SUMO蛋白在肝细胞癌中差异性调节p65的细胞内定位和稳定性以及影响p65与MANF的相互作用
发布时间：2018-07-26 20:04
【摘要】：SUMO (small ubiquitin-related modifier)蛋白是一类与泛素非常类似的小分子蛋白,参与重要的蛋白质翻译后的修饰——SUMO化修饰(SUMOylation)。SUMO化修饰调节机体的一系列生理学与病理学的过程,与很多疾病的发生发展都存在密切的联系,例如炎症性疾病和炎症相关性疾病,包括肝细胞癌(hepatocellular carcinoma, HCC)。p65是NF-κB家族最重要亚基之一,p65蛋白入核后激活NF-κB信号通路在肝细胞癌的发生发展过程中起重要作用。近年来已有文献证实,在人胚胎肾293T细胞和小鼠3T3纤维细胞中p65蛋白能被外源性的SUMO3蛋白进行SUMO化修饰,但是目前未见p65蛋白与SUMO1、SUMO2蛋白之间相互作用,以及p65蛋白发生SUMO化修饰之后对HCC影响的研究报道。本课题组前期证实MANF (Mesencephalic Astrocyte-derived Neurotrophic factor,中脑新型胶质细胞源性神经营养因子)能在细胞核内与p65存在相互作用从而抑制NF-κB的转录活性,但是SUMO蛋白是否影响p65与MANF的相互作用,未见报道。目的：研究SUMO蛋白在HCC中如何差异性地调节p65细胞内的水平和定位及是否影响p65与MANF的相互作用。方法：选取乙肝患者及HCC患者的肝组织固定石蜡包埋切片,采用免疫组化的方法在人的肝炎组织、HCC患者的癌组织和癌旁组织中检测p65、SUMO1及SUMO2/3蛋白的表达情况；在人肝癌组织及肝癌细胞株中采用免疫荧光双标检测p65与SUMO1或SUMO2/3是否存在细胞内的共定位现象；在人肝癌组织及肝癌细胞株中采用免疫共沉淀的方法验证p65与SUMO1或SUMO2/3之间是否存在相互作用以及p65蛋白是否能发生SUMO化修饰；Western blot在肝癌细胞株中观察肿瘤微环境(如：缺氧及炎症刺激)对p65蛋白的细胞内亚定位以及SUMO1和SUMO2/3蛋白水平的表达是否有影响；免疫共沉淀实验观察肝癌细胞株行缺氧及炎症刺激处理后p65的SUMO化修饰水平是否产生变化；在肝癌细胞株中转染外源性的SUMO1或SUMO2/3,采用Western blot及核浆分离实验观察p65蛋白水平及细胞内亚定位的变化；通过双荧光素酶报告基因实验检测过表达及沉默SUMO1对NF-κB转录活性的影响；在肝癌细胞株中,采用MTT、克隆形成实验、transwell小室实验观察SUMO1及SUMO2/3对肝癌细胞增殖及侵袭能力的影响,以及在人肝癌组织中行免疫荧光双标检测SUMO1与ki67蛋白之间的相关性,评价SUMO1蛋白是否与肝癌细胞的增殖有关；在肝癌细胞株中分别敲低内源性SUMO1或SUMO2/3,免疫共沉淀实验检测p65与MANF蛋白之间的相互作用是否受SUMO蛋白的影响。结果：1.在人肝组织中观察p65、SUMO1以及SUMO2/3蛋白的表达情况免疫组化结果显示,在肝炎组织中有少量SUMO1阳性表达细胞；肝癌组织中SUMO1表达量明显高于癌旁组织,且定位于细胞核中。SUMO2/3的表达与SUMO1不同,SUMO2/3在肝炎组织中有少量表达,细胞质和细胞核中均有分布；癌组织中SUMO2/3的表达明显低于癌旁组织,主要定位于细胞质中。p65蛋白的表达与SUMO2/3非常类似,肝炎组织中少量表达,癌组织低表达,癌旁组织高表达,但是在肝癌组织的胞核中有p65存在。2. SUMO1与p65的相互作用免疫荧光双标显示p65蛋白与SUMO1在HCC患者的癌组织以及肝癌细胞株中存在共定位的现象,主要共定位于细胞核中。在人肝癌组织及肝癌细胞株中,免疫共沉淀实验也发现SUMO1与p65的相互作用,并发现SUMO1可使p65蛋白发生SUMO化修饰。3. SUMO2/3与p65的相互作用人肝组织连续切片的DAB染色显示,p65蛋白与SUMO2/3的表达高度一致。进一步免疫荧光双标实验发现,p65与SUMO2/3共定位于细胞质中；同时SUMO2/3表达高的细胞中p65表达多,SUMO2/3不表达的细胞中,p65表达很少。在HCC患者的肝组织及肝癌细胞株中,免疫共沉淀实验进一步证实SUMO2/3与p65的相互作用,并发现SUMO2/3可使p65蛋白发生SUMO化修饰。4.缺氧及炎症刺激促进SUMO1和SUMO2/3蛋白的表达及p65的SUMO化肝癌细胞株经过缺糖缺氧处理150 min后可发现有部分p65蛋白入核,SUMO1及SUMO2/3蛋白表达量增加。肝癌细胞株经过TNF-a (10ng/ml)处理30 min,部分p65蛋白入核,细胞质中SUMO2/3蛋白水平明显增多,但SUMO1蛋白的水平没有明显变化。当TNF-a(10ng/ml)处理肝癌细胞株8 hrs时,细胞质中SUMO2/3蛋白的水平与TNF处理30 min时无明显变化,而细胞核中SUMO2/3蛋白水平明显增多,细胞质和细胞核中SUMO1蛋白的水平均明显增高。免疫共沉淀实验结果显示：细胞在转染SUMO化修饰E2酶(Ubc9)及外源性的SUMO质粒后24 hrs,缺糖缺氧处理150min能增加SUMO1蛋白相关的p65的SUMO化修饰水平,但并不影响SUMO2/3相关的p65的SUMO化修饰；细胞转染24 hrs后采用TNF-α(10ng/ml)刺激30 min,能同时增加SUMO1及SUMO2/3蛋白相关的p65蛋白SUMO化修饰水平。5. SUMO1蛋白参与调节p65蛋白的入核肝癌细胞株中过表达SUMO1并不影响p65蛋白水平。核浆分离实验发现,过表达SUMO1能增加缺糖缺氧诱导的p65蛋白入核,相反沉默内源性的SUMO1能抑制p65蛋白入核；同时沉默SUMO1能抑制TNF-α刺激诱导的p65蛋白入核,而过表达SUMO1对TNF-a诱导的p65蛋白入核影响不大。6.敲低内源性SUMO1蛋白抑制NF-κB的转录活性在肝癌细胞株中采用双荧光素酶报告基因实验方法,结果显示：正常培养的细胞以及细胞经过TNF-α刺激或缺糖缺氧处理,过表达SUMO1都能抑制NF-κB的转录活性；而敲低内源性的SUMO1也能抑制NF-κB的转录活性。7.敲低内源性SUMO1抑制肝癌细胞株的增殖与转移侵袭能力MTT、克隆形成实验证实：敲除内源性SUMO1抑制了细胞的增殖能力。于是我们进一步在人肝细胞癌的癌组织中采用免疫荧光双标观察ki67和SUMO1的相关性,发现：所有ki67阳性的细胞,SUMO1都是表达阳性。于是我们推测：SUMO1蛋白的表达可能与肝癌细胞的增殖能力有关。transwell实验显示,过表达SUMO1能增加肝癌细胞株的转移侵袭能力,沉默SUMO1抑制了细胞的转移侵袭能力。8. SUMO2/3稳定胞质中p65蛋白的水平与其拮抗p65的泛素化有关肝癌细胞株中过表达SUMO2/3发现,p65蛋白水平随转染SUMO2/3的DNA增多而增高。进一步核浆分离实验发现,SUMO2/3蛋白主要是维持p65蛋白在细胞质中的稳定性。在肝癌细胞株中过表达SUMO2/3检测p65的mRNA水平,发现SUMO2/3并不影响p65的转录水平。同时免疫共沉淀实验发现,当SUMO2/3蛋白相关的p65的SUMO化修饰增多时,p65蛋白的泛素化减少。该结果提示SUMO2/3蛋白可能是通过拮抗p65的泛素化,减少p65蛋白降解来稳定胞质中的p65蛋白水平。9.过表达SUMO2/3蛋白抑制肝癌细胞株的增殖MTT实验发现,在肝癌细胞株中过表达SUMO2/3抑制了肝癌细胞株的增殖能力,SUMO2/3并不影响肝癌细胞株的转移与侵袭能力。10. SUMO蛋白影响p65与MANF之间的相互作用免疫共沉淀实验发现,在肝癌细胞株中敲低内源性SUMO1或SUMO2/3, p65与MANF蛋白之间的相互作用减少。结论：1. SUMO1和SUMO2/3能与p65相互作用并对其进行SUMO化修饰；2.缺糖缺氧和TNF-α能增强SUMO蛋白与p65的相互作用；3.敲低SUMO1能抑制缺糖缺氧和TNF-α诱导的p65蛋白入核,并能抑制NF-κB的转录活性；4. SUMO2/3能稳定胞质中的p65蛋白水平；5.p65蛋白的泛素修饰拮抗它的SUMO化；6. SUMO1能促进肝癌细胞的增殖与转移侵袭能力；7.过表达SUMO2/3能抑制肝癌细胞的增殖；8.敲低SUMO1或SUMO2/3影响p65与MANF之间的相互作用。
[Abstract]:SUMO (small ubiquitin-related modifier) protein is a kind of small molecular protein that is very similar to ubiquitin, and participates in a series of physiological and pathological processes of SUMO modification (SUMOylation) modification (SUMOylation).SUMO modification, which is closely related to the occurrence and development of many diseases, for example. Hepatocellular carcinoma (HCC).P65 is one of the most important subunits of the NF- kappa B family, and the activation of NF- kappa B signaling pathway after p65 protein nucleation plays an important role in the development of hepatocellular carcinoma. In recent years, Yi Youwen has demonstrated that in human embryonic kidney 293T cells and mouse 3T3 fibers P65 protein in cells can be modified by exogenous SUMO3 protein by SUMO, but there is no interaction between p65 protein and SUMO1, SUMO2 protein and the effect of SUMO modification on p65 protein. This research group confirmed MANF (Mesencephalic Astrocyte-derived), new medium brain glue. The cytokine derived neurotrophic factor (PNF) can interact with p65 in the nucleus to inhibit the transcriptional activity of NF- kappa B, but it is not reported whether SUMO protein affects the interaction between p65 and MANF. The purpose of this study is to investigate how the SUMO protein can regulate the level and localization of p65 cells in HCC and whether p65 and MANF are influenced by the SUMO protein. Methods: the liver tissues of patients with hepatitis B and HCC were fixed in paraffin embedded section, and the expression of p65, SUMO1 and SUMO2/3 protein were detected by immunohistochemical method in human hepatitis tissue, cancer tissue and para cancerous tissue of HCC patients; p65 and SUMO were detected by double immunofluorescence in human liver cancer tissue and liver cancer cell lines. Whether there is a co localization phenomenon within 1 or SUMO2/3 cells; the interaction between p65 and SUMO1 or SUMO2/3 by immunoprecipitation in human hepatocellular carcinoma and hepatoma cell lines and whether the p65 protein can be modified by SUMO; Western blot is used to observe the microenvironment (such as hypoxia and inflammation) in the hepatocellular carcinoma cell lines. Whether the subcellular localization of p65 protein and the expression of SUMO1 and SUMO2/3 protein have an effect on the expression of SUMO1 and SUMO2/3 protein, and the changes in the SUMO modification level of the liver cancer cell lines after the treatment of hypoxia and inflammatory stimulation were observed. The transfection of exogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines was used to use Western blot. The changes of p65 protein level and subcellular localization were observed and the effects of SUMO1 on NF- kappa B transcriptional activity were detected by double luciferase reporter gene experiment. In the hepatocellular carcinoma cell lines, MTT, clone formation experiment, and Transwell chamber experiment were used to observe the proliferation and invasion of liver cancer cells by SUMO1 and SUMO2/3. The effect of the ability and the correlation between SUMO1 and Ki67 protein in the human liver cancer tissue, to evaluate whether the SUMO1 protein is related to the proliferation of hepatoma cells; to knock down the endogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines, and to detect the interaction between p65 and MANF protein by the immunoprecipitation test. Results: 1. in human liver tissue, the expression of p65, SUMO1 and SUMO2/3 protein was observed. The immunohistochemical results showed that there was a small number of SUMO1 positive cells in the hepatitis tissues, and the expression of SUMO1 in the liver tissues was significantly higher than that in the paracancerous tissues, and the expression of.SUMO2/3 in the nucleus was different from that of SUMO1. SUMO2/3 in hepatitis was in hepatitis. There were a small amount of expression in the tissue, cytoplasm and nucleus in the nucleus. The expression of SUMO2/3 in the cancer tissues was obviously lower than that in the paracancerous tissues. The expression of.P65 protein in the cytoplasm was very similar to that of SUMO2/3. A small amount of expression in the hepatitis tissue, the low expression of the cancer tissue and high expression of the para cancer tissues were found, but there were p65 in the nucleus of the liver cancer. The interaction of immunofluorescence between.2. SUMO1 and p65 showed that p65 protein was Co located with SUMO1 in the cancer tissue of HCC patients and in the hepatocellular carcinoma cell lines, and was mainly located in the nucleus. The interaction between SUMO1 and p65 was also found in the immunoprecipitation experiment in human liver cancer tissue and liver cancer cell lines, and found SUMO1 can make p65. The DAB staining of the interaction between.3. SUMO2/3 and p65 showed that the p65 protein was highly consistent with the expression of SUMO2/3. Further immunofluorescence double labeling experiment found that p65 and SUMO2/3 were Co located in the cytoplasm, and the p65 expression in the cells with high expression of SUMO2/3 was more than that in the cells that were not expressed in SUMO2/3. 5 there are few expressions. In the liver and liver cancer cell lines of HCC patients, the immunoprecipitation experiment further confirms the interaction between SUMO2/3 and p65, and it is found that SUMO2/3 can make p65 protein SUMO modified.4. anoxia and inflammatory stimulation to promote the expression of SUMO1 and SUMO2/3 protein and p65 SUMO hepatoma cell line through glucose deficiency anoxia treatment 150 min. After that, some p65 protein was found to be nucleed and the expression of SUMO1 and SUMO2/3 protein increased. The hepatocellular carcinoma cell line was treated with TNF-a (10ng/ml) 30 min, some of the p65 protein was nucleed, the level of SUMO2/3 protein in the cytoplasm increased obviously, but the level of SUMO1 protein was not obviously changed. When TNF-a (10ng/ml) treated the hepatocellular carcinoma cell line 8 hrs, the cytoplasm The level of protein and TNF treatment 30 min did not change obviously, but the level of SUMO2/3 protein in the nucleus increased obviously, and the level of SUMO1 protein in cytoplasm and nucleus increased obviously. The result of immunoprecipitation experiment showed that the cells were 24 hrs after transfection of SUMO modified E2 enzyme (Ubc9) and exogenous SUMO plasmid, and 150min could be increased by oxygen deficiency anoxic treatment 150min. The SUMO modification level of p65 associated with SUMO1 protein does not affect the SUMO modification of p65 related to SUMO2/3; cells transfected with TNF- a (10ng/ml) stimulates 30 min after transfection of 24 hrs, and can simultaneously increase the level of SUMO1 and SUMO2/3 protein related protein SUMO1 does not affect the level of p65 protein. The overexpression of SUMO1 can increase the nucleation of p65 protein induced by oxygen deficiency and hypoxia, but the silent endogenous SUMO1 can inhibit the nucleation of p65 protein, while silent SUMO1 can inhibit the nucleation of p65 protein induced by TNF- alpha, and the overexpression of SUMO1 has no effect on TNF-a induced nucleation of the p65 protein. The transcriptional activity of.6. knockout low endogenous SUMO1 protein inhibited NF- kappa B in the hepatocellular carcinoma cell lines using the double luciferase reporter gene test method. The results showed that the transcriptional activity of NF- kappa B could be inhibited by normal cultured cells and cells after TNF- alpha stimulation or glucose deficiency anoxia treatment, and the low endogenous SUMO1 could also be suppressed. NF- kappa B transcriptional activity.7. knocks low endogenous SUMO1 to inhibit the proliferation and metastasis of hepatocellular carcinoma cell line MTT. Cloning experiments confirmed that knockout endogenous SUMO1 inhibited cell proliferation. We further observed the correlation of Ki67 and SUMO1 by immunofluorescence in human hepatocellular carcinoma tissue. All Ki67 positive cells were positive for SUMO1, so we speculated that the expression of SUMO1 protein may be related to the proliferation ability of hepatoma cells in.Transwell experiments. Overexpression of SUMO1 can increase the metastasis and invasion ability of liver cancer cell lines, and silence SUMO1 inhibits the transformation and invasion of.8. SUMO2/3 stable cytoplasm in.8. SUMO2/3 The level of the overexpression of SUMO2/3 in the hepatoma cell line related to its antagonism to the ubiquitination of p65 found that the level of p65 protein increased with the increase of DNA in the transfected SUMO2/3. Further nuclear plasma separation experiments found that SUMO2/3 protein mainly maintained the stability of the p65 protein in the cytoplasm. In the liver cancer cell lines, the level of mRNA was expressed by SUMO2/3 to detect p65. SUMO2/3 does not affect the transcriptional level of p65. At the same time, the immunoprecipitation experiment shows that the ubiquitination of p65 protein decreases when the SUMO modification of SUMO2/3 protein related p65 increases. The results suggest that the SUMO2/3 protein may be by antagonizing the ubiquitination of p65, reducing the p65 protein degradation to stabilize the p65 protein level in the cytoplasm and.9. over expression SUMO2/3. The inhibitory effect of protein inhibition on the proliferation of hepatocellular carcinoma cell strain MTT showed that overexpression of SUMO2/3 in hepatoma cells inhibited the proliferation of hepatocellular carcinoma cell lines, SUMO2/3 did not affect the metastasis and invasion of hepatoma cell lines,.10. SUMO protein affected the interaction between p65 and MANF, and the immune co precipitation experiment was found in the liver cancer cell lines. The interaction between the source SUMO1 or SUMO2/3, p65 and MANF protein decreased. Conclusion: 1. SUMO1 and SUMO2/3 can interact with p65 and modify them with SUMO; 2. the glucose deficiency anoxia and TNF- alpha can enhance the interaction between SUMO protein and p65; 3. knock low SUMO1 can inhibit the lack of glucose and hypoxia and the nucleation of the protein induced by alpha. The transcriptional activity of 4. SUMO2/3 can stabilize the level of p65 protein in the cytoplasm; the ubiquitin modification of 5.p65 protein antagonists its SUMO; 6. SUMO1 can promote the proliferation and metastasis of hepatoma cells; 7. overexpression of SUMO2/3 can inhibit the proliferation of hepatoma cells; 8. knock low SUMO1 or SUMO2/3 to respond to the interaction between p65 and MANF.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
[Abstract]:SUMO (small ubiquitin-related modifier) protein is a kind of small molecular protein that is very similar to ubiquitin, and participates in a series of physiological and pathological processes of SUMO modification (SUMOylation) modification (SUMOylation).SUMO modification, which is closely related to the occurrence and development of many diseases, for example. Hepatocellular carcinoma (HCC).P65 is one of the most important subunits of the NF- kappa B family, and the activation of NF- kappa B signaling pathway after p65 protein nucleation plays an important role in the development of hepatocellular carcinoma. In recent years, Yi Youwen has demonstrated that in human embryonic kidney 293T cells and mouse 3T3 fibers P65 protein in cells can be modified by exogenous SUMO3 protein by SUMO, but there is no interaction between p65 protein and SUMO1, SUMO2 protein and the effect of SUMO modification on p65 protein. This research group confirmed MANF (Mesencephalic Astrocyte-derived), new medium brain glue. The cytokine derived neurotrophic factor (PNF) can interact with p65 in the nucleus to inhibit the transcriptional activity of NF- kappa B, but it is not reported whether SUMO protein affects the interaction between p65 and MANF. The purpose of this study is to investigate how the SUMO protein can regulate the level and localization of p65 cells in HCC and whether p65 and MANF are influenced by the SUMO protein. Methods: the liver tissues of patients with hepatitis B and HCC were fixed in paraffin embedded section, and the expression of p65, SUMO1 and SUMO2/3 protein were detected by immunohistochemical method in human hepatitis tissue, cancer tissue and para cancerous tissue of HCC patients; p65 and SUMO were detected by double immunofluorescence in human liver cancer tissue and liver cancer cell lines. Whether there is a co localization phenomenon within 1 or SUMO2/3 cells; the interaction between p65 and SUMO1 or SUMO2/3 by immunoprecipitation in human hepatocellular carcinoma and hepatoma cell lines and whether the p65 protein can be modified by SUMO; Western blot is used to observe the microenvironment (such as hypoxia and inflammation) in the hepatocellular carcinoma cell lines. Whether the subcellular localization of p65 protein and the expression of SUMO1 and SUMO2/3 protein have an effect on the expression of SUMO1 and SUMO2/3 protein, and the changes in the SUMO modification level of the liver cancer cell lines after the treatment of hypoxia and inflammatory stimulation were observed. The transfection of exogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines was used to use Western blot. The changes of p65 protein level and subcellular localization were observed and the effects of SUMO1 on NF- kappa B transcriptional activity were detected by double luciferase reporter gene experiment. In the hepatocellular carcinoma cell lines, MTT, clone formation experiment, and Transwell chamber experiment were used to observe the proliferation and invasion of liver cancer cells by SUMO1 and SUMO2/3. The effect of the ability and the correlation between SUMO1 and Ki67 protein in the human liver cancer tissue, to evaluate whether the SUMO1 protein is related to the proliferation of hepatoma cells; to knock down the endogenous SUMO1 or SUMO2/3 in the hepatocellular carcinoma cell lines, and to detect the interaction between p65 and MANF protein by the immunoprecipitation test. Results: 1. in human liver tissue, the expression of p65, SUMO1 and SUMO2/3 protein was observed. The immunohistochemical results showed that there was a small number of SUMO1 positive cells in the hepatitis tissues, and the expression of SUMO1 in the liver tissues was significantly higher than that in the paracancerous tissues, and the expression of.SUMO2/3 in the nucleus was different from that of SUMO1. SUMO2/3 in hepatitis was in hepatitis. There were a small amount of expression in the tissue, cytoplasm and nucleus in the nucleus. The expression of SUMO2/3 in the cancer tissues was obviously lower than that in the paracancerous tissues. The expression of.P65 protein in the cytoplasm was very similar to that of SUMO2/3. A small amount of expression in the hepatitis tissue, the low expression of the cancer tissue and high expression of the para cancer tissues were found, but there were p65 in the nucleus of the liver cancer. The interaction of immunofluorescence between.2. SUMO1 and p65 showed that p65 protein was Co located with SUMO1 in the cancer tissue of HCC patients and in the hepatocellular carcinoma cell lines, and was mainly located in the nucleus. The interaction between SUMO1 and p65 was also found in the immunoprecipitation experiment in human liver cancer tissue and liver cancer cell lines, and found SUMO1 can make p65. The DAB staining of the interaction between.3. SUMO2/3 and p65 showed that the p65 protein was highly consistent with the expression of SUMO2/3. Further immunofluorescence double labeling experiment found that p65 and SUMO2/3 were Co located in the cytoplasm, and the p65 expression in the cells with high expression of SUMO2/3 was more than that in the cells that were not expressed in SUMO2/3. 5 there are few expressions. In the liver and liver cancer cell lines of HCC patients, the immunoprecipitation experiment further confirms the interaction between SUMO2/3 and p65, and it is found that SUMO2/3 can make p65 protein SUMO modified.4. anoxia and inflammatory stimulation to promote the expression of SUMO1 and SUMO2/3 protein and p65 SUMO hepatoma cell line through glucose deficiency anoxia treatment 150 min. After that, some p65 protein was found to be nucleed and the expression of SUMO1 and SUMO2/3 protein increased. The hepatocellular carcinoma cell line was treated with TNF-a (10ng/ml) 30 min, some of the p65 protein was nucleed, the level of SUMO2/3 protein in the cytoplasm increased obviously, but the level of SUMO1 protein was not obviously changed. When TNF-a (10ng/ml) treated the hepatocellular carcinoma cell line 8 hrs, the cytoplasm The level of protein and TNF treatment 30 min did not change obviously, but the level of SUMO2/3 protein in the nucleus increased obviously, and the level of SUMO1 protein in cytoplasm and nucleus increased obviously. The result of immunoprecipitation experiment showed that the cells were 24 hrs after transfection of SUMO modified E2 enzyme (Ubc9) and exogenous SUMO plasmid, and 150min could be increased by oxygen deficiency anoxic treatment 150min. The SUMO modification level of p65 associated with SUMO1 protein does not affect the SUMO modification of p65 related to SUMO2/3; cells transfected with TNF- a (10ng/ml) stimulates 30 min after transfection of 24 hrs, and can simultaneously increase the level of SUMO1 and SUMO2/3 protein related protein SUMO1 does not affect the level of p65 protein. The overexpression of SUMO1 can increase the nucleation of p65 protein induced by oxygen deficiency and hypoxia, but the silent endogenous SUMO1 can inhibit the nucleation of p65 protein, while silent SUMO1 can inhibit the nucleation of p65 protein induced by TNF- alpha, and the overexpression of SUMO1 has no effect on TNF-a induced nucleation of the p65 protein. The transcriptional activity of.6. knockout low endogenous SUMO1 protein inhibited NF- kappa B in the hepatocellular carcinoma cell lines using the double luciferase reporter gene test method. The results showed that the transcriptional activity of NF- kappa B could be inhibited by normal cultured cells and cells after TNF- alpha stimulation or glucose deficiency anoxia treatment, and the low endogenous SUMO1 could also be suppressed. NF- kappa B transcriptional activity.7. knocks low endogenous SUMO1 to inhibit the proliferation and metastasis of hepatocellular carcinoma cell line MTT. Cloning experiments confirmed that knockout endogenous SUMO1 inhibited cell proliferation. We further observed the correlation of Ki67 and SUMO1 by immunofluorescence in human hepatocellular carcinoma tissue. All Ki67 positive cells were positive for SUMO1, so we speculated that the expression of SUMO1 protein may be related to the proliferation ability of hepatoma cells in.Transwell experiments. Overexpression of SUMO1 can increase the metastasis and invasion ability of liver cancer cell lines, and silence SUMO1 inhibits the transformation and invasion of.8. SUMO2/3 stable cytoplasm in.8. SUMO2/3 The level of the overexpression of SUMO2/3 in the hepatoma cell line related to its antagonism to the ubiquitination of p65 found that the level of p65 protein increased with the increase of DNA in the transfected SUMO2/3. Further nuclear plasma separation experiments found that SUMO2/3 protein mainly maintained the stability of the p65 protein in the cytoplasm. In the liver cancer cell lines, the level of mRNA was expressed by SUMO2/3 to detect p65. SUMO2/3 does not affect the transcriptional level of p65. At the same time, the immunoprecipitation experiment shows that the ubiquitination of p65 protein decreases when the SUMO modification of SUMO2/3 protein related p65 increases. The results suggest that the SUMO2/3 protein may be by antagonizing the ubiquitination of p65, reducing the p65 protein degradation to stabilize the p65 protein level in the cytoplasm and.9. over expression SUMO2/3. The inhibitory effect of protein inhibition on the proliferation of hepatocellular carcinoma cell strain MTT showed that overexpression of SUMO2/3 in hepatoma cells inhibited the proliferation of hepatocellular carcinoma cell lines, SUMO2/3 did not affect the metastasis and invasion of hepatoma cell lines,.10. SUMO protein affected the interaction between p65 and MANF, and the immune co precipitation experiment was found in the liver cancer cell lines. The interaction between the source SUMO1 or SUMO2/3, p65 and MANF protein decreased. Conclusion: 1. SUMO1 and SUMO2/3 can interact with p65 and modify them with SUMO; 2. the glucose deficiency anoxia and TNF- alpha can enhance the interaction between SUMO protein and p65; 3. knock low SUMO1 can inhibit the lack of glucose and hypoxia and the nucleation of the protein induced by alpha. The transcriptional activity of 4. SUMO2/3 can stabilize the level of p65 protein in the cytoplasm; the ubiquitin modification of 5.p65 protein antagonists its SUMO; 6. SUMO1 can promote the proliferation and metastasis of hepatoma cells; 7. overexpression of SUMO2/3 can inhibit the proliferation of hepatoma cells; 8. knock low SUMO1 or SUMO2/3 to respond to the interaction between p65 and MANF.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
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