KLF4抑制宫颈癌放疗敏感性及其分子机制研究
发布时间：2018-08-07 11:01
【摘要】：研究目的宫颈癌是妇科三大恶性肿瘤之一,最新统计预测:在美国一年新发的宫颈癌约有12360例。宫颈癌的治疗主要包括手术、放疗、化疗以及综合治疗等。放疗是宫颈癌治疗的重要手段之一,但放疗抵抗成为限制疗效的主要障碍。放疗抵抗的因素很多:凋亡、环氧合酶(cyclooxygenases,COXs)、血管新生、低氧以及温度作用等均可影响放疗效果。Kruppel样因子4(Krüppel-like factor 4,KLF4)是一转录因子,在人类组织中广泛表达,在生长发育、突变及维持正常组织稳态等生理活动中发挥着重要作用。研究发现KLF4在放疗损伤中表现出抗凋亡作用,通过协助DNA修复、降低细胞死亡、增加更新能力或三者共同作用等辅助细胞生存。体内研究发现KLF4在调节BMI1+小肠干细胞内稳态和放疗损伤后再生能力方面起着重要作用。目前有研究表明,放疗后KLF4可导致细胞周期捕获、抑制BAX表达,最终降低细胞凋亡能力;对小鼠放疗后KLF4也表现出抗凋亡作用。再者,我们课题申请前期预实验发现在HeLa细胞中KLF4表达与放疗敏感性相关。于是我们推测KLF4与宫颈癌的放疗敏感性可能相关,如果对调节KLF4表达水平,是否能对宫颈癌细胞的放疗敏感性产生影响,其分子机制如何?为了验证这一假设,我们将从临床资料回顾性研究、分子、细胞、以及动物几个层面进行研究讨论,这将为KLF4参与放疗敏感性的机制研究奠定基础,为提高临床放疗效果提供潜在的药物靶点。研究方法1.在组织层面,采用免疫组织化学技术检测KLF4表达,结合病例资料进行相关性统计分析。2.在分子层面,使用免疫荧光、qPCR和western-blot检测相关分子表达,应用RNAi技术和过表达技术构建慢病毒载体,人源表达谱芯片检测过表达KLF4后调节的下游基因,双荧光素酶报告基因检测启动子转录活性。3.在细胞层面,细胞流式术测细胞周期和凋亡,平板克隆测放疗敏感性。4.在动物层面,建立宫颈癌裸鼠皮下荷瘤模型,根据成瘤模型生长进一步观察KLF4在宫颈癌放疗中的作用,HE染色观察组织形态,免疫组织化学技术检测增殖、凋亡相关蛋白表达水平。研究结果1.放疗敏感性与FIGO分期、肿瘤直径大小、以及肿瘤分化程度相关;KLF4表达水平与FIGO分期以及肿瘤分化程度相关;KLF4表达水平与放疗敏感性相关,在放疗耐受组中表达高于敏感组。耐受组中KLF4的高表达与放疗无反应、放疗后原位复发、远处转移相关。KLF4高表达组总生存期和肿瘤无进展生存期显著低于低表达组。单/多因素回归分析提示:KLF4表达水平、FIGO分期、肿瘤直径大小、以及肿瘤分化程度是影响无瘤生存期和总生存时间的高风险因素。2.4株宫颈癌细胞中KLF4表达水平,其中以HeLa表达水平最高,C33A表达水平最低;比较HeLa和C33A的放疗敏感性,C33A放疗敏感性高于HeLa;成功构建KLF4 RNAi和过表达慢病毒;过表达KLF4降低宫颈癌细胞放疗敏感性,下调KLF4增强宫颈癌放疗敏感性。3.成功建立宫颈癌裸鼠皮下荷瘤模型,KLF4过表达增加宫颈癌瘤体组织的放疗抵抗性。下调KLF4增强宫颈癌瘤体组织的放疗敏感性。4.过表达KLF4上调IGF2基因;过表达KLF4增强IGF2启动子转录活性;过表达KLF4可激活IGF2作用于PI3K-AKT通路从而参与放疗抵抗作用。结论综上所述,高表达KLF4抑制宫颈癌放疗敏感性,干预KLF4表达水平可影响宫颈癌放疗敏感性,KLF4可通过提高IGF2水平,激活PI3K-AKT通路,从而参与宫颈癌放疗抵抗作用。实验为KLF4参与放疗敏感性的机制研究奠定基础,为提高临床放疗效果提供潜在的药物靶点。
[Abstract]:Research objective cervical cancer is one of the three major gynecologic malignancies. The latest statistical prediction is that there are about 12360 new cervical cancers in the United States. The treatment of cervical cancer mainly includes surgery, radiotherapy, chemotherapy and comprehensive treatment. Radiotherapy is one of the important methods for the treatment of cervical cancer, but radiation resistance is the main obstacle to the effect. There are many factors of resistance: apoptosis, cyclooxygenases (COXs), angiogenesis, hypoxia, and temperature action, which can affect the effect of radiation therapy,.Kruppel like factor 4 (Kr u ppel-like factor 4, KLF4), a transcription factor, which is widely expressed in human tissues, and plays a role in growth, mutation and maintenance of normal tissue homeostasis. Important role. The study found that KLF4 showed anti apoptotic effect in radiation injury and assisted cell survival by assisting DNA repair, reducing cell death, increasing the ability to renew or three common effects. In vivo studies have found that KLF4 plays an important role in regulating the homeostasis of BMI1+ small intestinal stem cells and regenerative ability after radiation injury. The study showed that KLF4 could lead to cell cycle capture, inhibit BAX expression, and eventually reduce cell apoptosis, and KLF4 also showed anti apoptosis effect on mice after radiotherapy. Furthermore, our preliminary preliminary preliminary experiment found that the expression of KLF4 in HeLa cells was related to the sensitivity of radiotherapy to radiotherapy, so we speculated that KLF4 was sensitive to radiotherapy of cervical cancer. What is the molecular mechanism of the effect of KLF4 expression level on radiation sensitivity of cervical cancer cells? In order to verify this hypothesis, we will discuss the clinical data in a retrospective study, molecular, cell, and animal studies, which will be a mechanism for the involvement of KLF4 in radiation sensitivity. Lay the foundation and provide potential drug targets for improving the effect of clinical radiotherapy. Method 1. at the tissue level, the expression of KLF4 was detected by immunohistochemical technique, and the correlation statistical analysis of the case data was carried out at the molecular level of.2.. Immunofluorescence, qPCR and Western-blot were used to detect the expression of related molecules, and RNAi technology and overwatch were used. The lentivirus vector was constructed by technology. The downstream gene was detected by human source expression chip, and the double luciferase reporter gene was used to detect the promoter transcriptional activity.3. at the cell level. Cell flow cytometry was used to detect cell cycle and apoptosis. The sensitivity of.4. at the animal level was measured by flat clones, and the subcutaneous tumor bearing model of cervical cancer in nude mice was established. The role of KLF4 in the radiotherapy of cervical cancer was further observed on the basis of the tumor growth model. HE staining was used to observe tissue morphology, immunohistochemical technique to detect proliferation and expression of apoptosis related proteins. Results 1. the results of radiotherapy sensitivity and FIGO staging, tumor diameter, and the degree of tumor differentiation were correlated with the level of KLF4 expression and FIGO staging. The level of tumor differentiation was related, the expression of KLF4 was associated with the sensitivity of radiotherapy. The expression of KLF4 was higher in the radiotherapy tolerance group than in the sensitive group. The high expression of the tumor in the tolerance group was not reacted with the radiotherapy, the recurrence in situ after radiotherapy, the total survival time and the progression free survival period of the distant metastasis related.KLF4 high expression group were significantly lower than that of the low expression group. The expression level of KLF4, FIGO stage, tumor diameter, and the degree of tumor differentiation were the high risk factors of KLF4 expression in.2.4 strain of.2.4 strain, which had the highest level of HeLa expression and the lowest level of C33A expression, compared with the radiotherapy sensitivity of HeLa and C33A, and the sensitivity of C33A radiotherapy was higher than that of HeLa and C33A. HeLa; successful construction of KLF4 RNAi and overexpression of lentivirus; overexpression of KLF4 to reduce the sensitivity of cervical cancer cell radiation, down regulation of KLF4 to enhance the sensitivity of cervical cancer radiation sensitivity.3. to establish a subcutaneous tumor bearing model of cervical cancer in nude mice, KLF4 overexpression to increase the radiation resistance of cervical cancer tissue. Down regulation of KLF4 to enhance the sensitivity of cervical cancer tissue to radiotherapy .4. overexpression of KLF4 up-regulated IGF2 gene, overexpressed KLF4 to enhance IGF2 promoter activity, overexpression of KLF4 can activate IGF2 to act on PI3K-AKT pathway and thus participate in radiation resistance. Conclusion high expression of KLF4 inhibits radiotherapy sensitivity of cervical cancer, and intervention of KLF4 expression can affect radiotherapy sensitivity of cervical cancer, KLF4 can be improved by improving The level of IGF2 activates the PI3K-AKT pathway and participates in the radiation resistance of cervical cancer. The experiment lays the foundation for the study of the mechanism of KLF4's involvement in radiation sensitivity, and provides potential drug targets for improving the effect of clinical radiotherapy.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33
，
本文编号：2169816
[Abstract]:Research objective cervical cancer is one of the three major gynecologic malignancies. The latest statistical prediction is that there are about 12360 new cervical cancers in the United States. The treatment of cervical cancer mainly includes surgery, radiotherapy, chemotherapy and comprehensive treatment. Radiotherapy is one of the important methods for the treatment of cervical cancer, but radiation resistance is the main obstacle to the effect. There are many factors of resistance: apoptosis, cyclooxygenases (COXs), angiogenesis, hypoxia, and temperature action, which can affect the effect of radiation therapy,.Kruppel like factor 4 (Kr u ppel-like factor 4, KLF4), a transcription factor, which is widely expressed in human tissues, and plays a role in growth, mutation and maintenance of normal tissue homeostasis. Important role. The study found that KLF4 showed anti apoptotic effect in radiation injury and assisted cell survival by assisting DNA repair, reducing cell death, increasing the ability to renew or three common effects. In vivo studies have found that KLF4 plays an important role in regulating the homeostasis of BMI1+ small intestinal stem cells and regenerative ability after radiation injury. The study showed that KLF4 could lead to cell cycle capture, inhibit BAX expression, and eventually reduce cell apoptosis, and KLF4 also showed anti apoptosis effect on mice after radiotherapy. Furthermore, our preliminary preliminary preliminary experiment found that the expression of KLF4 in HeLa cells was related to the sensitivity of radiotherapy to radiotherapy, so we speculated that KLF4 was sensitive to radiotherapy of cervical cancer. What is the molecular mechanism of the effect of KLF4 expression level on radiation sensitivity of cervical cancer cells? In order to verify this hypothesis, we will discuss the clinical data in a retrospective study, molecular, cell, and animal studies, which will be a mechanism for the involvement of KLF4 in radiation sensitivity. Lay the foundation and provide potential drug targets for improving the effect of clinical radiotherapy. Method 1. at the tissue level, the expression of KLF4 was detected by immunohistochemical technique, and the correlation statistical analysis of the case data was carried out at the molecular level of.2.. Immunofluorescence, qPCR and Western-blot were used to detect the expression of related molecules, and RNAi technology and overwatch were used. The lentivirus vector was constructed by technology. The downstream gene was detected by human source expression chip, and the double luciferase reporter gene was used to detect the promoter transcriptional activity.3. at the cell level. Cell flow cytometry was used to detect cell cycle and apoptosis. The sensitivity of.4. at the animal level was measured by flat clones, and the subcutaneous tumor bearing model of cervical cancer in nude mice was established. The role of KLF4 in the radiotherapy of cervical cancer was further observed on the basis of the tumor growth model. HE staining was used to observe tissue morphology, immunohistochemical technique to detect proliferation and expression of apoptosis related proteins. Results 1. the results of radiotherapy sensitivity and FIGO staging, tumor diameter, and the degree of tumor differentiation were correlated with the level of KLF4 expression and FIGO staging. The level of tumor differentiation was related, the expression of KLF4 was associated with the sensitivity of radiotherapy. The expression of KLF4 was higher in the radiotherapy tolerance group than in the sensitive group. The high expression of the tumor in the tolerance group was not reacted with the radiotherapy, the recurrence in situ after radiotherapy, the total survival time and the progression free survival period of the distant metastasis related.KLF4 high expression group were significantly lower than that of the low expression group. The expression level of KLF4, FIGO stage, tumor diameter, and the degree of tumor differentiation were the high risk factors of KLF4 expression in.2.4 strain of.2.4 strain, which had the highest level of HeLa expression and the lowest level of C33A expression, compared with the radiotherapy sensitivity of HeLa and C33A, and the sensitivity of C33A radiotherapy was higher than that of HeLa and C33A. HeLa; successful construction of KLF4 RNAi and overexpression of lentivirus; overexpression of KLF4 to reduce the sensitivity of cervical cancer cell radiation, down regulation of KLF4 to enhance the sensitivity of cervical cancer radiation sensitivity.3. to establish a subcutaneous tumor bearing model of cervical cancer in nude mice, KLF4 overexpression to increase the radiation resistance of cervical cancer tissue. Down regulation of KLF4 to enhance the sensitivity of cervical cancer tissue to radiotherapy .4. overexpression of KLF4 up-regulated IGF2 gene, overexpressed KLF4 to enhance IGF2 promoter activity, overexpression of KLF4 can activate IGF2 to act on PI3K-AKT pathway and thus participate in radiation resistance. Conclusion high expression of KLF4 inhibits radiotherapy sensitivity of cervical cancer, and intervention of KLF4 expression can affect radiotherapy sensitivity of cervical cancer, KLF4 can be improved by improving The level of IGF2 activates the PI3K-AKT pathway and participates in the radiation resistance of cervical cancer. The experiment lays the foundation for the study of the mechanism of KLF4's involvement in radiation sensitivity, and provides potential drug targets for improving the effect of clinical radiotherapy.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33
，
本文编号：2169816
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