STK33及NGFR在头颈鳞状细胞癌中的作用及机制研究

发布时间：2018-04-29 09:36

本文选题：STK33 + Fadu细胞　；参考：《山东大学》2016年博士论文

【摘要】：第一部分STK33促进下咽癌增殖与钙离子相关性研究背景：下咽鳞状细胞癌(hypopharyngeal squamous cell carcinoma, HSCC)是人类最常见的恶性肿瘤之一,多年来,虽然治疗手段不断发展,但患者的生存率未有显著提高。普遍认为大多数肿瘤是由于包括癌基因的激活以及抑癌基因的失活的多因素作用的结果。尽管HSCC的发生和发展包含许多基因与外源因素,但是参与这种肿瘤形成的分子机制知之甚少。蛋白激酶是研究比较透彻且具多样化特性的蛋白超级家族。丝氨酸/苏氨酸激酶(serine/threonine kinase, STK)作为这一超级家族的成员,其功能是使蛋白质上的丝氨酸和苏氨酸残基发生磷酸化,在调节诸如DNA复制、信号转导通路、细胞增殖、细胞分化、细胞死亡及肿瘤发生和发展等重要细胞进程中发挥关键性的作用。丝氨酸/苏氨酸激酶33 (STK33)调控与Ras基因存在交互依赖关系的肿瘤细胞的信号转导,从而在肿瘤增殖过程中发挥特殊作用。STK33属钙离子/钙调素依赖蛋白(calcium/calmodulin dependent kinase, CAMK)家族成员,目前数据表明,STK33通过特异性磷酸化波形蛋白从而参与细胞骨架解聚的动态过程,以及影响细胞的结构和功能。最近,有力的证据证明STK33参与肿瘤的形成。然而,STK33在HSCC中的精确功能和机制尚未清楚,仍需进一步研究。本文旨在在我们前期研究的基础上,进一步通过体内实验和Microarray分析来探索STK33的功能,重点探索STK33的改变与钙离子的可能关系。方法：通过含有针对STK33的shRNA的慢病毒载体,感染细胞,并使用PCR法检测慢病毒感染后Fadu细胞中STK33 mRNA表达的变化。进行Microarray分析,寻找STK33基因敲除后的表达谱变化。注射Fadu细胞,Mock空载体细胞,以及STK33基因敲除细胞于5周雄性裸鼠右侧腋下进行皮下注射,定期测量肿瘤大小,并计算肿瘤体积,待对照组的肿瘤直径达到1.5厘米时,收获肿瘤和肺组织。在进行苏木素伊红染色后,使用显微镜观察肿瘤和肺的形态学改变,以了解Fadu细胞在体内成瘤和侵袭转移的能力。进行免疫组化分析STK33蛋白的表达。使用MTT和吖啶橙染色检测Ionomycin对Fadu细胞存活力及形态学改变的影响。通过使用Fluo-3/AM染色,借助共聚焦显微镜对细胞内钙离子的浓度进行检测。使用实时荧光定量PCR及western blot检测相关基因的mRNA及蛋白的表达情况。结果：STK33-RNAi稳定表达的Fadu细胞,感染空载体的Fadu细胞和对照Fadu细胞皮下注射到雄性裸鼠右侧腋下后第14天对照Fadu细胞成瘤,共生长39天。空载体对照组和对照Fadu细胞组表现出相似的生长率,在第39天时,体积达到约570mm3。然而,稳定转染的STK33-RNAi表达的Fadu细胞在裸鼠体内生长缓慢,在39天仅形成较小的肿瘤,体积约为90 mm3。在STK33表达和不表达的细胞中肿瘤的体积有显著差异。这表明STK33促进HSCC在体内的形成。免疫组化结果显示STK33在STK33-RNAi细胞中的表达比载体对照组的细胞弱。注射对照组细胞的裸鼠的肺显示存在转移；相反,在注射STK33-RNAi细胞的裸鼠中未见转移。通过Microarray分析,在STK33-RNAi细胞中,我们发现了140个有显著差异的标记基因。这些基因显著(p0.05)上调(n=86)或者下调(n=54)。通过通路分析,我们发现STK33影响很多基因的表达,调节很多通路。从中我们发现了Calpainl(CAPN1),并用qRT-PCR确认CAPN1的mRNA表达水平。在STK33-RNAi Fadu细胞中CAPN1的表达显著下降(pO. 05)。Ionomycin可以迅速升高细胞内钙离子的浓度。在1.5 μM的Ionomycin作用6小时后进行吖啶橙染色发现未处理的细胞呈现多边形,然而,典型的凋亡特点出现在Ionomycin处理的细胞中,包括细胞形态不规则,细胞皱缩,染色质凝集,染色小体出现,表明Ionomycin可以诱导Fadu细胞凋亡。细胞存活率在Fadu细胞中显示出明显的随Ionomycin作用时间延长而降低的趋势。表明Ionomycin抑制细胞存活率呈现时间依赖性。在STK33-RNAi作用后细胞存活率显著下降。此外,在STK33-RNAi与Ionomycin同时作用后,细胞存活率下降程度低于仅Ionomycin作用(p0.05)。与未处理的Fadu细胞相比,STK33的蛋白表达在1.5 μM Ionomycin作用1,2,4,6小时后升高。统计分析表明STK33的蛋白表达在作用1,2,4,6小时后比对照组显著升高(p0.05)。这表明,Ionomycin可以增高STK33蛋白的表达。Fadu细胞中的CAPN1在1.5 μM Ionomycin作用1,2,4,6及24小时后CAPN1显著升高(p0.05)。但在STK33-RNAi组中,作用前后没有显著性差异。结论：shRNA介导的STK33基因的敲除抑制Fadu细胞在体内的成瘤能力,从而证明STK33本身对肿瘤生长的促进作用。表明STK33在肿瘤形成中发挥重要作用。此外,通过免疫组化发现STK33在STK33-RNAi组所成肿瘤中的表达明显下降。这表明STK33-RNAi仍然发挥它对STK33的敲除作用,藉以表明Fadu细胞在以STK33-RNAi抗肿瘤影响的基础上的增值能力的降低。总之,体内实验进一步证实了我们前期的从体外及临床切除样本中得到的STK33是一个潜在癌基因的研究结果。STK33基因通过多种信号通路在Fadu细胞中发挥作用。这个过程中包含大量决定肿瘤进展中的重要功能的基因。CAPN1,在STK33-RNAi的干扰下显著下调,正如本研究中进一步PCR所证明的。本研究表明CAPN1的活性受STK33-RNAi的影响,提示CAPN1涉及STK33在Fadu细胞中诱导肿瘤形成的作用。这个发现可能是一个很有吸引力的线索,在Fadu细胞中,STK33可能与钙离子有关。因为STK33属于CAMK家族。这促使我们关注STK33活性与Ionomycin作用后Fadu细胞内钙离子改变的关系。Ionomycin可以在体外抑制Fadu细胞的生长。这表明Ionomycin通过提高细胞内钙离子浓度来表现出它对Fadu细胞的毒性作用。有意思的是,STK33-RNAi可以在一定程度上抵消Ionomycin引起的细胞损伤。表明STK33敲除可能保护细胞不受Ionomycin触发的高细胞内钙离子浓度而引起的细胞损伤。这为细胞内游离钙离子可能通过STK33活性来影响CAPN1表达,从而影响一些与STK33在肿瘤发生中重要作用相关的重要的信号转导通路,提供了证据。总之,本研究进一步证明了STK33是一个潜在的癌基因,它通过调节众多基因在HSCC肿瘤发生中发挥重要作用。另外,在Fadu细胞内的STK33和钙离子存在相互的影响。第二部分NGFR在ESM1介导的口腔鳞状细胞癌肿瘤发生中的作用研究背景：口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)是人类最常见的恶性肿瘤之一。近年来,其生存率并没有随着治疗手段的迅速发展而明显改善。这种情况主要因为远处转移以及治疗耐受的复发。对于为什么某些细胞对化疗和放疗更加耐药的生物学解释来自支持在恶性肿瘤中有一组细胞赋予“干细胞”样属性的观点。我们实验室最近发现了一个在OSCC中的肿瘤起始细胞的新的标志,我们认为它可以解决我们刚刚提到的困境。这个标志就是低亲和力的神经生长因子受体(nerve growth factor receptor, NGFR), NGFR是头颈鳞状细胞癌进展、转移和生存中的肿瘤起始细胞的一个功能和可命中目标的标志物。它的激活导致NF-κB(核因子-K B),Jun激酶以及其他信号通路的活化,从而发挥重要的生物学功能。NGFR在OSCC中的确切功能和机制已经被研究了,但是还有不清楚的地方。本研究在我们前期研究的基础上,设计通过小鼠OSCC细胞的Microarray分析和体内外实验来进一步探索NGFR的功能。方法：使用实时荧光定量PCR、流式细胞术及ELISA实验检测相关基因的mRNA和蛋白的表达情况。通过慢病毒感染细胞,稳定过表达MOC2细胞中的NGFR(MOC2T),进行Microarray分析,寻找NGFR基因过表达后的表达谱变化。通过慢病毒感染细胞,稳定过表达MOC2细胞中的ESM1,敲除ESM1在MOC2和MOC2T细胞中的表达。使用MTT和Transwell侵袭转移实验分析ESM1口NGFR对细胞增殖和侵袭转移能力的影响。注射MOC2, MOC2-7, MOC2-10, MOC2-ESM1-SH, MOC2T和MOC2T-ESM1-SH细胞于6-11周B10; B6-Rag2-/-II2rg-/-小鼠右侧后腿背侧进行皮下注射,定期测量肿瘤大小,并计算肿瘤体积,收获肿瘤和肺组织。在进行苏木素伊红染色后,使用显微镜观察肿瘤和肺的形态学改变。检测ESM1是否参与血管形成,进行免疫荧光分析血管内皮生长因子(vascular endothelial growth factor, VEGF)的表达。结果：通过qRT-PCR检测发现,NGFR在小鼠OSCC细胞系(MOC2,MOC2-7和MOC2-10)中表达。通过慢病毒感染,形成NGFR稳定过表达的MOC2细胞株,通过Microarray分析寻找NGFR过表达后的基因表达谱改变。通过对Microarray结果的分析,我们发现了内皮细胞特异性分子-1(endothelial cell specific molecule 1, ESM1)。 ESM1是一种新型的内皮衍生的水溶性硫酸皮肤蛋白多糖,具有广泛结合与细胞信号和粘附相关的生物活性分子的能力,从而调节健康和疾病中的不同类型细胞的增殖,分化,转移和粘附。NGFR依赖的ESM1的mRNA水平的表达在NGFR过表达和NGF处理的细胞中得到验证。ESM1的表达水平与小鼠OSCC细胞系的增殖和侵袭转移能力成正相关。ESM1过表达诱导MOC2细胞活力,迁移和侵袭能力。相反,ESM1的敲除抑制MOC2细胞的增殖,迁移和侵袭能力。免疫缺陷小鼠皮下移植瘤表明ESM1的敲除减小肿瘤大小和肺转移的克隆数目。降低NGFR过表达的MOC2细胞(MOC2T)的ESM1的表达也可以在体内及体外降低MOC2T肿瘤增殖以及侵袭和转移能力。总之,这是第一次报告ESM1是NGFR的一个靶点,从而为NGFR和ESM1之间提供了一个新的互连。我们发现,ESM1敲除在体内和体外减少MOC2细胞的增殖和转移,因而提示NGFR通过它所调节的基因来诱导增殖和转移。此外,ESM1单独或与NGFR结合可能作为OSCC的新的预后生物标志物,也可能描绘了针对OSCC的创新的治疗方法。
[Abstract]:The first part STK33 promotes the relationship between the proliferation of hypopharyngeal carcinoma and the calcium ion: hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common malignant tumors in human. For many years, the survival rate of the patients has not been significantly improved. It includes the activation of oncogenes and the multifactor effects of inactivation of the tumor suppressor genes. Although the occurrence and development of HSCC include many genes and exogenous factors, little is known about the molecular mechanism involved in the formation of this tumor. Protein kinase is a more thorough and diversified protein superfamily. Serine / threonine excitation Serine/threonine kinase (STK), as a member of this superfamily, has the function of phosphorylating the serine and threonine residues on the protein and plays a key role in regulating important cell processes, such as DNA replication, signal transduction, cell proliferation, cell differentiation, cell death and tumorigenesis and development. Tyrosine / threonine kinase 33 (STK33) regulates the signal transduction of tumor cells interacting with the Ras gene, which plays a special role in the proliferation of.STK33 family members of the calcium/calmodulin dependent kinase (CAMK) family of calcium ion / calmodulin dependent protein (CAMK). The current data show that STK33 through specific phosphorylation Vimentin is involved in the dynamic process of cytoskeleton depolymerization, as well as the structure and function of cells. Recently, strong evidence has shown that STK33 is involved in the formation of tumors. However, the precise function and mechanism of STK33 in HSCC are still not clear, and further study is needed. Experiments and Microarray analysis to explore the function of STK33, focus on exploring the possible relationship between the changes of STK33 and calcium ions. Methods: by using the lentivirus vector containing shRNA for STK33, infected cells, and using PCR to detect the alteration of STK33 mRNA expression in Fadu cells after the infection of lentivirus infection. Microarray analysis, to find STK33 gene knockout. Fadu cells, Mock no-load cells, and STK33 gene knockout cells were injected subcutaneously at the right arm of the male nude mice in 5 weeks. The tumor size was measured regularly and the tumor volume was calculated. The tumor and lung tissue were harvested when the diameter of the control group reached 1.5 cm. The morphological changes of tumor and lung were observed by microscopes in order to understand the ability of Fadu cells to be tumorigenic and invasive in the body. Immunohistochemical analysis of the expression of STK33 protein. The effects of Ionomycin on the viability and morphological changes of Fadu cells were detected by MTT and acridine orange staining. By using Fluo-3/AM staining, confocal microscopy was used. Detection of intracellular calcium concentration. The expression of mRNA and protein of related genes was detected by real-time fluorescent quantitative PCR and Western blot. Results: STK33-RNAi stable Fadu cells, Fadu cells infected with empty carrier and control Fadu cells were injected subcutaneously into the right lateral axillary of male nude mice for fourteenth days to compare with Fadu cells to form a tumor. The growth rate was similar to that of the control group and the control Fadu cell group for 39 days. At thirty-ninth days, the volume reached about 570mm3., but the Fadu cells expressed by the stable transfected STK33-RNAi were slowly growing in the nude mice, and only a smaller tumor was formed on the 39 day, and the volume was about 90 mm3. in the STK33 and unexpressed cells. There were significant differences. This indicates that STK33 promotes the formation of HSCC in the body. The immunohistochemical results show that the expression of STK33 in STK33-RNAi cells is weaker than that in the carrier control group. The lung display of the nude mice injected with the control group is metastatic; on the contrary, there is no metastasis in the nude mice injected with STK33-RNAi cells. Microarray analysis, in STK33- In RNAi cells, we found 140 marked differentially marked genes. These genes were significantly (P0.05) up-regulated (n=86) or down down (n=54). Through pathway analysis, we found that STK33 affects many genes and regulates many pathways. We found Calpainl (CAPN1), and confirmed CAPN1's mRNA expression with qRT-PCR. In STK33-RNAi. The expression of CAPN1 in Fadu cells decreased significantly (pO. 05).Ionomycin could rapidly increase the concentration of intracellular calcium. After 6 hours of Ionomycin action of 1.5 mu M, acridine orange staining showed that the untreated cells presented polygons. However, typical apoptotic characteristics appeared in the cells treated with Ionomycin, including irregular cell morphology. Cell crinkle, chromatin agglutination, and dyed corpuscle appeared, indicating that Ionomycin could induce apoptosis of Fadu cells. The survival rate of cell in Fadu cells showed a tendency to decrease with the prolongation of the time of Ionomycin action. It showed that the survival rate of Ionomycin inhibited cells was time dependent. The survival rate of cells decreased significantly after the action of STK33-RNAi. In addition, after the simultaneous action of STK33-RNAi and Ionomycin, the decrease of cell survival rate was lower than that of Ionomycin only (P0.05). Compared with untreated Fadu cells, the protein expression of STK33 increased after 1.5 u M Ionomycin in 1,2,4,6 hours. The statistical analysis showed that the protein expression of STK33 was significantly higher than that of the control group after the action 1,2,4,6 hours (P0.05). This indicates that Ionomycin can increase the expression of CAPN1 in the expression of STK33 protein in.Fadu cells in 1,2,4,6 and 24 hours after 1,2,4,6 and CAPN1 significantly increased (P0.05). But in the STK33-RNAi group, there is no significant difference before and after action. Conclusion: shRNA mediated STK33 gene knockout inhibits the tumorigenicity of.Fadu cells in the body. STK33 itself promotes the growth of tumor. It shows that STK33 plays an important role in the formation of tumor. In addition, the expression of STK33 in the tumor of STK33-RNAi group is obviously decreased by immunohistochemistry. This indicates that STK33-RNAi still plays a knockout effect on STK33, which indicates that Fadu cells are on the basis of the anti tumor effect of STK33-RNAi. In conclusion, in vivo experiments further confirmed that our earlier STK33 from in vitro and clinically excised samples is a potential oncogene study that the.STK33 gene plays a role in Fadu cells through a variety of signaling pathways. This process includes a large number of important functions that determine the progression of cancer. Gene.CAPN1, down significantly under the interference of STK33-RNAi, as demonstrated by further PCR in this study. This study showed that the activity of CAPN1 was affected by STK33-RNAi, suggesting that CAPN1 involves the role of STK33 in inducing tumor formation in Fadu cells. This discovery may be a very attractive clue, in Fadu cells, STK33 may be associated with calcium. Ions are related, because STK33 belongs to the CAMK family. This has prompted us to focus on the relationship between STK33 activity and the changes in calcium ions in Fadu cells after the action of Ionomycin..Ionomycin can inhibit the growth of Fadu cells in vitro. This indicates that Ionomycin shows its toxic effect on Fadu cells by increasing intracellular calcium concentration. It is interesting, STK. 33-RNAi can counteract the cell damage caused by Ionomycin to a certain extent. It shows that STK33 knockout may protect cells from cell damage caused by high intracellular calcium ion concentration triggered by Ionomycin, which may affect the expression of CAPN1 through the activity of STK33 in the cell, which may affect some of the STK33 in the carcinogenesis of the tumor. The important signal transduction pathway related to the important role, provided evidence. In conclusion, this study further demonstrated that STK33 is a potential oncogene, which plays an important role in the development of HSCC tumors by regulating many genes. In addition, the STK33 and calcium ions in the Fadu cells are interacting with each other. The second part of NGFR is mediated by ESM1. The role of oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in human oral squamous cell carcinoma. In recent years, the survival rate has not improved obviously with the rapid development of treatment. This situation is mainly due to the distant metastasis and the recovery of the treatment tolerance. The biological explanation for why some cells are more resistant to chemotherapy and radiation comes from the view that a group of cells give a "stem cell" like attribute in the malignant tumor. Our laboratory recently discovered a new marker of the tumor starting cells in OSCC, and we think it can solve the difficulties we have just mentioned. This symbol is a low affinity nerve growth factor receptor (NGFR), and NGFR is a function and target marker for the progression, metastasis and survival of cancer starting cells in the head and neck squamous cell carcinoma. Its activation leads to the survival of NF- kappa B (nuclear factor -K B), Jun kinase, and other signaling pathways. The exact function and mechanism of the important biological function,.NGFR, has been studied in OSCC, but there is no clear place. On the basis of our previous study, this study was designed to further explore the function of NGFR through the Microarray analysis of mouse OSCC cells and in vivo and in vivo. Methods: using real time fluorescence determination. PCR, flow cytometry and ELISA test to detect the expression of mRNA and protein related genes. Through the lentivirus infected cells, stable overexpression of NGFR (MOC2T) in MOC2 cells, Microarray analysis was carried out to find the expression profiles of NGFR gene overexpression. Through the lentivirus infected cells, the ESM1 of the MOC2 cells was stably expressed and the ES was knocked out of ES. M1 expression in MOC2 and MOC2T cells. Use MTT and Transwell invasion and metastasis test to analyze the effect of ESM1 mouth NGFR on cell proliferation and invasion and metastasis. Injection MOC2, MOC2-7, MOC2-10, MOC2-ESM1-SH, MOC2T, and cells at 6-11 weeks; subcutaneous injection on the back of the right hind leg of the right mouse. The size of the tumor and the volume of the tumor were calculated and the tumor and lung tissue were harvested. After hematoxylin staining, the morphological changes of the tumor and lung were observed by microscope. Whether ESM1 was involved in angiogenesis and the expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) was analyzed by immunofluorescence. Results: through qRT-PC R detection showed that NGFR was expressed in the mouse OSCC cell line (MOC2, MOC2-7 and MOC2-10). Through the infection of the lentivirus, the MOC2 cell line that was stable and overexpressed in NGFR was formed, and the gene expression profiles after the NGFR overexpressed were found by Microarray analysis. Ell specific molecule 1, ESM1). ESM1 is a new type of endothelium derived water-soluble sulfonated skin proteoglycan that has the ability to combine with cell signaling and adhesion related bioactive molecules to regulate the proliferation, differentiation, transfer and adhesion of.NGFR dependent ESM1 mRNA levels in different types of cells in health and disease. Expression in NGFR overexpressed and NGF treated cells showed that the expression level of.ESM1 was positively related to the proliferation and invasion and metastasis of OSCC cell lines in mice..ESM1 overexpression induced MOC2 cell viability, migration and invasion ability. On the contrary, ESM1 knockout inhibited the proliferation, migration and invasion of MOC2 cells. Subcutaneous transplantation of immune deficient mice. The tumor indicates that the ESM1 knockout reduces the number of tumor size and the number of clones of the lung metastasis. The ESM1 expression of the MOC2 cells (MOC2T) that reduces the overexpression of NGFR can also reduce the proliferation and invasion and metastasis of MOC2T in vivo and in vitro. In a word, this is the first report that ESM1 is a target for NGFR, thus providing one between NGFR and ESM1. New interconnection. We have found that ESM1 knocks down the proliferation and metastasis of MOC2 cells in vivo and in vitro, thus suggesting that NGFR can induce proliferation and metastasis through its regulated genes. In addition, the combination of ESM1 alone or with NGFR may serve as a new prognostic biomarker for OSCC, and may also describe an innovative treatment for OSCC.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.91

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