胰腺癌发病中NF-κB与Hedgehog信号通路交互作用的K-ras基因突变依赖性的分子机制研究

发布时间：2018-06-27 02:16

本文选题：胰腺癌 + Hedgehog通路　；参考：《第二军医大学》2015年博士论文

【摘要】：研究背景及目的胰腺导管腺癌(pancreatic ductal adenocarcinoma, PDAC)是消化系统常见恶性肿瘤之一,约占胰腺恶性肿瘤的90%以上。既往研究证实,诸如Hedgehog (HH)通路异常活化、炎症和K-ras突变等因素均参与了PDAC的形成过程。HH信号通路参与了胰腺形态发生过程,而在大多数正常胰腺中该通路处于沉默状态。但HH通路异常活化在PDAC中十分常见,且在早期即有表现。HH通路活化机制可大致分为①配体过表达介导的经典活化通路②以信号通路成员突变为主的非经典活化通路,其中前者较为常见。在人类,经典活化过程由HH通路配体Sonic Hedgehog (Shh)引发,最终导致HH通路核因子glioma-associated oncogene(Gli)家族蛋白入核,启动对下游基因的调控。在Gli家族中,Gli1是最主要的活化因子,Gli1入核是HH通路活化的标志。研究证实,炎症和PDAC的发生密切相关。核因子-κB (nuclear factor-kappa B, NF-κB)是炎症反应的重要成员,它参与了PDAC的发生、发展过程。NF-κB入核,NF-κB通路活化,从而启动对下游基因的调控。K-ras突变发生于PDAC早期。在PDAC中K-ras突变率颇高,可达75～95%。临床中发现,K-ras基因突变存在多个位点,但主要集中在第12、13密码子。K-ras蛋白只有在和三磷酸鸟苷(guanosine triphosphate, GTP)结合后(K-ras-GTP)才能实现对细胞活性的调控。通常情况下K-ras-GTP蛋白会在GTP酶活化蛋白(GTPase-activating proteins, GAPs)的作用下迅速转变成无活性的K-ras-GDP,使K-ras蛋白处于失活状态。K-ras基因突变导致K-ras蛋白持续处于K-ras-GTP状态,使K-ras蛋白保持活化状态。既往研究证实上述三条通路参与PDAC的发病过程,HH通路和NF-κB间存在相关性,且NF-κB可活化HH通路,K-ras突变可导致HH通路和NF-κB的活化,但三者间在胰腺癌发生中相互作用及其机制却鲜见报道。探讨三者之间的相互作用机制,对深入了解PDAC发病的分子机制,寻求特异、有效的个体化治疗方法至关重要。据此,本课题开展了以下研究：第一部分PDAC组织和细胞株中HH与NF-κB通路主要成员表达和K-ras基因突变的检测、及三者之间相关性分析实验材料、方法：收集32例胰腺癌组织和3株PDAC细胞株,采用免疫组化、实时定量PC、western-blot和DNA测序技术,分别检测Shh蛋白和Gli1、NF-κB核蛋白的表达,以及K-ras基因12和13密码子突变情况。实验结果：在PDAC组织中存在Shh、Gli1和NF-κB表达量之间的正相关性,同时这种正相关性在K-ras突变的状态下显著存在,表明在PDAC发病中HH和NF-κB通路活化具有显著正相关性,并且这种相关性具有一定的K-ras突变依赖性；PDAC细胞中可能也存在HH通路和NF-κB通路活化的正相关性,并且这种相关性也可能具有一定的K-ras突变依赖性。第二部分PDAC细胞株中活化NF-κB通路对HH通路的调控作用观察,并分析此调控作用对K-ras突变的依赖性实验材料、方法：选取K-ras 基因野生(BxPC-3)和突变(Panc-1/12密码子突变；SW1990/13密码子突变)PDAC细胞进行干预,活化NF-κB通路的作用研究。对上述细胞株分别进行①白介素-1p(interleukin-1β, IL-1β)②肿瘤坏死因子-α(tumor necrosis factor-a, TNF-a)刺激干预(以单纯培养基处理组作为对照),采用q-PCR、Westernblot方法检测干预后Shh, Glil和NF-kB的mRNA和蛋白质表达变化,同时平行检测干预后细胞增殖、凋亡等变化,观察干预方法对NF-κB通路本身活化作用以及促进HH通路活化的作用效果,并分析这种作用效果与K-ras突变的相关性。实验结果：在K-ras突变的PDAC细胞株中,干预活化NF-κB通路后可促进HH通路进一步高度活化,并同时增强了PDAC细胞株的恶性细胞行为特征；但在K-ras野生的PDAC细胞株中,这种干预活化的作用效果不显著。研究表明,在PDAC发病中NF-κB通路活化可Crosstalk(串话)促进HH通路活化,并且此Crosstalk作用具有一定的K-ras突变依赖性。第三部分PDAC细胞株中活化-HH通路对NF-κB通路的调控作用观察、并分析此调控作用对K-ras突变的依赖性实验材料、方法：同第二部分研究,选取K-ras基因野生(BxPC-3)和突变(Panc-1/12密码子突变；SW1990/13密码子突变)的PDAC细胞株,分别进行①Gli1 cDNA转染过表达、②配体Shh刺激干预(以单纯培养基处理组作为对照),采用q-PCR、 Westernblot方法检测干预后Shh、Gli1和NF-κB的mRNA和蛋白质表达变化,同时平行检测干预后细胞增殖、凋亡等变化,观察干预方法对HH通路本身的活化作用以及促进NF-κB通路活化的作用效果,并分析这种作用效果与K-ras突变的关系。实验结果：在K-ras突变的PDAC细胞株中,干预活化HH通路后可促进NF-κB通路进一步高度活化,并同时增强了PDAC细胞株的恶性细胞行为特征；但在K-ras野生的PDAC细胞株中,这种干预活化的作用效果不显著。研究表明,在PDAC发病中HH通路活化可Crosstalk (串话)促进NF-κB通路活化,并此Crosstalk作用具有一定的K-ras突变依赖性。第四部分PDAC中HH通路和NF-κB通路之间Crosstalk的K-ras突变依赖性的分子机制研究实验材料、方法：在上述第二和三部分的细胞干预实验基础上,进一步检测细胞中K-ras蛋白及活性表达变化,检钡K-ras基因下游基因p-/t-ERK1/2,和p-/t-AKTl的表达变化,并利用Ras活性检测试剂盒检测Ras活性变化,综合判断K-ras通路的活化水平变化；同时分别采用RNA干扰技术(si-Kras)阻断PDAC细胞内K-ras基因的表达,和采用G12D突变型K-ras基因cDNA质粒转染野生型细胞,观察这些阻断和补救的反向措施对K-ras本身活性的干预成功性,及对Crosstalk相互促进活化的影响,进一步确定HH和NF-κB通路之间相互促进活化的Crosstalk的K-ras突变依赖性的分子机制。实验结果：K-ras活性改变是HH和NF-κB通路活化及二者间Corsstalk对K-ras突变依赖的主要分子机制。第五部分PDAC中HH通路和NF-κB通路之间Crosstalk的K-ras突变依赖性的动物研究实验材料、方法：在细胞学研究基础上,我们进行动物实验,以深入探讨HH通路和NF-κB通路之间Crosstalk的K-ras突变依赖性。我们利用①Glil cDNA稳转的SW1990细胞和普通SW1990细胞种植于胸腺缺失的裸鼠,检测Gli1和NF-κB核蛋白表达,观察Gli1过表达,HH通路活化对NF-κB通路的影响；同时平行检测肿瘤大小,验证HH通路活化对肿瘤生长的影响；②分别以K-ras基因野生(BxPC-3)和突变(Panc-1/12密码子突变；SW1990/13密码子突变)的PDAC细胞种植于胸腺缺失的裸鼠,成瘤后分别以PBS(对照)、Shh、IL-1β和TNF-a刺激裸鼠,检测Shh蛋白表达以及Gli1和NF-κB核蛋白表达,观察不同K-ras表型细胞对HH通路和NF-κB通路活化,以及二者间的crosstalk;同时平行检测肿瘤大小,验证HH通路和NF-κB通路活化对肿瘤生长的影响。实验结果：①K-ras突变的PDAC细胞中,Gli1过表达、HH通路活化可促进NF-κB通路活化,并促进肿瘤生长；②K-ras突变的PDAC细胞中,存在HH通路和NF-κB通路的crosstalk。研究提示,动物体中,也存在HH通路和NF-κB通路的Crosstalk的K-ras突变依赖性。全文结论1、PDAC中,K-ras基因12、13密码子突变在PDAC组织中发生率高(81.25%)；HH通路和NF-κB通路活化间的显著正相关关系存在K-ras突变依赖性；2、PDAC中,NF-κB通路活化,以及其对HH通路的活化作用存在K-ras突变依赖性；3、PDAC中,HH经典活化通路,以及其对NF-κB通路的活化作用存在K-ras突变依赖性；4. PDAC中,K-ras活性改变是PDAC中HH通路和NF-κB活化及二者间相互活化作用K-ras突变依赖性的主要机制；5、动物实验证实HH经典活化通路,NF-κB通路活化,以及二者间相互活化作用的K-ras依赖性。
[Abstract]:Background and objective pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the digestive system, accounting for more than 90% of the malignant tumors of the pancreas. Previous studies have confirmed that such as the abnormal activation of the Hedgehog (HH) pathway and the involvement of the.HH signaling pathway in the formation of PDAC, such as inflammation and K-ras mutations, are involved in the process. The process of pancreatic morphogenesis is in silence in most normal pancreas. However, the abnormal activation of HH pathway is very common in PDAC, and in the early stage, the activation mechanism of.HH pathway may be roughly divided into the classical activation pathways mediated by ligand overexpression, the non classical activation pathway, which is the main signal transduction pathway. The former is more common in the former. In humans, the classical activation process is triggered by the HH pathway ligand Sonic Hedgehog (Shh), which eventually leads to the nuclear factor glioma-associated oncogene (Gli) family protein of the HH pathway and the regulation of the downstream genes. In the Gli family, Gli1 is the most important activating factor. Gli1 nucleation is the sign of the activation of the HH pathway. In fact, inflammation is closely related to the occurrence of PDAC. Nuclear factor kappa B (nuclear factor-kappa B, NF- kappa B) is an important member of the inflammatory response. It participates in the occurrence of PDAC. The development process of.NF- kappa B enters the nucleus and NF- kappa B pathway is activated, thus starting to regulate the downstream genes in the early stage. The mutation rate is quite high, up to 75~95 It is found that there are several loci in the K-ras gene mutation, but it is mainly concentrated in the 12,13 codon.K-ras protein only after combining with guanosine triphosphate (GTP) (K-ras-GTP) to realize the regulation of cell activity. In general, K-ras-GTP egg white will be in GTP enzyme activated protein (GTPase-activating proteins, GA). Ps changed rapidly into inactive K-ras-GDP, which made the K-ras protein in the inactivation state of.K-ras gene mutation leading to the continuous K-ras-GTP state of the K-ras protein and the active state of the K-ras protein. The previous study confirmed that the above three pathways were involved in the pathogenesis of PDAC, and there was a correlation between HH path and NF- kappa B, and NF- kappa was activated. K-ras mutation can lead to the activation of HH pathway and NF- kappa B, but the interaction between the three and the pathogenesis of pancreatic cancer is rarely reported. It is very important to explore the mechanism of the interaction between the three and to find out the molecular mechanism of the pathogenesis of PDAC, and to seek specific and effective individualized treatment methods. The first part was the detection of the main members of HH and NF- kappa B pathway in PDAC tissue and cell lines, and the detection of K-ras gene mutation, and the correlation analysis between the three experimental materials. Methods: 32 cases of pancreatic cancer and 3 PDAC cell lines were collected. Immunohistochemistry, real-time quantitative PC, Western-blot and DNA sequencing techniques were used to detect Shh protein and Gli, respectively. 1, NF- kappa B nucleoprotein expression, and mutation of K-ras gene 12 and 13 codon. Experimental results: there is a positive correlation between Shh, Gli1 and NF- kappa B expression in PDAC tissue, and this positive correlation exists in the state of K-ras mutation, indicating that HH and NF- kappa pathway activation has a significant positive correlation in the pathogenesis of PDAC, and This correlation has a certain K-ras mutation dependence, and there may be a positive correlation between the activation of the HH pathway and the NF- kappa B pathway in the PDAC cells, and this correlation may also have a certain K-ras mutation dependence. The regulation of the NF- kappa B pathway in the second part of the PDAC cell line is observed and the regulatory role is analyzed for K. -ras mutation dependent experimental materials, methods: select the K-ras gene wild (BxPC-3) and mutation (Panc-1/12 codon mutation, SW1990/13 codon mutation) PDAC cells to intervene and activate the NF- kappa B pathway. Rosis factor-A, TNF-a) stimulation intervention (with simple culture medium treatment group as control), q-PCR and Westernblot methods were used to detect the changes of mRNA and protein expression in Shh, Glil and NF-kB, parallel detection of cell proliferation, apoptosis and other changes after intervention. The activation of NF- kappa B pathway and the promotion of HH pathway activation were observed. The effect of this effect was analyzed and the correlation between the effect and the K-ras mutation was analyzed. Experimental results: in the PDAC cell line of the K-ras mutation, the intervention of the activation of the NF- kappa B pathway could promote the further activation of the HH pathway and enhance the behavior of the malignant cells of the PDAC cell line, but in the PDAC cell line of the wild K-ras, this intervention The effect of activation was not significant. The study showed that the activation of the NF- kappa B pathway in the pathogenesis of PDAC could promote the activation of the HH pathway, and the action of the Crosstalk had a certain K-ras mutation dependence. The regulation of the -HH pathway in the third part of the PDAC cell line was observed and the regulation of this regulation on K-ras mutation was analyzed. Dependent experimental materials, methods: the same second part of the study, select the K-ras gene wild (BxPC-3) and mutation (Panc-1/12 codon mutation; SW1990/13 codon mutation) PDAC cell lines, respectively, Gli1 cDNA transfection, and ligand Shh stimulation intervention (single pure culture group as the control), q-PCR, Westernblot prescription The changes of mRNA and protein expression of Shh, Gli1 and NF- kappa B were detected after the intervention, and the changes of cell proliferation and apoptosis after intervention were detected parallel. The effect of intervention on the activation of HH pathway itself and the effect of promoting the activation of NF- kappa B pathway were observed, and the relationship between the effect fruit and K-ras mutation was analyzed. The experimental results were in K-ras mutation. In the PDAC cell line, the activation of the activation of the HH pathway promoted the further activation of the NF- kappa B pathway and enhanced the behavioral characteristics of the malignant cells of the PDAC cell line, but the effect of this intervention was not significant in the PDAC cell line of the K-ras wild. The study showed that the HH pathway activation of Crosstalk (crosstalk) promoted NF in the pathogenesis of PDAC. Activation of the kappa B pathway, and this Crosstalk action has a certain K-ras mutation dependence. Fourth part PDAC HH pathway and NF- kappa B pathway in the K-ras mutation dependence of the molecular mechanism of molecular mechanisms to study the experimental materials. Methods: on the basis of the second and third part of the cell intervention experiments, further detection of the cell K-ras protein and activity in the cell The expression changes, the changes in the expression of the downstream gene p-/t-ERK1/2, and p-/t-AKTl of the barium K-ras gene were detected, and the changes in the activity of Ras were detected by the Ras activity detection kit and the activation level of the K-ras pathway was judged, and the RNA interference technique (si-Kras) was used to block the expression of the K-ras gene in the PDAC cell, and the G12D mutant K-ras gene was used. The cDNA plasmid transfected wild type cells, observed the success of the intervention of these blocking and remedial actions on the intervention of K-ras's own activity, and the effect on the mutual promotion of Crosstalk, and further determined the molecular mechanism of K-ras mutation dependence between the HH and the NF- kappa B pathway, which promoted the activation of Crosstalk. The experimental results: K-ras activity change is H. H and NF- kappa B pathway activation and the main molecular mechanism of Corsstalk dependence on K-ras mutation between the two. Fifth the K-ras mutation dependence of Crosstalk between the HH pathway and NF- kappa B pathway in PDAC. Methods: on the basis of cytology, we carried out animal tests to explore the relationship between the HH pathway and the nuclear kappa pathway. Crosstalk K-ras mutation dependence. We use Glil cDNA stable SW1990 cells and common SW1990 cells to grow the nude mice with thymus deletion, detect the expression of Gli1 and NF- kappa B nuclear protein, observe the effect of Gli1 overexpression, HH pathway activation on the NF- kappa pathway, simultaneously detect the size of the tumor and verify the effect of the activation of the pathway on the growth of the tumor. (2) the PDAC cells of K-ras gene wild (BxPC-3) and mutation (Panc-1/12 codon mutation; SW1990/13 codon mutation) were planted in the nude mice of the thymus deletion, and the expression of Shh protein was detected by PBS (control), Shh, IL-1 beta and TNF-a respectively. The expression of Shh protein and the expression of Gli1 and NF- kappa nuclear proteins were detected and the different phenotype cells were observed. The activation of H pathway and NF- kappa B pathway, as well as the crosstalk between the two, simultaneously detected the tumor size, and verified the effect of the activation of the HH pathway and NF- kappa B pathway on the growth of the tumor. In the cell, the crosstalk. study of the HH pathway and the NF- kappa B pathway suggests that there is also a K-ras mutation dependence of Crosstalk in the HH pathway and the NF- kappa B pathway in the animal body. Conclusion 1, PDAC, K-ras gene 12,13 codon mutation occurs in a high rate (81.25%), and there is a significant positive correlation between the activation and the activation of the pathway. Ras mutation dependence; 2, PDAC, the activation of NF- kappa B pathway, and the K-ras mutation dependence on the activation of HH pathway; 3, PDAC, HH classical activation pathway, and the existence of K-ras mutation dependence on the activation of NF- kappa B pathway; in 4. PDAC, the activity change is the activation and the mutual activation of the two. The main mechanisms of K-ras mutation dependence; 5, animal experiments confirmed that the HH classical activation pathway, the activation of the NF- kappa B pathway, and the K-ras dependence of the mutual activation among the two.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.9

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