肺癌组织与细胞中Cullin7的表达及临床意义的研究
发布时间：2018-07-17 19:28
【摘要】：肺癌已成为全世界癌症死亡的首要原因,每年超过一百万的人从这种疾病死亡,肺癌死亡率40年间上升近10倍。全世界肺癌每年新增病例约180万,其中三分之二发现时已处于晚期,肺癌确诊后患者5年生存率只有约10%。肺癌最主要的治疗手段是以手术、放疗、化疗和分子靶向治疗为主的综合治疗。其中65-70%发现已为晚期失去手术机会,对于此类患者如果可以采用化疗或放疗,5年生存率仅为7-17%,如果患者不能耐受或失去放化疗机会,5年生存率仅为2%。因此,探寻更为有效的肺癌治疗策略尤其是肺癌早期诊断早期治疗具有重要意义。对了解新的发病机制和确定新的肿瘤标志物方面将有很多空间。最新研究提出,如能及早发现肺癌,把它扼杀在萌芽状态阻止其沿多条进化路径发展,情况可能会变得乐观,将会有更多肺癌患者存活。人体内Cullin7(一种多功能泛素连接酶亚基)的高表达是否就是肺癌发生的众多机制之一,肺癌的产生是否就与人体内或肺癌组织中的Cullin7的异常表达高度相关,值得肯定的是Cullin7也许会是探索肺癌发病原因及治疗研究中的一个新方向。Cullin7(CUL7)是将包含F-box蛋白Fbw8、Skpl和ROC1 RING指形蛋白的E3泛素连接酶组织在一起的分子骨架。CUL7 E3连接酶失调与人类遗传疾病直接相关,因为在常染色体隐性遗传3M综合征和雅库特人矮小综合征患者中观察到Cullin7生殖细胞系突变,其特征为严重的出生前和出生后生长迟缓。另外,小鼠Cullin7基因切除会导致子宫内生长迟缓以及围产期幼仔死亡,强调了其对于生长调节的重要意义。最近识别出胰岛素受体底物1-种胰岛素和胰岛素样生长因子-1信号传导的重要介质,是CUL7 E3连接酶的蛋白水解靶标,这揭示了Cullin7与充分确立的生长调节途径间的分子联系。该结果连同其它证明CUL7与p53肿瘤抑制蛋白,以及猴病毒40大T抗原肿瘤蛋白间存在相互作用的研究均进一步提示了CUL7是生长控制中的一个新角色。曾有研究表明Cullin7对于癌症萌发过程中的血管生成至关重要,最新的研究表明Cullin7可在人和小鼠肿瘤内皮细胞中表达。通过Guo Hongsheng等人研究Cullin7在乳腺癌细胞中的表达,认为HCC1937细胞中的Cullin7在细胞增殖中异位表达明显增加,而敲除BT474细胞中的Cullin7会导致生长速率减缓。这些结果表明Cullin7会刺激乳腺癌细胞增殖。同时首次证明Cullin7可在蛋白水平上(而非mRNA水平上)调节乳腺癌细胞中的p53表达,这表明Cullin7采用替代机制来提高乳腺癌细胞的侵袭性。然而,Cullin7对其他原癌基因和抑癌基因水平的影响需要进一步的研究才能确定。第一部分我们通过免疫组织化学染色检测人正常肺组织以及肺癌组织中Cullin7的表达差异。染色结果显示在正常肺组织中Cullin7蛋白表达仅见于少量细胞胞质中,且着色较浅；肺癌组织中Cullin7蛋白在多数细胞的胞质和胞核中呈强着色,且胞核着色相比胞质颜色更深。显微镜下观察发现癌组织中Cullin7蛋白染色阳性细胞明显多于正常组织,计数结果t检验显示差异具有统计学意义(P0.01),该结果表明肺癌组织Cullin7蛋白表达明显高于正常肺组织。我们采用Cullin7-siRNA干扰肺癌A427、H460、A549、H1299细胞中Cullin7的表达,并通过半定量RT-PCR检测siRNA干扰效率。琼脂糖凝胶电泳结果显示A427、H460、A549对照组细胞中Cullin7基因表达量较高,而H1299细胞中Cullin7基因表达量稍低。干扰后的A427细胞样品中Cullin7基因RNA表达量弱于对照组,干扰效率相对较低；H460、A549、H1299田胞样品中Cullin7基因RNA表达量明显弱于对照组,siRNA的干扰效率在A549、H1299细胞中尤其显著。为了确定Cullin7对肺癌细胞增殖的影响,我们对转染Cullin7-siRNA后的A427、H460、A549、H1299四种肺癌细胞进行MTT检测,实验结果显示,A427、H460、A549、H1299四种肺癌细胞在干扰Cullin7表达后,吸光度(OD值)比对照组分别降低30%、39%、57%和60%,降低幅度与其基因干扰效率高低趋势一致,且具有统计学意义(P0.05)。实验结果提示干扰Cullin7表达之后对肺癌细胞的增殖能力具有抑制作用。为了进一步阐明Cullin7对肺癌细胞增殖能力的影响,我们对转染后的A427、H460、A549和H1299四种细胞进行克隆形成实验,显微镜下观察,最终实验结果表明,A427、H460、A549和H1299四种肺癌细胞在干扰Cullin7表达后,其克隆形成数目与对照组相比均有显著减少。结果表明,干扰Cullin7表达可以抑制肺癌细胞形成克隆。为验证Cullin7表达降低对肺癌细胞体外增殖能力的影响,我们采取异种移植瘤模型实验(裸鼠成瘤实验)进一步检测Cullin7表达降低是否能够影响肺癌细胞体外增殖能力。裸鼠成瘤实验结果表明,在肺癌细胞中低表达Cullin7能够抑制体内肿瘤的形成,表现在成瘤的体积和成瘤的重量均明显低于肺癌细胞对照组,t检验差异具有统计学意义(P0.01),上述体内实验证实了Cullin7低表达能够抑制肺癌细胞的成瘤能力。第二部分为进一步确定Cullin7参与的调控通路,我们采用Western-B lot检测干扰Cullin7表达后相关重要调控蛋白表达量的变化。结果显示在A427、H460、A549和H1299四种肺癌细胞系中干扰Cullin7基因表达后,Cullin7蛋白量显著下降,并且p53、p27和p21三种蛋白的表达量与对照组相比均有上升,并且在A427与A549两种细胞中三种蛋白表达量的上升水平与对照组相比更加显著。实验结果提示Cullin7蛋白可能是p53、p27和p21蛋白表达的一个负调控因子。在肿瘤细胞中,p27与p21是p53调控的下游基因,所以我们进一步验证了Cullin7与p53的调控关系。由于A549肺癌细胞株中p53、p27和p21三种蛋白表达量变化水平与对照组相比更加显著,我们选取A549用于后续实验,对该细胞株进行C ullin7siRNA的干扰及Cullin7siRNA与p53siRNA的共同干扰,观察A549细胞的克隆形成数目变化。实验结果表明,在Cullin7siRNA干扰Cullin7的表达后,A549细胞的克隆形成数目显著减少；同时干扰Cullin7与p53的表达时,A549细胞的克隆形成效率有显著恢复。实验结果提示Cullin7可能通过抑制p53表达来调控肺癌细胞增殖。为进一步确定Cullin7作用于p53基因的调控机制,在A549细胞中,我们通过Western-Blot实验检测p53调控的H2AX磷酸化(γ-H2 X)蛋白的表达。Western-Blot实验结果显示,当干扰Cullin7的表达时,p53蛋白表达量升高,同时γ-H2AX蛋白相对于对照组出现明显增加,p21和p27蛋白表达量亦有明显上升。然而同时干扰Cullin7与p53表达时,γ-H2AX蛋白表达相对于对照组变化并不明显,p21和p27蛋白表达量亦未有明显变化。免疫荧光染色结果也表明,干扰Cullin7表达会导致细胞γ-H2AX蛋白表达量增加,而同时干扰Cullin7及p53表达时并不会造成γ-H2AX蛋白表达量出现显著变化。上述实验结果提示,Cullin7可能是通过p53蛋白参与到γ-H2AX修饰及相关的基因修复途径对肺癌细胞进行调控。
[Abstract]:Lung cancer has become the leading cause of cancer death in the world. More than one million of the people are dying from this disease every year, and the mortality rate of lung cancer has risen by nearly 10 times in 40 years. The number of new cases of lung cancer in the world is about 1 million 800 thousand a year, of which 2/3 has been found at a late stage. The 5 year survival rate of patients with lung cancer is only about 10%.'s most important treatment hand for lung cancer. The segment is a comprehensive treatment based on surgery, radiotherapy, chemotherapy and molecular targeting. 65-70% has been found to be a late loss of operation. If chemotherapy or radiotherapy can be used for such patients, the 5 year survival rate is only 7-17%. If the patient is unable to tolerate or lose the chemoradiation machine, the 5 year survival rate is only 2%., so the search is more effective. The treatment strategy of lung cancer, especially early diagnosis of lung cancer, is of great significance. There will be a lot of room for understanding the new pathogenesis and identifying new tumor markers. The latest research suggests that if we can detect lung cancer early and stifle it in the bud to prevent it from developing along multiple pathways, the situation may become optimistic. The high expression of Cullin7 (a multifunction ubiquitin ligase subunit) in the human body is one of the many mechanisms of lung cancer. Whether the production of lung cancer is highly related to the abnormal expression of Cullin7 in the human body or lung tissue, it is worth affirming that Cullin7 may be the cause of exploring the cause of lung cancer and A new direction in the treatment study,.Cullin7 (CUL7), is a direct correlation between the.CUL7 E3 ligase dysfunction of the E3 ubiquitin ligase containing the F-box protein Fbw8, Skpl and ROC1 RING finger proteins, which is directly related to human genetic disease, because it is observed in the autosomal recessive hereditary 3M syndrome and the ikustan dwarf syndrome The mutation of the Cullin7 germ cell line is characterized by severe pre birth and postnatal growth retardation. In addition, the Cullin7 gene removal in mice will lead to intrauterine growth retardation and perinatal infant death, emphasizing its importance for growth regulation. Recently, the insulin receptor substrate 1- species insulin and insulin like growth are identified. The important medium for the conduction of the sub-1 signal is the proteolysis target of the CUL7 E3 ligase, which reveals the molecular linkage between Cullin7 and the fully established pathway of growth regulation. The results, together with other studies demonstrating the interaction between CUL7 and p53 tumor suppressor, and the 40 big T antigen tumor proteins of monkey virus, further hint CUL7 It is a new role in growth control. Some studies have shown that Cullin7 is essential for angiogenesis during the course of cancer germination. The latest research shows that Cullin7 can be expressed in human and mouse tumor endothelial cells. The expression of Cullin7 in breast cancer cells is studied by Guo Hongsheng et al., and the Cullin7 in HCC1937 cells is thinner. Ectopic expression in cell proliferation is significantly increased, while Cullin7 in BT474 cells may lead to a slow growth rate. These results suggest that Cullin7 stimulates the proliferation of breast cancer cells. At the same time, it is the first time that Cullin7 can regulate the expression of p53 in breast cancer cells at the protein level (not mRNA level), suggesting that Cullin7 is used as an alternative mechanism to improve the expression of p53 in breast cancer cells. The invasiveness of high breast cancer cells. However, the effect of Cullin7 on other proto oncogenes and tumor suppressor genes needs further study. Part 1 we detected the difference in Cullin7 expression in normal lung tissue and lung cancer tissues by immunohistochemical staining. The color staining results showed that Cullin7 eggs were in normal lung tissue. The white expression was found only in a small number of cytoplasm and shallower. The Cullin7 protein in the lung cancer tissues was strongly colored in the cytoplasm and nucleus of most of the cells, and the coloration of the nucleus was deeper than that of the cytoplasm. Under the microscope, it was found that the Cullin7 protein staining positive cells in the cancer tissues were much more than those of the normal tissues. The results of the t test showed the difference. The results showed that the expression of Cullin7 protein in lung cancer tissues was significantly higher than that of normal lung tissue. We used Cullin7-siRNA to interfere with the expression of Cullin7 in A427, H460, A549, H1299 cells in lung cancer and detected siRNA interference efficiency by semi quantitative RT-PCR. The results of agar gel electrophoresis showed A427, H460, and control cells. The expression of Cullin7 gene was higher and the expression of Cullin7 gene in H1299 cells was slightly lower. The RNA expression of Cullin7 gene in the A427 cell samples after interference was weaker than that of the control group, and the interference efficiency was relatively low. The RNA expression of Cullin7 gene in H460, A549, H1299 field cell samples was obviously weaker than that of the control group. To determine the effect of Cullin7 on the proliferation of lung cancer cells, we performed MTT tests on four lung cancer cells of A427, H460, A549 and H1299 after transfection of Cullin7-siRNA. The results showed that the absorbance of A427, H460, A549, H1299 four lung cancer cells was 30%, 39%, 57% and 60% lower than the control group after interference of Cullin7. The experimental results suggest that the interference of Cullin7 expression can inhibit the proliferation of lung cancer cells. In order to further clarify the effect of Cullin7 on the proliferation of lung cancer cells, we cloned four cells of A427, H460, A549 and H1299 after transfection. The results showed that the number of four lung cancer cells in A427, H460, A549 and H1299 decreased significantly compared with the control group after interfering Cullin7 expression. The results showed that the interference of Cullin7 expression could inhibit the cell form cloning of lung cancer. The results showed that the expression of Cullin7 was reduced to the lung cancer cells in vitro. The effect of proliferation ability, we take the xenograft tumor model test (nude mouse tumorigenesis experiment) to further detect whether the decrease of Cullin7 expression can affect the proliferation ability of lung cancer cells in vitro. The results of tumor formation in nude mice show that the low expression of Cullin7 in lung cancer cells can inhibit the formation of tumor in vivo, and is manifested in the volume and tumor formation of the tumor. The weight of the t test was significantly lower than that of the lung cancer cell control group, and the difference of the t test was statistically significant (P0.01). The above experiments confirmed that the low expression of Cullin7 could inhibit the tumor formation of lung cancer cells. The second part was to further determine the regulatory pathway of Cullin7 participation, and we used Western-B lot to detect the important regulation after interference of Cullin7 expression. The changes in protein expression showed that after the interference of Cullin7 gene expression in four lung cancer cell lines of A427, H460, A549 and H1299, the amount of Cullin7 protein decreased significantly, and the expression of the three proteins of p53, p27 and p21 increased compared with those of the control group, and the increased levels of the three protein expressions in the A427 and A549 two cells were compared with those of the control. The experimental results suggest that Cullin7 protein may be a negative regulator of the expression of p53, p27 and p21. In tumor cells, p27 and p21 are downstream genes of p53 regulation, so we further verified the regulatory relationship between Cullin7 and p53. P27 and three protein expressions of A549 changes water due to p53 in the A549 lung cancer cell lines. A549 was more significant compared with the control group. We selected A549 for subsequent experiments. The interference of C ullin7siRNA and the common interference between Cullin7siRNA and p53siRNA were used to observe the changes in the number of cloning and formation of A549 cells. The experimental results showed that the number of the clone formation of A549 cells decreased significantly after the Cullin7siRNA interfered with the expression of Cullin7. Less; when the expression of Cullin7 and p53 is interfered with the expression of the A549 cells, the cloning efficiency of the A549 cells can be recovered significantly. The experimental results suggest that Cullin7 may regulate the proliferation of lung cancer cells by inhibiting the expression of p53. To further determine the regulation mechanism of the Cullin7 action on the p53 gene, we detect p53 H2AX by Western-Blot in A549 cells. The expression of phosphorylation (gamma -H2 X) protein expression.Western-Blot showed that when the expression of Cullin7 was disturbed, the expression of p53 protein increased, and the expression of gamma -H2AX protein increased significantly compared to the control group, and the expression of p21 and p27 protein increased significantly. However, when the expression of Cullin7 and p53 was disturbed, the expression of gamma -H2AX protein was relative to the control group. There was no obvious change in the expression of p21 and p27 protein. The results of immunofluorescence staining also showed that interference of Cullin7 expression could lead to increased expression of gamma -H2AX protein, while interference of Cullin7 and p53 expression did not cause significant changes in the expression of gamma -H2AX protein at the same time. The results suggested that Cullin7 may be passed. P53 protein is involved in gamma -H2AX modification and related gene repair pathways to regulate lung cancer cells.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2
本文编号：2130695
[Abstract]:Lung cancer has become the leading cause of cancer death in the world. More than one million of the people are dying from this disease every year, and the mortality rate of lung cancer has risen by nearly 10 times in 40 years. The number of new cases of lung cancer in the world is about 1 million 800 thousand a year, of which 2/3 has been found at a late stage. The 5 year survival rate of patients with lung cancer is only about 10%.'s most important treatment hand for lung cancer. The segment is a comprehensive treatment based on surgery, radiotherapy, chemotherapy and molecular targeting. 65-70% has been found to be a late loss of operation. If chemotherapy or radiotherapy can be used for such patients, the 5 year survival rate is only 7-17%. If the patient is unable to tolerate or lose the chemoradiation machine, the 5 year survival rate is only 2%., so the search is more effective. The treatment strategy of lung cancer, especially early diagnosis of lung cancer, is of great significance. There will be a lot of room for understanding the new pathogenesis and identifying new tumor markers. The latest research suggests that if we can detect lung cancer early and stifle it in the bud to prevent it from developing along multiple pathways, the situation may become optimistic. The high expression of Cullin7 (a multifunction ubiquitin ligase subunit) in the human body is one of the many mechanisms of lung cancer. Whether the production of lung cancer is highly related to the abnormal expression of Cullin7 in the human body or lung tissue, it is worth affirming that Cullin7 may be the cause of exploring the cause of lung cancer and A new direction in the treatment study,.Cullin7 (CUL7), is a direct correlation between the.CUL7 E3 ligase dysfunction of the E3 ubiquitin ligase containing the F-box protein Fbw8, Skpl and ROC1 RING finger proteins, which is directly related to human genetic disease, because it is observed in the autosomal recessive hereditary 3M syndrome and the ikustan dwarf syndrome The mutation of the Cullin7 germ cell line is characterized by severe pre birth and postnatal growth retardation. In addition, the Cullin7 gene removal in mice will lead to intrauterine growth retardation and perinatal infant death, emphasizing its importance for growth regulation. Recently, the insulin receptor substrate 1- species insulin and insulin like growth are identified. The important medium for the conduction of the sub-1 signal is the proteolysis target of the CUL7 E3 ligase, which reveals the molecular linkage between Cullin7 and the fully established pathway of growth regulation. The results, together with other studies demonstrating the interaction between CUL7 and p53 tumor suppressor, and the 40 big T antigen tumor proteins of monkey virus, further hint CUL7 It is a new role in growth control. Some studies have shown that Cullin7 is essential for angiogenesis during the course of cancer germination. The latest research shows that Cullin7 can be expressed in human and mouse tumor endothelial cells. The expression of Cullin7 in breast cancer cells is studied by Guo Hongsheng et al., and the Cullin7 in HCC1937 cells is thinner. Ectopic expression in cell proliferation is significantly increased, while Cullin7 in BT474 cells may lead to a slow growth rate. These results suggest that Cullin7 stimulates the proliferation of breast cancer cells. At the same time, it is the first time that Cullin7 can regulate the expression of p53 in breast cancer cells at the protein level (not mRNA level), suggesting that Cullin7 is used as an alternative mechanism to improve the expression of p53 in breast cancer cells. The invasiveness of high breast cancer cells. However, the effect of Cullin7 on other proto oncogenes and tumor suppressor genes needs further study. Part 1 we detected the difference in Cullin7 expression in normal lung tissue and lung cancer tissues by immunohistochemical staining. The color staining results showed that Cullin7 eggs were in normal lung tissue. The white expression was found only in a small number of cytoplasm and shallower. The Cullin7 protein in the lung cancer tissues was strongly colored in the cytoplasm and nucleus of most of the cells, and the coloration of the nucleus was deeper than that of the cytoplasm. Under the microscope, it was found that the Cullin7 protein staining positive cells in the cancer tissues were much more than those of the normal tissues. The results of the t test showed the difference. The results showed that the expression of Cullin7 protein in lung cancer tissues was significantly higher than that of normal lung tissue. We used Cullin7-siRNA to interfere with the expression of Cullin7 in A427, H460, A549, H1299 cells in lung cancer and detected siRNA interference efficiency by semi quantitative RT-PCR. The results of agar gel electrophoresis showed A427, H460, and control cells. The expression of Cullin7 gene was higher and the expression of Cullin7 gene in H1299 cells was slightly lower. The RNA expression of Cullin7 gene in the A427 cell samples after interference was weaker than that of the control group, and the interference efficiency was relatively low. The RNA expression of Cullin7 gene in H460, A549, H1299 field cell samples was obviously weaker than that of the control group. To determine the effect of Cullin7 on the proliferation of lung cancer cells, we performed MTT tests on four lung cancer cells of A427, H460, A549 and H1299 after transfection of Cullin7-siRNA. The results showed that the absorbance of A427, H460, A549, H1299 four lung cancer cells was 30%, 39%, 57% and 60% lower than the control group after interference of Cullin7. The experimental results suggest that the interference of Cullin7 expression can inhibit the proliferation of lung cancer cells. In order to further clarify the effect of Cullin7 on the proliferation of lung cancer cells, we cloned four cells of A427, H460, A549 and H1299 after transfection. The results showed that the number of four lung cancer cells in A427, H460, A549 and H1299 decreased significantly compared with the control group after interfering Cullin7 expression. The results showed that the interference of Cullin7 expression could inhibit the cell form cloning of lung cancer. The results showed that the expression of Cullin7 was reduced to the lung cancer cells in vitro. The effect of proliferation ability, we take the xenograft tumor model test (nude mouse tumorigenesis experiment) to further detect whether the decrease of Cullin7 expression can affect the proliferation ability of lung cancer cells in vitro. The results of tumor formation in nude mice show that the low expression of Cullin7 in lung cancer cells can inhibit the formation of tumor in vivo, and is manifested in the volume and tumor formation of the tumor. The weight of the t test was significantly lower than that of the lung cancer cell control group, and the difference of the t test was statistically significant (P0.01). The above experiments confirmed that the low expression of Cullin7 could inhibit the tumor formation of lung cancer cells. The second part was to further determine the regulatory pathway of Cullin7 participation, and we used Western-B lot to detect the important regulation after interference of Cullin7 expression. The changes in protein expression showed that after the interference of Cullin7 gene expression in four lung cancer cell lines of A427, H460, A549 and H1299, the amount of Cullin7 protein decreased significantly, and the expression of the three proteins of p53, p27 and p21 increased compared with those of the control group, and the increased levels of the three protein expressions in the A427 and A549 two cells were compared with those of the control. The experimental results suggest that Cullin7 protein may be a negative regulator of the expression of p53, p27 and p21. In tumor cells, p27 and p21 are downstream genes of p53 regulation, so we further verified the regulatory relationship between Cullin7 and p53. P27 and three protein expressions of A549 changes water due to p53 in the A549 lung cancer cell lines. A549 was more significant compared with the control group. We selected A549 for subsequent experiments. The interference of C ullin7siRNA and the common interference between Cullin7siRNA and p53siRNA were used to observe the changes in the number of cloning and formation of A549 cells. The experimental results showed that the number of the clone formation of A549 cells decreased significantly after the Cullin7siRNA interfered with the expression of Cullin7. Less; when the expression of Cullin7 and p53 is interfered with the expression of the A549 cells, the cloning efficiency of the A549 cells can be recovered significantly. The experimental results suggest that Cullin7 may regulate the proliferation of lung cancer cells by inhibiting the expression of p53. To further determine the regulation mechanism of the Cullin7 action on the p53 gene, we detect p53 H2AX by Western-Blot in A549 cells. The expression of phosphorylation (gamma -H2 X) protein expression.Western-Blot showed that when the expression of Cullin7 was disturbed, the expression of p53 protein increased, and the expression of gamma -H2AX protein increased significantly compared to the control group, and the expression of p21 and p27 protein increased significantly. However, when the expression of Cullin7 and p53 was disturbed, the expression of gamma -H2AX protein was relative to the control group. There was no obvious change in the expression of p21 and p27 protein. The results of immunofluorescence staining also showed that interference of Cullin7 expression could lead to increased expression of gamma -H2AX protein, while interference of Cullin7 and p53 expression did not cause significant changes in the expression of gamma -H2AX protein at the same time. The results suggested that Cullin7 may be passed. P53 protein is involved in gamma -H2AX modification and related gene repair pathways to regulate lung cancer cells.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2
【参考文献】
相关期刊论文 前1条
1 刘民培,吴祖泽;肿瘤基因的一种新的功能分类与概念[J];中国肿瘤临床;2004年21期
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