix adjacent to cancer, and the relationship between the expression of hPOT1 and the clinicopathological features of cervical cancer, and explore the role of hPOT1 in the development of cervical cancer.
Methods: the expression of hPOT1 protein in 94 cervical cancer tissues and 12 normal cervical tissues was detected by immunohistochemistry. The hPOT1 positive index (Positive Index, PI) was used to determine the results with reference to the literature method. The difference between the hPOT1 positive index in the cervical cancer tissue and the normal tissue adjacent to the uterus was compared. And according to hPOT, the difference between the positive index of the positive index of the cervical cancer and the normal tissues near the uterus was compared. In the 1 positive index, 94 cases of cervical cancer were divided into low expression group (less than 30%) and high expression group (30%). The statistical differences in age distribution, pathological type, pathological grade, T staging and lymph node metastasis were analyzed in the two groups.
Results: the results of immunohistochemical staining showed that hPOT1 protein was mainly expressed in the nucleus of cervical cancer and normal cervical tissue. The positive index of brown yellow granules.HPOT1 protein in cervical cancer tissues was 31.52 + 8.95%, which was significantly higher than that of the positive index (15.50 + 4.26%, P0.001) of.HPOT1 protein in the para cancerous tissue. The positive index of the expression group was 23.04 + 6.36% and 40.76 + 6.49% (P0.001). The age distribution of the two groups, pathological type (cervical squamous cell carcinoma and non squamous cell carcinoma), pathological classification (G1-2/G3), lymph node metastasis (N0/N+) had no significant difference (P0.05). However, the proportion of T2-3 phase (29/45) in the high expression group of hPOT1 was significantly higher than that of T1 phase (16/45), in hPOT1. The T1 phase ratio (35/49) in the low expression group was significantly higher than that in the T2-3 phase (14/49), and the difference between the two groups was statistically significant (P0.001), indicating that the later the T stage, the higher the hPOT1 expression level.
Conclusion: the increased expression of telomere protection protein hPOT1 is closely related to the occurrence and development of cervical cancer. HPOT1 may be a potential target for anti-cancer therapy.
Second, inhibition of hPOT1 expression on radiosensitivity of cervical cancer cells.
Objective: to establish a C33A cell model to stabilize the hPOT1-shRNA plasmid transfected with lentivirus carrying body weight group, in order to study the effect of inhibition of hPOT1 expression on the radiosensitivity of C33A cells and to explore the possibility of hPOT1 as a radiosensitizing target of cervical cancer.
Methods: the recombinant lentivirus vector pGMLV-SC5-hPOT1-shRN A and the corresponding negative control empty plasmid vector pGMLV-SC5-Neg-shRNA were constructed, and the C33A cells were transfected stably. Then the changes of hPOT1mRNA content and protein expression level were detected by qRT-PCR and Western blot methods, and the experiment was carried out by cloning and using Graph. Pad Prism5.0 software combined with click multiple target model formula to fit cell survival curve, get SF2, Do, Dq, N value, and calculate radiosensitization ratio SERSF2 and SERDq..
Results: the recombinant lentivirus vector targeting hPOT1 was successfully constructed, and C33A cells were successfully transfected. The cell model C33A/hPOT1-shRNA of the stable transfected experimental group and the corresponding negative control group cell model C33A/Neg-shRNA. were verified by qRT-PCR and Western blot methods, and the hPOT1mRNA of C33A/hPOT1-shRNA cells was relatively contained. The expression level of the quantity and protein was significantly decreased (p0.001). The results of the clone formation experiment showed that the SF2 of C33A/hPOT1-shRNA and C33A/Neg-shRNA cells were 0.380 + 0.059 and 0.563 + 0.072 (P=0.0003), Do was 1.428 and 1.658 respectively. Dq was 0.983 and 2.393. inhibited hPOT1, respectively, and SERSF2 and SERDq were 1.482,2.432..
Conclusion: targeted inhibition of hPOT1 expression can increase the sensitivity of cervical cancer C33A cells to radiation, and hPOT1 may become a target for radiosensitization of cervical cancer.
The third part is to inhibit the radiosensitization mechanism of hPOT1 expression on cervical cancer cells.
Objective: to further explore the mechanism of inhibiting the expression of hPOT1 and enhancing the radiosensitivity of C33A cells.
Methods: after the target inhibition of C33A cell hPOT1 expression, the average telomere length of the stable transfected cells to 8-10 generations was detected by Southern Blot method. The activity of telomerase was detected by TRAP-PCR-ELISA, and the changes of cell apoptosis and cell cycle distribution at different time points after X-ray irradiation were detected by flow cytometry. Cell immunofluorescence combined with confocal laser scanning microscopy was used to detect the changes of the gamma -H2AX foci in the nucleus after irradiation (reflecting DNA damage and repair).
Results: the expression of hPOT1 protein in C33A cells had no significant effect on telomerase activity, but the average telomere prolonged (8.78 + 0.726kb ratio 7.20 + 0.551kb, p=0.0055) in the progeny cells, and increased the spontaneous apoptosis rate of C33A cells (4.07 + 0.52% to 5.16 + 0.67%, t=2.57, p=0.042) and radiation induced apoptosis rate (43.16+3.11) Compared with 34.95%+4.56, t=2.97, p=0.025), the proportion of G2/M block in cell cycle after radiation was reduced (12h point t=5.995, p0.001; 24h point t=9.527, p0.001; 48h t=12.81, etc.), which shortened the duration of the arrest period. White expression led to a significant increase in 0.5h gamma -H2AX focal points (P0.001) after 5Gy X-ray irradiation. After irradiation, the residual gamma -H2AX focal points in the nucleus of 12h and C33A/hPOT1-shRNA were still more than C33A/Neg-shRNA in the negative control group, indicating that the target inhibition hPOT1 led to the increase of the DSB damage induced by the radiation and the decline or delay of the DSB repair ability.
Conclusion: the mechanism of targeting inhibition of hPOT1 expression to increase the radiosensitivity of C33A cells is not related to the changes of telomerase activity, but may be related to telomere extension but increased telomere instability. Inhibition of hPOT1 expression is reduced and G2/M block is shortened, which may lead to unrepaired or repair of incomplete cells into mitosis, and further increase The instability of telomere increases cell apoptosis and causes radiation sensitization. In addition, the increase of DSB damage after radiation and the decrease of DSB repair ability or repair delay may also be related to the radiosensitization of hPOT1. In conclusion, inhibition of hPOT1 expression leads to increased radiosensitivity of C33A cells, and hPOT1 may be an important target for radiosensitization of cervical cancer. This study provides a theoretical basis for finding new targets for radiosensitization.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

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1 王忠民;姜继勇;乔新民;;复发宫颈癌发病因素及其不同治疗方式对预后的影响[J];中国实用妇科与产科杂志;2010年03期

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